INTRODUCTION: Ovarian metastases from colorectal cancer (CRC) are uncommon but are associated with poor prognosis and therapeutic challenges. These lesions may exhibit discordant responses to systemic therapy, suggesting site-specific biological heterogeneity. However, the molecular characteristics of CRC-associated ovarian metastases remain insufficiently defined. We aimed to characterize the circulating tumor deoxyribonucleic acid (ctDNA) landscape of ovarian metastatic CRC using a plasma-based deep sequencing approach. METHODS: Plasma samples from patients with CRC and ovarian metastases were analyzed using cancer personalized profiling by deep sequencing. Pathogenic mutations and copy-number alterations were identified to delineate the genomic features of this metastatic subset. RESULTS: Pathogenic genomic alterations were detected in the majority of patients. Canonical CRC driver mutations, including TP53, KRAS, and APC, were observed at frequencies comparable to those reported in large-scale genomic datasets such as The Cancer Genome Atlas. In addition, recurrent amplification of growth factor receptor genes, including EGFR, MET, and ERBB2, was identified. Detectable tumor-derived alterations in plasma indicate active ctDNA shedding from ovarian lesions, supporting the feasibility of noninvasive genomic profiling in this setting. CONCLUSIONS: CRC-associated ovarian metastases retain the core genomic architecture of colorectal tumorigenesis while demonstrating recurrent receptor tyrosine kinase amplifications that may reflect adaptive clonal evolution within the ovarian microenvironment. Plasma-based ctDNA profiling provides a feasible approach for molecular characterization of this rare metastatic phenotype and may contribute to improved understanding of site-specific tumor biology.

Intra-abdominal epithelioid neoplasm with EWSR1/FUS::CREB fusions is an emerging entity characterized by a broad age distribution, epithelioid morphology, variable epithelial marker expression, prominent lymphoplasmacytic infiltration, and systemic inflammation. A few ovarian cases have been reported. We describe a 63-yr-old woman who presented with anemia and elevated C-reactive protein. She underwent surgery for a 14-cm right ovarian mass. Grossly, the tumor was solid with cystic change and hemorrhage, and had a light tan cut surface. Histologically, it consisted of uniform sheets of epithelioid cells with ample pale eosinophilic cytoplasm, divided by fibrotic septa with dense lymphoplasmacytic infiltration. Immunohistochemically, the tumor was positive for EMA, WT1, and vimentin; focally positive for CAM5.2; and negative for AE1/AE3, estrogen and progesterone receptors, PAX8, sex cord markers, desmin, HMB45, and Melan A. The Ki-67 labeling index was 20%. The differential diagnoses, including poorly differentiated carcinoma, sex cord-stromal tumors, perivascular epithelioid cell tumor, and inflammatory myofibroblastic tumor, were considered. Whole-genome sequencing revealed a FUS::CREM gene fusion. Based on clinicopathologic and genomic features, the tumor was classified as an ovarian example of EWSR1/FUS::CREB fusion-associated epithelioid neoplasm. Inflammation-related laboratory abnormalities resolved postoperatively. No adjuvant therapy was administered, and the patient remained disease-free at 12 mo. This represents the third reported ovarian tumor with FUS::CREM fusion and the seventh adnexal tumor with EWSR1/FUS::CREB family fusion. Prognostic information on these adnexal tumors is limited, but given the aggressive nature of analogous extra-adnexal and testicular tumors, cautious management and further studies are warranted.

BACKGROUND: In HER2-positive advanced or recurrent gastric cancer, tissue-based HER2 assessment has been established; however, the clinical relevance of ERBB2 copy number (CN) measured from plasma cell-free DNA (cfDNA), particularly its longitudinal change during treatment, remains unclear. METHODS: This was a prespecified ancillary correlative analysis of the phase II KSCC-TROX trial (jRCTs071190007; UMIN000036867), evaluating whether changes in cfDNA ERBB2 CN (ΔCN) reflect clinical outcomes. Of the 66 patients included in the TROX trial analysis set, 55 with baseline blood samples comprised Cohort A, and 34 with paired follow-up samples comprised Cohort B. ERBB2 CN was quantified using droplet digital PCR. RESULTS: Baseline ERBB2 CN was associated with tumor HER2 immunohistochemistry score but not with progression-free survival (PFS) or overall survival (OS) when dichotomized at a cut-off of 2.5. In Cohort B, an increase in ERBB2 CN (ΔCN > 0) was significantly associated with shorter PFS compared with ΔCN ≤ 0 (p = 0.008), which was maintained in multivariable analysis (p = 0.001). OS was also significantly shorter in patients with ΔCN > 0 (p = 0.024). CONCLUSIONS: An increase in cfDNA ERBB2 CN during treatment was independently associated with shorter PFS in HER2-positive advanced gastric cancer. Assessment of ERBB2 CN change may provide a useful biomarker for treatment monitoring and prognostic stratification.

Sarcopenia has been reported that as a prognostic factor in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs), mainly based on retrospective, imaging-based analyses. However, its prognostic value has not been prospectively evaluated. In this prospective study we investigated the clinical impact of sarcopenia and explored circulating proteins associated with muscle loss and ICI outcomes. Patients with advanced NSCLC who received ICIs as first-line therapy were enrolled. Before treatment initiation, muscle mass was assessed using bioelectrical impedance analysis, together with grip strength, walking speed, and physical activity. Pretreatment plasma samples were subjected to comprehensive protein profiling. Between October 2019 and August 2022, 41 patients were enrolled. Bothe overall survival and progression-free survival were significantly shorter in patients with sarcopenia. Notably, patients with pre-sarcopenia, defined as reduced muscle mass without impairment of muscle strength or physical performance, also exhibited poor clinical outcomes. Protein analysis identified penraxin-3 as being significantly associated with muscle loss and overall survival. These findings demonstrate, for the first time in a prospective setting, that even pre-sarcopenia is associated with poor prognosis in NSCLC patients treated with ICIs, and suggest that pentraxin-3 may serve as a biomarker linking sarcopenia to unfavorable outcomes.

INTRODUCTION: A previous analysis of the RELAY phase 3 liquid biopsy addendum demonstrated suppressed EGFR-activating mutation allele count, increased total cell-free DNA (cfDNA) concentration, and shortened cfDNA fragment size with ramucirumab (RAM) plus erlotinib (ERL) versus placebo (PL) plus ERL in patients with EGFR-mutated NSCLC. We report updated data by EGFR mutation subtypes. METHODS: Patients were randomized 1:1 to RAM plus ERL or PL plus ERL. Liquid biopsy samples were collected at baseline, during treatment, and at post-progression 30-day follow-up. EGFR-activating mutation allele count, plasma cfDNA concentration, cfDNA fragment size, and treatment-emergent T790M were analyzed in patients with a valid baseline sample (N = 131) by EGFR mutation subgroups (ex19del; L858R). RESULTS: Within both ex19del and L858R subgroups, EGFR-activating mutation allele count decreased from baseline to cycle 4 in both treatment arms. Total cfDNA concentration significantly increased from baseline to cycle 4 (p < 0.0001) and was sustained to follow-up in the RAM plus ERL arm within the L858R subgroup only. cfDNA fragment size decreased from baseline to cycle 4 in the RAM plus ERL arm within both subgroups. T790M rates in the RAM plus ERL and PL plus ERL arms were 22.2% (four of 18) and 52.9% (nine of 17), respectively, in the ex19del subgroup by cycle less than or equal to 155 and 31.8% (seven of 22) and 18.8% (three of 16), respectively, in the L858R subgroup by cycle less than or equal to 176. CONCLUSIONS: This updated analysis revealed differences in total cfDNA concentration and T790M mutation rates between ex19del and L858R. RAM plus ERL may exhibit greater antitumor effects on L858R, supporting the favorable survival benefits observed previously in patients with a L858R.

UNLABELLED: KRASG12C inhibitors, such as sotorasib, show clinical efficacy for non-small cell lung cancer (NSCLC) positive for the G12C mutations of KRAS, but primary and acquired resistance to these drugs remains a clinical problem. In this study, we show that the development of resistance to sotorasib in KRASG12C-positive NSCLC cells was mediated by constitutive activation of EGFR resulting from downregulation of the protein tyrosine phosphatase receptor type R (PTPRR). PTPRR has been identified as a physiologic regulator of ERK signaling in several cancer types. In our study, PTPRR was demonstrated to bind directly to EGFR, facilitating its dephosphorylation on tyrosine residues. Resumption of PTPRR expression in the resistant cells attenuated EGFR phosphorylation and restored sotorasib sensitivity. PTPRR downregulation was associated with gene promoter hypermethylation in the sotorasib-resistant cells and NSCLC tissue samples. Furthermore, low PTPRR expression in tumor specimens was associated with shorter progression-free and overall survival for patients with NSCLC treated with sotorasib. In contrast to sotorasib, high PTPRR expression was associated with a poor response to EGFR tyrosine kinase inhibitors in EGFR-mutated NSCLC, suggesting that PTPRR may broadly regulate EGFR dependence in NSCLC. Finally, dual blockade of KRASG12C and EGFR showed a substantial antitumor effect in a xenograft model of sotorasib-resistant NSCLC. This approach is therefore a rational therapeutic strategy for KRASG12C-positive NSCLC, especially for tumors showing PTPRR downregulation. SIGNIFICANCE: The current study shows that downregulation of PTPRR induces EGFR activation and resistance to KRASG12C inhibitors in NSCLC, suggesting dual KRAS-EGFR blockade as a rational therapy. PTPRR may help identify patient subgroups that would benefit from the addition of EGFR inhibitors to KRASG12C-targeted therapies.

BACKGROUND: Liquid biopsy using circulating tumor DNA (ctDNA) is a minimally invasive approach for detecting tumor-associated genomic alterations. Although ctDNA analysis has been widely explored in solid tumors, its application to cervical cancer remains limited. Cancer personalized profiling by deep sequencing (CAPP-Seq) enables sensitive ctDNA profiling via molecular barcoding and digital error suppression. METHODS: We evaluated the feasibility of ctDNA-based mutation profiling in cervical cancer using the CAPP-Seq platform by analyzing plasma samples from 38 patients. RESULTS: The cohort included three patients with stage I disease, nine with stage II, 19 with stage III, and seven with stage IV. Somatic gene alterations were detected in 33 of the 38 cases (87%), including squamous cell carcinoma (27/29 [93%]) and adenocarcinoma (6/9 [67%]). Non-synonymous mutations were identified in 23 patients (59%), with PIK3CA being the most frequently mutated gene [13/38 (34%)]. Copy number gains of EGFR, MET, and ERBB2 were observed in 24%, 11%, and 5% of cases, respectively. The median blood tumor mutational burden was 17.7 mutations/Mb, and 50% of the patients exhibited a hypermutated phenotype. In a subset of four patients who received concurrent chemoradiotherapy, longitudinal changes in ctDNA mutation profiles between pre- and post-treatment samples were associated with treatment response. CONCLUSIONS: This study demonstrates the feasibility of ctDNA-based mutation profiling using CAPP-Seq in cervical cancer, with a high detection rate of tumor-associated genomic alterations across histological subtypes. ctDNA analysis may represent a minimally invasive approach for the molecular characterization and disease monitoring of cervical cancer.

BACKGROUND: RNA-binding motif 10 (RBM10) mutation in non-small-cell lung cancer (NSCLC) is associated with decreased sensitivity to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in vitro and in patients who received osimertinib as neoadjuvant treatment or palliative systemic therapy. The incidence of this mutation in early-stage NSCLC and the relationship with other mutations are unknown. MATERIALS AND METHODS: We analyzed the clinical and genomic data of 190 patients with NSCLC who underwent surgical resection between June 2022 and April 2024. Genomic data were obtained from whole-exome sequencing performed in an ongoing multicenter prospective observational study. RESULTS: RBM10 mutation was detected in 17 of 152 patients with nonsquamous NSCLC (11%) and not detected in 38 patients with squamous cell carcinomas. The incidences of RBM10 mutation were higher in tumors with EGFR mutation (21%) and tumors with KRAS mutation (12%) compared with those without EGFR/KRAS mutations (2%, P < .001 and P = .07, respectively). In tumors with EGFR mutation (N = 68), RBM10 mutation was significantly associated with age (> 76 years, P < .01), the presence of ground-glass opacity (P < 0.05), and histological grade 1 (P < .05). We observed mutually exclusive relationships between RBM10 mutation, TP53 mutation, and MDM2 gene amplification, and a high incidence of RBM10 mutation or MDM2 gene amplification in tumors with EGFR L858R mutation/uncommon mutation. CONCLUSION: RBM10 mutation is frequent in Japanese patients with NSCLC with EGFR mutation, especially those with L858R or uncommon mutations, and was associated with late-onset and features of indolent tumor growth.

BACKGROUNDImmune checkpoint inhibitors (ICIs) targeting the programmed cell death 1 axis have revolutionized metastatic non-small cell lung cancer (mNSCLC) treatment. However, disease progression remains a concern, and the role of the complex tumor microenvironment (TME) in treatment failure is not fully understood.METHODSIn this biomarker study involving 103 patients with mNSCLC, including 81 patients who received ICI treatment, we evaluated the association between heterogeneous immune cell subsets and ICI efficacy through single-cell spatial profiling of pretreatment tumor tissue, using a 29-marker multiplex IHC platform built for in-depth dissection of the TME.RESULTSAmong various types of intratumoral lymphocytes, including Th1, Treg, and NK cells, only CD8+ T cells (tumor-infiltrating lymphocytes [TILs]) were associated with ICI efficacy. Computational tissue segmentation underscored the importance of direct physical interactions between CD8+ TILs and cancer cells for ICI efficacy. TIL phenotyping identified CD39/CD103/Ki-67 positivity as a hallmark of exhausted yet functional tumor-reactive CD8+ TILs. Immunosuppressive tumor-associated macrophages (TAMs) and cancer-associated fibroblasts were independent unfavorable adversaries. High CD73 expression on cancer cells was suggested to confer tolerance to ICI in EGFR/ALK-oncogene+ NSCLC, potentially through M2-TAM accumulation and aberrant angiogenesis.CONCLUSIONOur study delineates the clinical relevance of heterogeneous immune cell subsets in ICI-treated mNSCLC, aiding the development of targeted therapeutic strategies.FUNDINGOsaka Cancer Society, KANAE Foundation for the Promotion of Medical Science, SGH Foundation, and YOKOYAMA Foundation for Clinical Pharmacology.

IntroductionGlycolysis-driven hepatocellular carcinoma (HCC) has been associated with resistance to immune checkpoint inhibitors; however, clinical implementation of glycolysis-targeted therapies remains limited by the reliance on transcriptomic subclassification.MethodsWe developed a logistic regression model using clinical and pathological features to predict transcriptome-defined glycolysis subclass (training cohort, n = 113). An integer-based scoring system was derived for bedside application. Model performance was assessed using classification metrics, with external validation in TCGA-LIHC cohort. Imaging correlations were analyzed with 18F-fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography/computed tomography (PET/CT) and magnetic resonance imaging (MRI) in a clinical cohort (n = 133) lacking transcriptomic data. In addition, pathology-free LASSO model was also developed using 18F-FDG PET/CT, contrast-enhanced CT/Gd-MRI, and serum biomarkers.Results The integer logistic model (cutoff ≥3), incorporating serum α-fetoprotein, serum albumin, arterial enhancement pattern, and histopathological phenotypes (macrotrabecular-massive, compact, and pseudoglandular), achieved an AUROC of 0.905, accuracy of 0.850, and F1 score of 0.80 in the training cohort, with consistent performance across subgroups. External validation using the fully annotated subset of the TCGA-LIHC cohort (n = 70) demonstrated preserved model performance (AUROC = 0.825). When applied to the clinical cohort, predicted glycolysis-enhanced HCCs demonstrated significantly higher 18F-FDG uptake and distinct hepatobiliary phase defect on Gd-EOB-DTPA–enhanced MRI. Patients classified as the glycolysis subclass showed a trend toward shorter overall survival (median 95 months, p = 0.079) and significantly shorter recurrence-free survival (median 14.4 vs. 37.7 months, p = 0.017). Visual 18F-FDG PET positivity was strongly associated with the glycolysis subclass (p < 0.001) and corresponded to a tumor-to-liver uptake ratio ≥ 1.51. A pathology-free LASSO model using four non-invasive variables demonstrated robust predictive performance (AUROC = 0.893). Elevated glycolytic activity was exploratorily correlated with lower CD8⁺ T-cell infiltration and increased PD-1⁺CTLA-4⁺CD8⁺ T cells.Conclusion We provide a transcriptomics-independent logistic regression model for identifying glycolysis-enhanced HCC. This approach enables practical metabolic subclassification of HCC using routinely available clinical data. Macrotrabecular-massive and compact histopathological patterns, inhomogeneous arterial hyperenhancement, elevated serum α-fetoprotein levels, and 18F-FDG PET positivity are characteristic features of this subclass and may serve as potential indicators to inform the applicability of glycolysis–targeting therapies.

BACKGROUND: Intratumor heterogeneity (ITH), characterized by the coexistence of genetically distinct subclones within a tumor, has been associated with adverse clinical outcomes in various cancers. However, the clinical and biological implications of ITH in diffuse large B-cell lymphoma (DLBCL) are still incompletely understood. MATERIALS & METHODS: In this study, we applied a SNP-array-based approach to assess the clonal complexity in formalin-fixed, paraffin-embedded tumor specimens obtained from newly diagnosed patients with advanced-stage DLBCL (n = 74) by calculating the clonal composition (CC) number. RSEULTS: Patients with Poly-CC tumors (CC ≥ 1), which accounted for 79.7% of the cases, had a 5-year event-free survival rate of 38.9%, compared with 69.1% in those with Mono-CC tumors (CC = 0) (Log-rank p = 0.0520). This association reached statistical significance in the activated B-cell (ABC) subtype (n = 35, Log-rank p = 0.0450) but not in the germinal center B-cell (GCB) subtype (n = 30, Log-rank p = 0.910). Gene set enrichment analysis revealed upregulation of cell cycle-related pathways in Poly-CC tumors, consistent with the significantly higher Ki-67 positivity rate than in Mono-CC tumors, as confirmed by immunohistochemistry (p = 0.00227). Within the ABC subtype, Poly-CC (Poly-ABC) tumors exhibited more differentiated transcriptional states and enrichment of IRF4-associated gene signatures as compared with Mono-CC (Mono-ABC) tumors. Conversely, IFN-γ and IFN-α response pathways and the IL-6/JAK-STAT3 signaling pathway were markedly suppressed in the Poly-ABC tumors. Furthermore, Poly-ABC tumors carried a significantly higher number of pathogenic mutations as compared with Mono-ABC tumors (p = 0.0147). CONCLUSION: These results suggest that clonal complexity captures tumor-intrinsic features and biological diversity in DLBCL, especially in the ABC subtype, offering novel insights into the disease pathogenesis.

Endometrial cancer is generally associated with a favorable prognosis when detected at an early stage and treated appropriately, especially in young women receiving fertility-sparing therapy. However, rare cases may exhibit unexpected aggressive progression driven by novel genetic alterations.A 38-year-old woman was diagnosed with stage IA endometrial endometrioid carcinoma grade 1, based on pathologic examination by endometrial curettage, magnetic resonance imaging, and computed tomography scans. The tumour progressed systemically during fertility-sparing treatment and she died on day 112. Pathologic examination of the liver metastasis revealed that the tumour was morphologically different from the endometrial tumour and was oestrogen receptor-negative, chromogranin A-positive, and synaptophysin-positive on immunohistochemistry, leading to the diagnosis of neuroendocrine carcinoma. Gene panel testing identified TMEM178B-BRAF fusion and subclonal PIK3CA mutations in the liver metastasis, in addition to ARID1A and CTNNB1 mutations that were shared with the endometrial tumour. This is the first report of an endometrioid carcinoma transforming into a high-grade neuroendocrine tumour associated with TMEM178B-BRAF fusion.

INTRODUCTION: Ramucirumab (RAM) plus gefitinib (GEF) exhibited favorable efficacy and safety as first-line treatment in the primary analysis of the RELAY+ study of East Asian patients with metastatic EGFR-mutated NSCLC. We report the final overall survival (OS) and safety. METHODS: This open-label, two-period, single-arm exploratory study included patients with untreated NSCLC having EGFR ex19del or L858R mutations and no central nervous system metastasis or EGFR T790M mutation. Patients received RAM (10 mg/kg every 2 wk) plus GEF (250 mg once daily) until disease progression (period 1); patients with disease progression who acquired a T790M mutation received RAM plus osimertinib (80 mg once daily) (period 2). RESULTS: At final OS data cutoff (October 20, 2023; median follow-up: 42.5 mo), median (95% confidence interval [CI]) OS was 47.4 (35.9‒57.6) months, and the 3-year OS rate was 61.8%. In the L858R and ex19del subgroups, median OS was 51.9 and 38.4 months, and 3-year OS rates were 70.2% and 52.8%, respectively. Overall, 85.4% of patients received subsequent systemic therapy post study treatment discontinuation. Grade 3 or higher treatment-related adverse events (AEs) were reported by 51 (62.2%) patients. The most common grade 3 or higher treatment-emergent AE of special interest was hypertension (25.6%; grade 3 event only). Treatment-emergent T790M rate post progression was 81.3%. CONCLUSIONS: RELAY+ revealed a favorable benefit-risk profile for RAM plus GEF in East Asian patients with untreated EGFR-mutated metastatic NSCLC, supporting RAM plus GEF as an alternative first-line treatment option, particularly in those with an L858R mutation.

Trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate comprising trastuzumab linked to a payload DXd, has been extensively used to treat various cancers harboring HER2 overexpression. However, resistance development has been a major challenge to T-DXd treatment. To explore treatment strategies for T-DXd-resistant cancers, we examined two T-DXd-resistant cells named DSR32 and DSR4, which were obtained from HER2 gene-amplified H2170 lung cancer and N87 gastric cancer cells, respectively. The uptake of T-DXd and its transport to lysosomes in DSR32 cells were reduced. Subsequently, HER2 gene copy number (from 54 to 12) was reduced, which decreased HER2 expression on the cell surface. Thus, T-DXd-resistance might be observed due to the reduced T-DXd uptake caused by decreased HER2 expression in DSR32 cells. In DSR4 cells, no change was observed in HER2 expression and in the uptake and transport of T-DXd. The reduced linker cleavage activity may be associated with T-DXd resistance in DSR4 cells. Meanwhile, both DSR32 and DSR4 cells maintained HER2 activation; thus, zongertinib, a HER2-selective tyrosine kinase inhibitor, blocked the HER2 pathway, induced apoptosis, and inhibited colony formation. Overall, zongertinib can provide therapeutic relief to patients with HER2-overexpressing cancer who have developed resistance to T-DXd.

BACKGROUND: Osimertinib is commonly used as a first-line treatment for EGFR-mutated advanced non-small cell lung cancer (NSCLC). However, the optimal treatment strategy following osimertinib failure remains unclear. METHODS: Patients with EGFR-mutated NSCLC and exon 19 deletion or L858R mutation after failure of osimertinib treatment were assigned to a regimen of afatinib (20 mg daily) combined with carboplatin (AUC 5 mg/mL/min) and pemetrexed (500 mg/m2) every 3 weeks followed by maintenance treatment with afatinib plus pemetrexed, until disease progression or unacceptable toxicity was noted. The primary endpoint was the rate of progression-free survival (PFS) at 6 months after initiation of treatment (6M-PFSR). RESULTS: Thirty-six patients were enrolled. One patient met the exclusion criteria, and the efficacy was evaluated in the remaining 35 patients. The 6M-PFSR, the primary endpoint, was 57.1 %. The confidence interval of 39.3-71.5 % exceeded the predefined threshold of 35 %, thereby meeting its primary endpoint. Notably, 28.6 % of patients achieved a long-term PFS of 1 year or longer. The objective response rate (ORR) was 51.4 %, the disease control rate (DCR) was 88.6 %, the median PFS was 8.2 months, the median duration of response (DOR) was 5.6 months, and the median overall survival (OS) was 22.5 months. The most common adverse events were diarrhea (52.8 %) and anorexia (47.2 %), both of which were predictable and manageable. Interstitial pneumonia developed in three patients (8.3 %), one of whom died. Another patient died from sepsis, bringing the total number of deaths in the study to two. CONCLUSIONS: The combination of afatinib and the platinum doublet demonstrated satisfactory efficacy with manageable adverse events in tumors refractory to osimertinib, meeting its primary endpoint. TRIAL REGISTRATION NUMBER: jRCTs021200005.

AIM: The global phase III RELAY trial demonstrated the efficacy of ramucirumab (RAM) plus erlotinib (ERL) in patients with untreated metastatic epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). We present safety and efficacy profiles of RAM+ERL in Japanese elderly (aged ≥ 75 years) patients in RELAY. METHODS: Patients were randomized (1:1) to RAM (10 mg/kg) or placebo (PL) intravenously every 2 weeks plus 150 mg/day ERL orally. Primary endpoint was progression-free survival (reported elsewhere). This report describes response rates, study drug exposure, dose adjustments, safety, and post-study treatment discontinuation therapies in Japanese elderly patients (RAM+ERL, n = 12; PL+ERL, n = 17). RESULTS: Overall response rate was similar in RAM+ERL (83.3%) and PL+ERL (82.4%) arms. Median treatment duration of RAM and ERL was 6.0 and 15.4 months, respectively. Most patients had RAM and/or ERL dose adjustments. Adverse events, including grade ≥ 3 events, were similar in both treatment arms, although proteinuria occurred exclusively in the RAM+ERL arm (six patients; no grade ≥ 3 events). All patients received subsequent therapy after first-line study treatment; various subsequent therapies were used. CONCLUSION: These results suggest that RAM+ERL may be a suitable first-line treatment option for elderly patients with EGFR-mutated NSCLC. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov identifier is NCT02411448.

BACKGROUND: The aim of this study was to explore the biological impact of chemotherapy during epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment in individuals with non-small cell lung cancer (NSCLC). METHODS: Plasma and tumor tissue specimens were prospectively collected from NSCLC patients with EGFR activating mutations who participated in a randomized phase III study comparing EGFR-TKI therapy with or without inserted chemotherapy (JCOG1404/WJOG8214L). The specimens were analyzed for genetic mutations by droplet digital polymerase chain reaction analysis and next-generation sequencing. RESULTS: Two hundred patients were enrolled in this biomarker study, with 113 and 87 individuals receiving EGFR-TKI monotherapy and EGFR-TKI treatment plus inserted chemotherapy, respectively. Although progression-free survival (PFS) for EGFR-TKI monotherapy was shorter in patients with than in those without detectable EGFR activating mutations in cell-free DNA at baseline, inserted chemotherapy improved PFS compared with EGFR-TKI monotherapy for the former patients (median, 17.5 vs. 11.8 months). Inserted chemotherapy suppressed the number of alleles positive for activating and T790M mutations of EGFR in cell-free DNA at disease progression. The benefit of inserted chemotherapy relative to EGFR-TKI monotherapy was more apparent in patients with (median PFS, 18.8 vs. 13.5 months) than in those without detectable concurrent mutations of TP53. CONCLUSIONS: Chemotherapy has the potential to suppress the development of EGFR-TKI resistance as well as to inhibit tumor growth for NSCLC positive for EGFR activating mutations. Our results provide biological insight into combination treatment with EGFR-TKIs and chemotherapy for such patients.

BACKGROUND: The prognosis for recurrent ovarian cancer is poor, but a small percentage of patients can be cured. The aim of this study was to clarify the criteria for being cured and the characteristics of cured cases. METHODS: Ovarian cancer cases at 2 university hospitals and 8 community hospitals were analyzed to identify patients who were considered cured after complete remission (CR) following recurrence. Analyses of the tumors were performed and included BRCA1/2 mutation analysis. RESULTS: Of the 157 cases of recurrence, 21 (13%) showed no evidence of disease (NED). NED cases had a lower rate of ascites at the initial diagnosis, longer disease-free survival, a higher rate of solitary lesions, and a higher rate of secondary debulking surgery. All CR cases except for one showed no further recurrence when DFS reached 4 years, which was considered a criterion for being cured. The case of relapse occurred after long-term treatment with bevacizumab. Furthermore, 19.4% of the CR cases achieved 4-year DFS, which represents 9.3% of the cases of recurrent ovarian cancer and 2.3% of all cases of ovarian cancer. BRCA mutation analysis of the tumor was possible in 17 of the 30 cases of recurrent ovarian cancer that achieved a 4-year DFS. Pathogenic variants of BRCA were found in 5 of the 11 cases of high-grade serous carcinoma. CONCLUSIONS: Approximately 10% of patients with recurrent ovarian cancer achieved a 4-year DFS and were mostly cured. The curing of cases not involving high-grade serous carcinoma (HGSC) was unrelated to the presence of pathogenic BRCA variants.

Afatinib and osimertinib are current treatment options for non-small cell lung cancer (NSCLC) patients with uncommon epidermal growth factor receptor (EGFR) mutations, although their efficacy is limited. To explore potentially effective drugs for these patients, we evaluated the efficacy of conventional EGFR tyrosine kinase inhibitors (TKIs) and novel third-generation (3G) TKIs using in vitro models. Ba/F3 cells transformed with each of the five most frequent uncommon EGFR mutations, Del18 (delE709_T710insD), E709K, G719A, S768I, and L861Q, were used. The growth inhibitory effects of five novel 3G-TKIs, almonertinib, lazertinib, furmonertinib, rezivertinib, and befotertinib, in addition to currently available TKIs, were evaluated. We also explored for secondary resistant mutations to afatinib or osimertinib and TKIs that can overcome these resistances. Afatinib was active against all uncommon EGFR mutations tested. The 3G-TKIs were all active against the L861Q mutation and were inactive against the S768I mutation. Furmonertinib and befotertinib showed efficacy against exon 18 mutations (Del18, E709K, and G719A). In the acquired resistance models to afatinib or osimertinib, we found T790M or a novel T725M secondary mutation, respectively, both of which could be overcome by lazertinib. However, some afatinib-resistant cells acquired V769L/M secondary mutations that were refractory to all EGFR-TKIs tested. In conclusion, afatinib exhibited broad activity and some 3G-TKIs showed promising efficacy in the front-line setting. Lazertinib is a potential second-line option after acquisition of resistance to afatinib or osimertinib.

BACKGROUND: B7-H3 and delta-like ligand 3 (DLL3) are novel therapeutic targets in extensive-stage small cell lung cancer (ES-SCLC). We aimed to assess the impact of B7-H3 and DLL3 expression on the tumor immune microenvironment (TME) and on the therapeutic efficacy of programmed cell death-ligand 1 (PD-L1) blockade for ES-SCLC. PATIENTS AND METHODS: A total of 146 ES-SCLC patients who received platinum-based chemotherapy either with (Chemo + ICI cohort) or without (Chemo cohort) an immune checkpoint inhibitor was analyzed. Progression-free survival (PFS) and overall survival (OS) were evaluated in each cohort according to B7-H3 or DLL3 expression status as detected by immunohistochemistry. The relation of B7-H3 or DLL3 expression to characteristics of the TME was assessed by immune-related gene expression profiling (irGEP). RESULTS: In the Chemo + ICI cohort, patients with high B7-H3 expression showed a shorter PFS (median of 4.3 vs. 5.4 months; HR of 2.11 with a 95 % Cl of 1.08-4.10; P = 0.03) and OS (median of 8.4 vs. 14.2 months; HR of 1.83 with a 95 % CI of 0.91-3.69; P = 0.09) than those with low B7-H3 expression. In the Chemo cohort, there was no apparent difference in survival outcomes between the high and low B7-H3 expression groups. The irGEP analysis revealed that the effector function of CD8+ T cells was impaired in tumors with high B7-H3 expression. No clear association was apparent between therapeutic efficacy and DLL3 expression status. CONCLUSIONS: High B7-H3 expression may serve as a resistance mechanism for PD-L1 antibody therapy by promoting T cell dysfunction in ES-SCLC.

BACKGROUND/AIM: Photodynamic diagnosis (PDD) with 5-aminolevulinic acid (5-ALA) can be used to detect bladder cancer lesions, but false-positive results without pathological malignancy could sometimes occur. However, a false-positive lesion might be the precancerous lesion. Telomerase reverse transcriptase (TERT) promoter mutations are commonly found in the tumors and urine of bladder cancer patients. TERT promoter mutations have been detected in normal mucosa and have been associated with subsequent bladder cancer development. In this study, we evaluated TERT promoter mutations in PDD false-positive samples in patients with non-muscle-invasive bladder cancer (NMIBC) who underwent PDD-assisted transurethral resection of bladder tumor (PDD-TURBT) after oral 5-ALA administration. MATERIALS AND METHODS: Among 117 patients who underwent PDD-TURBT for NMIBC, 18 NMIBC patients who had PDD with 5-ALA positive samples, which were pathologically diagnosed as no malignant diseases, were included in this study. TERT promoter mutations were evaluated in 24 PDD false-positive samples. Droplet digital PCR (ddPCR) was employed to detect C228T and C250T mutations in the TERT promoter region using genomic DNA. The relationship between mutations in the TERT promoter and recurrence of bladder tumors was assessed using the Kaplan-Meier survival analysis. RESULTS:  TERT promoter mutations in PDD false positives were found in six (33.3%) patients. C228T mutation was found in five (27.8%) patients, and C250T mutation was found in one (5.5%) patient. TERT promoter mutations were significantly associated with the presence of two or more tumors (P=0.033) and with an European Organisation for Research and Treatment of Cancer (EORTC) risk score of 5 or higher (P=0.010) in PDD false-positive cases. Two-year recurrence-free survival (RFS) rate was 33.3% in TERT promoter mutation-positive cases and 50% in negative cases. There was no significant difference in RFS between TERT promoter-positive cases and negative cases (P=0.698). CONCLUSIONS:  TERT promoter mutations were frequently found in PDD false-positive samples in patients with NMIBC who underwent PDD-TURBT. While limited in extent, PDD false-positive samples in patients with TERT promoter mutation positivity in the non-malignant urothelium might be precancerous lesions.

Radiotherapy (RT) for prostate cancer increases the risk of bladder cancer. The genomic landscape of bladder cancer following RT for prostate cancer and its differentiation from bladder cancers that develop without a history of pelvic RT remains unclear. We examined gene mutations in bladder cancers that developed following RT and those that developed without prior RT. Fourteen patients who developed primary bladder cancer following brachytherapy were categorized into radiation-associated bladder tumor (RA-BT) group, whereas 33 patients diagnosed with primary bladder cancer without a history of pelvic RT were classified into the bladder tumor (BT) group. The frequency of TERT promoter mutations was 35.7% and 63.6% in the RA-BT and BT groups, respectively (p = 0.112). Among the other characteristic mutations, FGFR3 and TP53 were frequently observed in both groups (FGFR3: RA-BT vs. BT, 14.3 vs. 42.4%; TP53: RA-BT vs. BT, 50 vs. 33.3%). Rare mutations in bladder cancer were more frequently observed in the RA-BT group, including ADGRB3 (28.6%), CBL (21.4%), TGM7 (21.4%), and BTK (14.3%). There were significantly more C → T substitutions in the RA-BT group than in the BT group. In our study, the genetic mutations in the RA-BT group had distinct features from those in the BT group.

Recent next-generation sequencing (NGS) studies have shown that synchronous endometrial and ovarian cancers (SEOCs) are often derived from the same clone. However, the clonal relationship in Lynch syndrome remains unclear. A 45-year-old woman was diagnosed with grade 2 endometrial endometrioid carcinoma and an ovarian yolk sac tumor with clear cell carcinoma. Genetic testing revealed different MLH1 variants in the endometrial and ovarian cancers, and peripheral blood analysis identified an exon 5 deletion in MLH1, confirming Lynch syndrome. The somatic variants in the tumors were distinct. A review of the literature found six cases of SEOC in Lynch syndrome with NGS-based clonal analysis, four of which (67%) showed independent cancers with different somatic profiles. These findings suggest that, unlike sporadic SEOC, synchronous cancers in Lynch syndrome are more likely to arise from separate clones.

The biological basis of the development of cancer of unknown primary (CUP) remains largely unknown, with no evidence of whether a common biological basis exists at present. Our previous multicenter clinical study predicted the primary site of CUP for site‑specific therapy. Concomitantly with the study, a microarray analysis of tumor mRNA samples obtained from 60 participants of the study with CUP was performed, and a gene expression profile specific to CUP was constructed. Several of the genes identified as being upregulated/downregulated in CUP could potentially be clinically useful common biomarkers of CUP. In the present study, to identify genes that may be more closely related to the development of CUP (characterized by its metastatic potential) among the upregulated genes, cell‑based small interfering RNA screening was performed in vitro, and two genes, protein kinase DNA‑activated catalytic subunit (PRKDC) and proteasome subunit β type‑4 (PSMB4), were identified to be possibly involved in the metastatic ability of CUP, since knockdown of these genes resulted in reduced migration of A549 cells. These genes were further knocked down in A549 cells using short hairpin RNAs (shRNAs) and the cells were implanted into the footpad of mice. Marked suppression of the metastatic ability of implanted cells from the footpad to the popliteal lymph node (LN) was observed in cells transfected with the shRNAs for PRKDC and PSMB4. In addition, bortezomib, a proteasome inhibitor, markedly reduced the ability of cells implanted into the footpad to metastasize to the LNs, as well as cell growth at the metastatic site, compared with vehicle or NU7447 (inhibitor of PRKDC). These findings indicated that proteasomal function activation augmented the metastatic ability of malignant CUP cells.

INTRODUCTION: Significant improvement in progression-free survival (PFS; primary end point) was reported in the phase 3 RELAY study with ramucirumab (RAM) plus erlotinib (ERL) versus placebo (PL) in untreated EGFR-mutated NSCLC (hazard ratio [HR] = 0.59, 95% confidence interval [CI]: 0.46-0.76, p < 0.0001), including in the Japanese subset. We report updated PFS and final overall survival (OS) for the Japanese subset. METHODS: Patients (no central nervous system metastases) were randomized 1:1 (stratification included EGFR leucine to arginine substitution [L858R]/exon 19 deletion [ex19del]) to ERL (150 mg/d) with RAM (10 mg/kg; n = 106) or PL (n = 105) intravenously every 2 weeks. The study was not powered for OS. RESULTS: At final OS data cutoff (median follow-up = 48.2 mo), PFS benefit was sustained with RAM plus ERL versus PL plus ERL (median [m] PFS: 19.4 versus 11.2 mo; HR = 0.69, 95% CI: 0.51-0.93); the mOS was 54.3 and 46.0 months (HR = 0.91, 95% CI: 0.65-1.26), respectively. In L858R (n = 110) and ex19del (n = 100) subgroups, the mOS was 54.3 versus 43.2 months (HR = 0.63, 95% CI: 0.40-0.99) and 53.9 versus 62.1 months (HR = 1.40, 95% CI: 0.86-2.28), respectively. T790M rates post-progression were 52.0% versus 51.1%, respectively. Osimertinib as subsequent therapy was received by 61.0% versus 55.2% patients (L858R: 58.2% versus 48.1%; ex19del: 65.3% versus 62.7%); the median (range) osimertinib treatment duration was 16.8 (0.7-58.3) versus 20.1 (2.1-77.2) months. Safety was consistent with known RAM and ERL profiles, with no increased toxicity over time. CONCLUSIONS: The Japanese subset reported that RAM plus ERL improved PFS, and a mOS greater than 50 months was achieved. OS differed by EGFR mutation type, with an indication of benefit for patients with L858R. TRIAL REGISTRATION: NCT02411448.

BACKGROUND/AIMS: Previously, we advocated the importance of classifying hepatocellular carcinoma (HCC) based on physiological functions. This study aims to classify HCC by focusing on liver-intrinsic metabolism and glycolytic pathway in cancer cells. METHODS: Comprehensive RNA/DNA sequencing, immunohistochemistry, and radiological evaluations were performed on HCC tissues from the training cohort (n=136) and validated in 916 public samples. HCC was classified using hierarchical clustering and compared with previous molecular, histopathological, and hemodynamic classifications. RESULTS: Liver-specific metabolism and glycolysis are mutually exclusive and were divided into two major subclasses: The "rich metabolism" subclass (60.3%) is characterized by enhanced bile acid and fatty acid metabolism, well-to-moderate differentiation, microtrabecular or pseudoglandular pattern, and homogeneous arterial-phase hyperenhancement (APHE), corresponding to Hoshida S3 with favorable prognosis. In IL6-JAK-STAT3-high (25.0%) conditions, upregulated ALB expression, enhanced gluconeogenesis and urea cycle activity, and an inflammatory-microenvironment are observed. Conversely, the Wnt/β-catenin-high environment (19.9%) features elevated GLUL, APOB and CYP3A4 expression, frequent CTNNB1 (D32-S37) mutations, and an immune-desert/excluded phenotype. The "glycolysis" subclass (39.7%), characterized by histopathological dedifferentiation and downregulated liver-specific metabolism, encompasses subclasses with PI3K/mTOR (20.6%) and NOTCH/TGF-β (19.1%) signaling. These often exhibit TP53 mutations, macrotrabecular massive or compact patterns, inhomogeneous/rim-APHE, and high expression of hypoxia-inducible factors and glucose transporters, corresponding to Hoshida S1/2 with poor prognosis. CONCLUSION: The loss of liver-specific metabolism correlates with morphological dedifferentiation, indicating cellular dedifferentiation may exhibit both physiological and pathological duality. Key signaling pathways involved in the maturation process from fetal to adult liver and zonation program may play a critical role in defining HCC diversity.

BACKGROUND: The RELAY-Brain trial examined the clinical utility and survival impacts of ramucirumab (RAM) combined with epidermal growth factor receptor (EGFR)-TKI in patients with EGFR-mutated non-small-cell lung cancer (NSCLC) with brain metastases. Although RAM combined with erlotinib (ERL) is known to have clinical benefits, the benefits in patients with baseline brain metastases remain unclear. This report examined the long-term follow-up data (Japan Registry of Clinical Trials: jRCTs2051190027) of the same patients, analyzing relevant biomarkers from tumor and plasma samples. PATIENTS AND METHODS: The six patients enrolled in the RELAY-Brain trial received RAM plus ERL or osimertinib (OSM). Our long-term follow-up observational study assessed patient survival, treatment status, and genetic biomarkers. Tumor and plasma samples were analyzed at baseline, during treatment, and at disease progression using next-generation sequencing and droplet digital PCR, to identify gene alterations and EGFR-TKI-resistant mutations. RESULTS: After median follow-up of 44.2 months, the first site of disease progression in three of the patients was the brain metastases. In the RAM + ERL group, two patients with the T790M mutation subsequently received OSM. Progression-free survival (PFS) and overall survival ranged from 10.46 to 42.07 months and from 30.14 to 52.25 months, respectively. Gene alterations included TP53 mutations in three patients and ERBB2 and PIK3CA mutations in two. TP53 mutations were associated with shorter PFS. CONCLUSION: RAM with ERL or OSM achieved significant clinical benefits for patients with EGFR-mutated NSCLC with asymptomatic brain metastases. These positive outcomes and the identification of related biomarkers support the therapeutic potential of these combinations.

BACKGROUND: Recognizing rare molecular variants of driver mutations poses a challenge in precision oncology, particularly for treatment of non-small cell lung cancer (NSCLC). In this study, we aimed to determine whether Oncomine Dx Target Test Multi-CDx System (ODxTT), the most widely used genetic test for NSCLC in Japan, potentially overlooks druggable EGFR mutations. MATERIALS AND METHODS: Among 418 patients who underwent molecular testing using ODxTT at our hospital, 267 were diagnosed with adenocarcinoma. No mutations were reported in 82 of these cases. For these 82 cases, we searched for EGFR mutations in exons 18-21 by examining the binary alignment map file. Once a mutation was identified, its pathological significance was evaluated using the ClinVar database to determine whether ODxTT had overlooked any actionable EGFR mutations. RESULTS: Mutations in EGFR exons 19 and 18 were identified in six and four cases, respectively. Three, six, and none of these variants were detectable using the Cobas EGFR Mutation Test v2, Lung Cancer Compact Panel, and Amoy Dx, respectively. Of the 10 patients, five were subsequently treated with EGFR TKI; three showed partial response, one had stable disease, and one had progressive disease. CONCLUSIONS: ODxTT failed to identify 10 actionable EGFR mutations, accounting for 12.2% (10/82) of the cases initially reported as not carrying actionable mutations. Therefore, comprehensive genomic profiling should be actively performed early in cases with high clinical suspicion of EGFR mutations.

We have been exploring biomarkers that could help physicians select the appropriate opioid for individualized treatment of cancer pain. Recently, we identified a single nucleotide polymorphism (SNP) of CCL11 (rs17809012) as one such biomarker that was significantly associated with the analgesic effect of morphine. The current study measured the plasma concentrations of chemokines/cytokines in pre-treatment plasma samples of a total of 138 patients who were randomized to receive morphine (n=70) or oxycodone (n=68). Based on the results, one cytokine, IL-16, was identified whose plasma concentrations showed a clear bias between patients with cancer pain who responded well and responded poorly to oxycodone. A genotypic analysis also identified a SNP of the IL-16 gene (rs4778889) as being significantly associated with the analgesic effect of oxycodone. Whether both of the SNPs identified as being significant (CCL11 rs17809012 and IL-16 rs4778889) could be used in combination to accurately predict which opioid might be the most suitable to provide pain relief in patients with cancer was assessed. Morphine tended to provide superior analgesic effect over oxycodone in patients with the IL-16 rs4778889 TT genotype and the CCL11 rs17809012 AG/GG genotype (n=45), while a trend towards a greater analgesic effect of oxycodone was observed in patients with other genotype combinations of these SNPs (n=93) (P=0.0012 for the interaction), suggesting that the IL-16 rs4778889 and CCL11 rs17809012 SNPs could serve as a potential dual-biomarker combination for personalized analgesic therapy in patients with cancer pain.

Transformation to small cell lung cancer (SCLC) is a resistance mechanism in epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) after EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. The efficacy of immune checkpoint inhibitor (ICI) in transformed SCLC remains to be elucidated. The present case report highlights a patient whose tumor underwent transformation to SCLC after developing resistance to an EGFR-TKI treatment. The patient subsequently achieved long-term remission lasting more than 5 years through treatment with an anti-PD-1 antibody nivolumab. Generally, the efficacy of ICI is inferior in EGFR-mutated NSCLC compared to those with EGFR wild-type NSCLC. However, some cases that have transformed to SCLC may be sensitive to ICI treatment. Further investigation is necessary to determine the efficacy of ICI in cases that have undergone transformation to SCLC.

INTRODUCTION: Many clinical studies showed a synergy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) and vascular endothelial growth factor inhibitors. We hypothesized afatinib plus bevacizumab exerts clinical potency after developing various osimertinib resistant mechanisms. METHODS: EGFR-mutant non-small cell lung cancer patients were enrolled after osimertinib resistance. Afatinib at 30-40 mg/day and bevacizumab at 15 mg/kg tri-weekly were administered until progression. Plasma/histologic rebiopsied samples after osimertinib failure were analyzed to examine resistant mechanisms: gene alterations/copy-number gain using cancer personalized profiling by deep sequencing. RESULTS: Between January 2018 and October 2020, 28 patients were enrolled. Response and disease control rates were 17.9 % and 78.6 %, respectively. Median duration of response was 9.0 (range, 4.2-22.3) months. Median progression-free and overall survivals were 2.7 and 9.3 months, respectively. Twenty-eight (100 %) plasma and/or 21 (75 %) histologic rebiopsies identified: 17 (61 %) TP53; 15 (54 %) T790M; 9 (32 %) uncommon EGFR; 9 (32 %) MET; 6 (21 %) C797S; 3 (11 %) BRAF; 2 (7 %) HER2; 2 (7 %) KRAS; and 2 (7 %) PI3K mutations. One (17 %) of 6 C797S patients showed complete response. Three (33 %) of 9 uncommon EGFR-mutated patients achieved radiographic response. Neither 15 T790M-positive nor 6 EGFR downstream signaling mutations: BRAF; KRAS; or PI3K-positive patients responded, but 5 (38 %) of 13 T790M-negative patients responded. Adverse events ≥ grade 3 and incidence ≥ 5 % were: hypertension (29 %); proteinuria (7 %); and diarrhea (7 %). There were neither treatment-related death nor interstitial lung disease. CONCLUSIONS: Selected population could obtain clinical benefit from afatinib plus bevacizumab, based on rebiopsy results after osimertinib resistance.

INTRODUCTION: EGFR tyrosine kinase inhibitors have been the standard treatment for patients with NSCLC who have sensitive EGFR mutations. This study revealed final analysis survival data, biomarkers, and resistance mechanisms of osimertinib plus bevacizumab or osimertinib monotherapy in previously untreated patients with advanced EGFR-positive nonsquamous NSCLC. METHODS: We previously reported the primary results of a randomized, open-label, phase 2 study comparing osimertinib plus bevacizumab with osimertinib monotherapy for this population. In this exploratory analysis using tissue and plasma samples, we evaluated gene profiles at baseline and disease progression or the last dose using targeted deep sequencing. RESULTS: The median progression-free survival (PFS) by the blinded independent central reviewer was 22.1 months for the osimertinib plus bevacizumab arm and 20.2 months for the osimertinib arm (hazard ratio [HR] = 0.864, 95% confidence interval [CI]: 0.549-1.359). The 3-year overall survival was not different between the two arms (osimertinib plus bevacizumab: 57.1%; osimertinib monotherapy: 65.0%; HR 1.271, 95% CI: 0.727-2.223). A total of 94 patients had assessable plasma samples at baseline, and 40 had assessable pretreatment tissue samples. EGFR mutations (76.6%) and TP53 mutations (44.7%) were detected in plasma samples at baseline. In patients with plasma TP53 mutations (n = 42), the median PFS by blinded independent central reviewer was 19.8 months for the osimertinib plus bevacizumab arm and 20.2 months for the osimertinib arm (HR = 1.107, 95% CI: 0.534-2.297). CONCLUSIONS: There was also no significant difference in the PFS between the two arms, even in patients with TP53 mutations.

AIM: Although immune checkpoint inhibitors (ICPi) for salivary gland cancer (SGC) have been investigated in clinical trials, details of the tumor immune microenvironment (TIME) remain unclear. This research aimed to elucidate the TIME of SGC and its relationship with tumor mutation burden (TMB) and to explore the rationale for the applicability of ICPi. MATERIALS AND METHODS: We selected five pathological types, namely adenoid cystic carcinoma (ACC); adenocarcinoma, not otherwise specified (ANOS); salivary duct carcinoma (SDC); and low/high-grade mucoepidermoid carcinoma (MEClow/high). We investigated the TIME and TMB of each pathological type. TIME was evaluated by multiplexed fluorescent immunohistochemistry. TMB was measured by next-generation sequencing. RESULTS: ACC and MEChigh showed the lowest and highest infiltration of immune effector and suppressor cells in both tumor and stroma. ANOS, SDC, and MEClow showed modest infiltration of immune effector cells in tumors. Correlation analysis showed a positive correlation between CD3+CD8+ T cells in tumor and TMB (r = 0.647). CD3+CD8+ T cells in tumors showed a positive correlation with programmed cell death-ligand 1 expression in tumor cells (r = 0.513) and a weak positive correlation with CD3+CD4+Foxp3+ cells in tumors (r = 0.399). However, no correlation was observed between CD3+CD8+ T cells and CD204+ cells in tumors (r = -0.049). CONCLUSION: The TIME of ACC was the so-called immune desert type, which may explain the mechanisms of the poor response to ICPi in previous clinical trials. On the other hand, MEChigh was the immune-inflamed type, and this may support the rationale of ICPi for this pathological subtype.

Chronic systemic inflammation caused by diseases such as ulcerative colitis (UC) and Crohn's disease (CD) increases the risk of developing colorectal cancer (CRC). Recent evidence indicates that patients with UC are more susceptible to prostate cancer (PCa), and individuals with PCa may also be at a higher risk of developing CRC. However, these relationships are not well defined. A better understanding of this phenomenon could improve the identification of high-risk populations. In this study, we characterized these relationships with experiments using preclinical mouse models of dextran sulfate sodium (DSS)-induced colitis (DSS-UC) and DSS/azoxymethane (AOM)-induced CRC (DSS/AOM-CRC) in wild-type and conditional transgenic mice of PCa. We showed that DSS-induced UC was more severe in mice with PCa and resulted in the development of CRC in the absence of AOM. We further showed that PCa-free mice that developed DSS-induced UC also showed histological changes in the normal prostate that resembled proliferative inflammatory atrophy. Finally, we used immunohistochemical immune profiling to show that mice with PCa-induced chronic systemic inflammation accumulated Gr1+ myeloid cells in the normal colon and exposure to DSS further enriched these cells in active colitis regions and colon tumors. Our study provides evidence to support a link between systemic chronic inflammation and cancer.

BACKGROUND: Prostate cancer is a complex disease that develops over time and is influenced by several lifestyle factors that also impact gut microbes. Gut dysbiosis is intricately linked to prostate carcinogenesis, but the precise mechanisms remain poorly understood. Mice are crucial for studying the relationships between gut microbes and prostate cancer, but discovering similarities between humans and mice may aid in elucidating new mechanisms. METHODS: We used 16s rRNA sequencing data from stool samples of tumor-bearing prostate-specific conditional Pten-knockout mice, disease-free wildtype mice, and a human cohort suspected of having prostate cancer to conduct taxonomic and metagenomic profiling. Features were associated with prostate cancer status and low risk (a negative biopsy of Gleason grade <2) or high risk (Gleason grade ≥2) in humans. RESULTS: In both humans and mice, community composition differed between individuals with and without prostate cancer. Odoribacter spp. and Desulfovibrio spp. were taxa associated with prostate cancer in mice and humans. Metabolic pathways associated with cofactor and vitamin synthesis were common in mouse and human prostate cancer, including bacterial synthesis of folate (vitamin B9), ubiquinone (CoQ10), phylloquinone (vitamin K1), menaquinone (vitamin K2), and tocopherol (vitamin E). CONCLUSIONS: Our study provides valuable data that can help bridge the gap between human and mouse microbiomes. Our findings provide evidence to support the notion that certain bacterial-derived metabolites may promote prostate cancer, as well as a preclinical model that can be used to characterize biological mechanisms and develop preventive interventions.

INTRODUCTION: Immunotherapy is becoming a promising approach for unresectable-hepatocellular carcinoma (HCC); the anti-tumor response is affected by the tumor microenvironment (TME). Although Wnt/β-catenin mutations are reported to cause non-inflamed phenotype, their role on TME remains controversial. We aimed to clarify the heterogeneity of immunophenotype in HCC with Wnt/β-catenin mutations. METHODS: This study includes 152 resected HCCs; mutations in the catenin beta-1, adenomatous polyposis coli, or AXIN1, or AXIN2 genes were defined as Wnt/β-catenin mutations. With hierarchical cluster analyses, TME was classified into inflamed or non-inflamed classes based on the gene expressions associated with T-cell activation. Expression profiles of molecules related to cell differentiation and biliary-stem cell markers were compared between the TME classes to investigate whether differences in tumor traits were associated with TME. RESULTS: Forty of 152 (26.3%) HCCs carried the Wnt/β-catenin mutations. Of these, 33 were classified as non-inflamed (33/40, 82.5%) and 7 as inflamed (7/40, 17.5%). Non-inflamed class was characterized by low number of CD3+, CD4+, and CD8+ cells on immunostaining, and high mRNA expressions of AXIN2 and GLUL, which are involved in the canonical Wnt/β-catenin signaling and hepatocyte differentiation, respectively. Non-inflamed tumors showed higher enhancement on the hepatobiliary-phase of gadolinium-ethoxybenzyl-diethylenetriamine (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) compared to inflamed tumors. HCCs classified as inflamed class are revealed to have high numbers of CD3+, CD4+, and CD8+ tumor infiltrating lymphocytes on immunostaining. This class is associated with increased expression of anti-epithelial cell adhesion molecule and FOXM1 accompanied by upregulation of genes related to interferon-gamma signaling, dendritic cell migration, regulatory T cells, and myeloid-derived suppressor cell activation and recognized as low enhancement nodule on Gd-EOB-DTPA-enhanced MRI. CONCLUSION: Heterogeneity of tumor traits and TME was observed in HCC with Wnt/β-catenin mutation. The potential was indicated that tumor traits and TME are determined not only by the activation of the HNF4A but also by FOXM1, both of which are downstream transcription factor of the Wnt/β-catenin signaling pathway.

BACKGROUND: Immune-related adverse events (irAEs) have been found to predict PD-L1 inhibitor efficacy in metastatic NSCLC. However, the relation of irAEs to clinical outcome for nonmetastatic NSCLC has remained unknown. METHODS: In this multicenter prospective study of Stage III NSCLC treated with PACIFIC regimen, the relation of irAEs to PFS was evaluated by 8-week landmark analysis to minimise lead-time bias as well as by multivariable analysis adjusted for baseline factors. irAEs were categorised as mild or nonmild according to whether they were treated with systemic steroid. RESULTS: Median PFS was 16.0 months, not reached, and 9.7 months for patients without (85 cases) or with mild (21 cases) or nonmild (21 cases) irAEs, respectively. Multivariable analysis indicated that nonmild irAEs were associated with poor PFS, with HRs of 3.86 (95% CI, 1.31-11.38) compared with no irAEs and 11.58 (95% CI, 2.11-63.63) compared with mild irAEs. This pattern was consistent after irAE grade, the number of durvalumab doses and immune profiles (PD-L1 score, CD8+ tumour-infiltrating lymphocyte density, and tumour mutation burden) were taken into consideration. CONCLUSIONS: The development of mild irAEs might predict a better survival outcome, whereas immunosuppressive steroid-treated irAEs were associated with a worse outcome, regardless of baseline clinical and immune profiles.

INTRODUCTION: Immune checkpoint inhibitors have recently been approved for the treatment of early-stage NSCLC in the perioperative setting on the basis of phase 3 trials. However, the characteristics of such patients who are susceptible to recurrence after adjuvant chemotherapy or who are likely to benefit from postoperative immunotherapy have remained unclear. METHODS: This biomarker study (WJOG12219LTR) was designed to evaluate cancer stem cell markers (CD44 and CD133), programmed death-ligand 1 (PD-L1) expression on tumor cells, CD8 expression on tumor-infiltrating lymphocytes, and tumor mutation burden in completely resected stage II to IIIA NSCLC with the use of archived DNA and tissue samples from the prospective WJOG4107 trial. Tumors were classified as inflamed or noninflamed on the basis of the PD-L1 tumor proportion score and CD8+ tumor-infiltrating lymphocyte density. The association between each potential biomarker and relapse-free survival (RFS) during adjuvant chemotherapy was assessed by Kaplan-Meier analysis. RESULTS: A total of 117 patients were included in this study. The median RFS was not reached (95% confidence intervals [CI]: 22.4 mo-not reached; n = 39) and 23.7 months (95% CI: 14.5-43.6; n = 41) in patients with inflamed or noninflamed adenocarcinoma, respectively (log-rank p = 0.02, hazard ratio of 0.52 [95% CI: 0.29-0.93]). Analysis of the combination of tumor inflammation category and TP53 mutation status revealed that inflamed tumors without TP53 mutations were associated with the longest RFS. CONCLUSIONS: PD-L1 expression on tumor cells, CD8+ T cell infiltration, and TP53 mutation status may help identify patients with early-stage NSCLC susceptible to recurrence after adjuvant chemotherapy.

BACKGROUND/AIM: The prognostic significance of androgen receptor amplification (AR amp) in cell-free DNA (cfDNA) was studied in Japanese patients with castration-resistant prostate cancer (CRPC). PATIENTS AND METHODS: A total of 120 serum samples were obtained from 38 patients with CRPC. Serum cfDNA was purified and the AR copy number was determined. Factors associated with progression-free survival (PFS) and overall survival (OS) were statistically investigated. RESULTS: The number of patients administered enzalutamide (Enza)/abiraterone (Abi)/docetaxel (DTX) was 33/25/11, respectively. The median PSA was 16.5 ng/ml. Thirty patients (79%) had bone metastases and three patients (7.9%) had lung metastases. The median follow-up was 655 days. The median initial AR copy number was 1.27 (1.10-11.50); an AR copy number of 1.27 or higher was defined as an AR-amp. Regarding PFS, the presence of AR-amp, Gleason score (GS), and ALP were significant factors in univariate analysis. In multivariate analysis, AR amplification was an independent prognostic factor (hazard ratio=7.7, p=0.0035). For OS, PSA and AR-amp were significant factors. In multivariate analysis, AR-amp (hazard ratio=4.65, p=0.0188) was the only independent prognostic factor. CONCLUSION: AR-amp was associated with high nadir PSA and low iPSA/PSA ratio. AR-amp was significantly associated with poor prognosis in Japanese patients with CRPC.

Sarcomatoid hepatocellular carcinoma (SHCC) is a rare and highly lethal subtype of HCC. The present study aimed to explore the unique markers of SHCC using whole gene expression analysis. Subsequently, gene expression analysis was performed using five sarcomatoid and seven carcinomatoid components of seven tissues from patients with SHCC. The results demonstrated a significant downregulation of polybromo 1 (PBRM1) gene expression in the sarcomatoid components. Immunohistochemical staining also indicated a decreased expression of PBRM1 in the sarcomatoid components. Moreover, gene ontology enrichment analysis revealed that most of the 336 differentially expressed genes between the sarcomatoid and carcinomatoid components were involved in functions associated with DNA replication and histone methylation, which was consistent with the loss of function of PBRM1 which encodes Switch/sucrose-non-fermentable chromatin remodeling complex protein. Therefore, the results of the present study suggested that PBRM1 may be a candidate biomarker for the evaluation of SHCC.

The extracellular signal-regulated kinase (ERK) MAPK pathway is dysregulated in various human cancers and is considered an attractive therapeutic target for cancer. Therefore, several inhibitors of this pathway are being developed, and some are already used in the clinic. We have previously identified an anticancer compound, ACA-28, with a unique property to preferentially induce ERK-dependent apoptosis in melanoma cells. To comprehensively understand the biological cellular impact induced by ACA-28, we performed a global gene expression analysis of human melanoma SK-MEL-28 cells exposed to ACA-28 using a DNA microarray. The transcriptome analysis identified nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcription factor that combats oxidative stress, as the most upregulated genetic pathway after ACA-28 treatment. Consistently, ACA-28 showed properties to increase the levels of reactive oxygen species (ROS) as well as Nrf2 protein, which is normally repressed by proteasomal degradation and activated in response to oxidative stresses. Furthermore, the ROS scavenger N-acetyl cysteine significantly attenuated the anticancer activity of ACA-28. Thus, ACA-28 activates Nrf2 signaling and exerts anticancer activity partly via its ROS-stimulating property. Interestingly, human A549 cancer cells with constitutively high levels of Nrf2 protein showed resistance to ACA-28, as compared with SK-MEL-28. Transient overexpression of Nrf2 also increased the resistance of cells to ACA-28, while knockdown of Nrf2 exerted the opposite effect. Thus, upregulation of Nrf2 signaling protects cancer cells from ACA-28-mediated cell death. Notably, the Nrf2 inhibitor ML385 substantially enhanced the cell death-inducing property of ACA-28 in pancreatic cancer cells, T3M4 and PANC-1. Our data suggest that Nrf2 plays a key role in determining cancer cell susceptibility to ACA-28 and provides a novel strategy for cancer therapy to combine the Nrf2 inhibitor and ACA-28.

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development.

The phase III IMPACT study (UMIN000044738) compared adjuvant gefitinib with cisplatin plus vinorelbine (cis/vin) in completely resected epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). Although the primary endpoint of disease-free survival (DFS) was not met, we searched for molecular predictors of adjuvant gefitinib efficacy. Of 234 patients enrolled in the IMPACT study, 202 patients were analyzed for 409 cancer-related gene mutations and tumor mutation burden using resected lung cancer specimens. Frequent somatic mutations included tumor protein p53 (TP53; 58.4%), CUB and Sushi multiple domains 3 (CSMD3; 11.8%), and NOTCH1 (9.9%). Multivariate analysis showed that NOTCH1 co-mutation was a significant poor prognostic factor for overall survival (OS) in the gefitinib group and cAMP response element binding protein (CREBBP) co-mutation for DFS and OS in the cis/vin group. In patients with NOTCH1 co-mutations, gefitinib group had a shorter OS than cis/vin group (Hazard ratio 5.49, 95% CI 1.07-28.00), with a significant interaction (P for interaction = 0.039). In patients with CREBBP co-mutations, the gefitinib group had a longer DFS than the cis/vin group, with a significant interaction (P for interaction = 0.058). In completely resected EGFR-mutated NSCLC, NOTCH1 and CREBBP mutations might predict poor outcome in patients treated with gefitinib and cis/vin, respectively.

BACKGROUND: An exploratory, proof-of-concept, liquid biopsy addendum to examine biomarkers within cell-free DNA (cfDNA) in the RELAY phase 3, randomized, double-blind, placebo-controlled study was conducted. RELAY showed improved progression-free survival (PFS) with ramucirumab (RAM), a human immunoglobulin G1 vascular endothelial growth factor receptor 2 antagonist, plus erlotinib (ERL), a tyrosine kinase inhibitor, compared with placebo (PL) plus ERL. METHODS: Treatment-naïve patients with endothelial growth factor receptor (EGFR)-mutated metastatic non-small cell lung cancer were randomized (1:1) to RAM + ERL or PL + ERL. Plasma samples were collected at baseline, on treatment, and at 30-day post-study treatment discontinuation follow-up. Baseline and treatment-emergent gene alterations and EGFR-activating mutation allele counts were investigated by next-generation sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR), respectively. cfDNA concentration and fragment size were evaluated by real-time polymerase chain reaction and the BioAnalyzer. Patients with a valid baseline plasma sample were included (70 RAM + ERL, 61 PL + ERL). RESULTS: TP53 mutation was the most frequently co-occurring baseline gene alteration (43%). Post-study treatment discontinuation EGFR T790M mutation rates were 54.5% (6/11) and 41.2% (7/17) by ddPCR, and 22.2% (2/9) and 29.4% (5/17) by NGS, in the RAM + ERL and PL + ERL arms, respectively. EGFR-activating mutation allele count decreased at Cycle 4 in both treatment arms and was sustained at follow-up with RAM + ERL. PFS improved for patients with no detectable EGFR-activating mutation at Cycle 4 vs. those with detectable EGFR-activating mutation. Total cfDNA concentration increased from baseline at Cycle 4 and through to follow-up with RAM + ERL. cfDNA fragment size was similar between treatment arms at baseline [mean (standard deviation) base pairs: RAM + ERL, 173.4 (2.6); PL + ERL, 172.9 (3.2)] and was shorter at Cycle 4 with RAM + ERL vs. PL + ERL [169.5 (2.8) vs. 174.1 (3.3), respectively; P<0.0001]. Baseline vs. Cycle 4 paired analysis showed a decrease in cfDNA fragment size for 84% (48/57) and 23% (11/47) of patient samples in the RAM + ERL and PL + ERL arms, respectively. CONCLUSIONS: EGFR-activating mutation allele count was suppressed, total cfDNA concentration increased, and short fragment-sized cfDNA increased with RAM + ERL, suggesting the additional anti-tumor effect of RAM may contribute to the PFS benefit observed in RELAY with RAM + ERL vs. PL + ERL. TRIAL REGISTRATION: ClinicalTrials.gov; identifier: NCT02411448.

Intracranial metastases are common in nonsmall-cell lung cancer (NSCLC) patients, whose prognosis is very poor. In addition, intracranial progression is common during systemic treatments due to the inability to penetrate central nervous system (CNS) barriers, whereas the intracranial effects of cancer immunotherapies remain unclear. We analyzed clinical data to evaluate the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies compared with those treated without PD-1 blockade therapies, and found that the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies was significantly lower than that in patients treated with cytotoxic chemotherapies. In murine models, intracranial rechallenged tumors after initial rejection by PD-1 blockade were suppressed. Accordingly, long-lived memory precursor effector T cells and antigen-specific T cells were increased by PD-1 blockade in intracranial lesions. However, intracranial rechallenged different tumors are not suppressed. Our results indicate that cancer immunotherapies can prevent intracranial progression, maintaining long-term effects intracranially as well as systemically. If intracranial recurrence occurs during the treatment with PD-1 blockade therapies, aggressive local therapies could be worthwhile.

BACKGROUND: This prospective cohort study evaluated the feasibility of using endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) samples for comprehensive mutational analysis of cancer-related genes using microtissues. METHODS: Fifty patients with suspected pancreatic cancer presenting consecutively at the Kindai University Hospital between January 2018 and January 2019 were enrolled. Cancerous tissues from EUS-FNB were obtained from each tumor and subjected to histological examination and mutational analysis. The primary endpoint was the collection rate of EUS-FNB specimens suitable for comprehensive cancer panels using deep sequencing. Clinical history and genetic variations between the disease control and progressive disease groups of patients on chemotherapy were evaluated as secondary endpoints. RESULTS: The collection rate of EUS-FNB specimens suitable for comprehensive cancer panels using deep sequencing was 93.6%. The cancer panel was sequenced for 25 patients with pancreatic cancer treated initially with systemic chemotherapy. Mutation in p53 and Smad4 were positively and negatively associated, respectively, with disease control at the initial evaluation. The median time to progression in 15 patients with p53 and without Smad4 mutations was 182.0 days; whereas, it was 92.5 days in other 10 patients; this difference was significant (p = 0.020). CONCLUSIONS: Tissue samples from EUS-FNB were suitable for mutational analysis. Pancreatic cancers with p53 and without Smad4 mutations responded better to chemotherapy and had a better prognosis than those others.

There have been few studies on predictive biomarkers that may be useful to select the most suitable opioids to optimize therapeutic efficacy in individual patients with cancer pain. We recently investigated the efficacy of morphine and oxycodone using single nucleotide polymorphisms (SNPs) of the catechol-O-methyltransferase (COMT) rs4680 gene as a biomarker (RELIEF study). To explore additional biomarkers that may enable the selection of an appropriate opioid for individual patients with cancer pain, three SNPs were examined: C-C motif chemokine ligand 11 (CCL11; rs17809012), histamine N-methyltransferase (HNMT; rs1050891) and transient receptor potential V1 (TRPV1; rs222749), which were screened from 74 pain-related SNPs. These SNPs, which were identified as being significantly associated with the analgesic effect of morphine, were then used to genotype the 135 patients in the RELIEF study who had been randomized into a morphine group (n=69) or an oxycodone group (n=66). The present study then assessed whether the SNPs could also be used as selective biomarkers to predict which opioid(s) might be the most suitable to provide pain relief for patients with cancer. Oxycodone tended to provide superior analgesic effects over morphine in patients carrying the genotype AA for the CCL11 rs17809012 SNP (P=0.012 for interaction), suggesting that it could serve as a potential biomarker for personalized analgesic therapy for patients suffering with cancer pain.

BACKGROUND: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), such as erlotinib, are standard-of-care for patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC), but most patients progress within 1 year. Previously, we demonstrated that erlotinib plus bevacizumab (EB) improved progression-free survival (PFS) in patients with EGFR-positive non-squamous NSCLC in the randomized JO25567 study. To understand this effect, we conducted comprehensive exploratory biomarker analyses. METHODS: Using blood and tissue specimens from patients enrolled in the JO25567 study, angiogenesis-related serum factors, plasma vascular endothelial growth factor-A (pVEGFA), angiogenesis-related gene polymorphisms, and messenger RNAs (mRNAs) in tumor tissue were analyzed. Interactions between potential predictors and treatment effect on PFS were analyzed in a Cox model. Continuous variable predictors were evaluated by multivariate fractional polynomial interaction methodology and subpopulation treatment effect pattern plotting (STEPP). RESULTS: Overall, 152 patients treated with EB or erlotinib alone (E) were included in the analysis. Among 26 factors analyzed in 134 baseline serum samples, high follistatin and low leptin were identified as potential biomarkers for worse and better outcomes with EB, with interaction P values of 0.0168 and 0.0049, respectively. Serum concentrations of 12 angiogenic factors were significantly higher in patients with high follistatin. Low pVEGFA levels related to better outcomes with EB, interaction P=0.033. VEGF-A165a was the only predictive tissue mRNA, showing a similar trend to pVEGFA. No valid results were obtained in 13 polymorphisms of eight genes. CONCLUSIONS: EB treatment showed better treatment outcomes in patients with low pVEGFA and serum leptin, and limited efficacy in patients with high serum follistatin.

INTRODUCTION: The PACIFIC regimen of consolidation therapy with the programmed cell death-ligand 1 inhibitor durvalumab after definitive concurrent chemoradiation therapy has become a standard of care for individuals with unresectable stage III NSCLC. Nevertheless, approximately half of the treated patients experience disease progression within 1 year, with the mechanisms of treatment resistance being poorly understood. We here performed a nationwide prospective biomarker study to explore the resistance mechanisms (WJOG11518L:SUBMARINE). METHODS: A total of 135 patients with unresectable stage III NSCLC who received the PACIFIC regimen were included for comprehensive profiling of the tumor microenvironment by immunohistochemistry, transcriptome analysis, and genomic sequencing of pretreatment tumor tissue and flow cytometric analysis of circulating immune cells. Progression-free survival was compared on the basis of these biomarkers. RESULTS: The importance of preexisting effective adaptive immunity in tumors was revealed for treatment benefit regardless of genomic features. We also identified CD73 expression by cancer cells as a mechanism of resistance to the PACIFIC regimen. Multivariable analysis of immunohistochemistry data with key clinical factors as covariables indicated that low CD8+ tumor-infiltrating lymphocyte density and the high CD73+ cancer cells were independently associated with poor durvalumab outcome (hazard ratios = 4.05 [95% confidence interval: 1.17-14.04] for CD8+ tumor-infiltrating lymphocytes; 4.79 [95% confidence interval: 1.12-20.58] for CD73). In addition, whole-exome sequencing of paired tumor samples suggested that cancer cells eventually escaped immune pressure as a result of neoantigen plasticity. CONCLUSIONS: Our study emphasizes the importance of functional adaptive immunity in stage III NSCLC and implicates CD73 as a promising treatment target, thus providing insight forming a basis for development of a new treatment approach in NSCLC.

BACKGROUND: Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has limited treatment options for patients with EGFR-mutated non-small-cell lung cancer (NSCLC). Although osimertinib or afatinib alone induced drug-resistant clones with EGFR secondary mutation in a preclinical model, its combination prevented the appearance of these mutations. We investigated alternating-dose therapy of osimertinib and afatinib in patients with EGFR-mutant NSCLC in a single-arm Phase II trial. METHODS: Treatment-naïve patients with stage IV NSCLC harboring an activating EGFR mutation were enrolled. Alternating cycles of osimertinib (80 mg/day) followed by afatinib (20 mg/day) were administered every 8 weeks. Genomic analysis was performed using circulating tumor DNA obtained before and after the treatment. RESULTS: Among the 46 enrolled patients, the median progression-free survival was 20.2 months. The overall response rate was 69.6%. The median overall survival was not reached. Among the 26 plasma samples obtained after the acquisition of resistance, 3 showed an increased MET gene copy number, and 1 showed BRAF mutation. Meanwhile, no EGFR secondary mutation was detected. CONCLUSION: The efficacy of our treatment was not significantly different from osimertinib alone, as reported previously in untreated advanced NSCLC patients with EGFR mutations. Although the sample size was limited, this treatment may prevent the emergence of EGFR secondary mutations that trigger drug resistance. Further studies are warranted to establish the significance of this treatment. CLINICAL TRIAL REGISTRATION: jRCTs051180009.

Resistance to ROS1 tyrosine kinase inhibitors is inevitable, but it has been unclear whether crizotinib might be effective after the development of entrectinib resistance. We here present a case of ROS1-rearranged NSCLC that responded to crizotinib after tumor progression due to MET polysomy during entrectinib treatment. This case suggests that crizotinib is an effective option for patients with MET polysomy, even after disease progression on entrectinib.

BACKGROUND: The Idylla™ EGFR Mutation Test is an ultra-rapid single-gene test that detects epidermal growth factor receptor (EGFR) mutations using formalin-fixed paraffin-embedded specimens. Here, we compared the performance of the Idylla EGFR Mutation Test with the Cobas® EGFR Mutation Test v2. METHODS: Surgically resected NSCLC specimens obtained at two Japanese institutions (N = 170) were examined. The Idylla EGFR Mutation Test and the Cobas EGFR Mutation Test v2 were performed independently and the results were compared. For discordant cases, the Ion AmpliSeq Colon and Lung Cancer Research Panel V2 was performed. RESULTS: After the exclusion of five inadequate/invalid samples, 165 cases were evaluated. EGFR mutation analysis revealed 52 were positive and 107 were negative for EGFR mutation in both assays (overall concordance rate: 96.4%). Analyses of the six discordant cases revealed that the Idylla EGFR Mutation Test was correct in four and the Cobas EGFR Mutation Test v2 was correct in two. In a trial calculation, the combination of the Idylla EGFR Mutation Test followed by a multi-gene panel test will reduce molecular screening expenses if applied to a cohort with EGFR mutation frequency >17.9%. CONCLUSIONS: We demonstrated the accuracy and potential clinical utility of the Idylla EGFR Mutation Test as a molecular screening platform in terms of turnaround time and molecular testing cost if applied to a cohort with a high EGFR mutation incidence (>17.9%).

BACKGROUND: Trifluridine/tipiracil (FTD/TPI) improved the overall survival in patients with metastatic colorectal cancer (mCRC) who had previously received standard chemotherapies; however, the clinical outcomes remain poor. OBJECTIVE: A multicenter phase II study aimed to assess the efficacy and safety of FTD/TPI plus cetuximab rechallenge. PATIENTS AND METHODS: Patients with histologically confirmed RAS wild-type mCRC refractory to prior anti-epidermal growth factor receptor (anti-EGFR) antibody were enrolled and treated with FTD/TPI (35 mg/m2 twice daily on days 1-5 and 8-12) plus cetuximab (initially 400 mg/m2, followed by weekly 250 mg/m2) every 4 weeks. The primary endpoint was disease control rate (DCR), expecting a target DCR of 65% and null hypothesis of 45% with 90% power and 10% one-sided alpha error. Gene alterations of RAS, BRAF, EGFR, PIK3CA, ERBB2, and MET in pre-treatment circulating tumor DNA were evaluated using the Guardant360 assay. RESULTS: A total of 56 patients (median age 60 years; left-sided tumors 91%; objective partial or complete response during the prior anti-EGFR therapy 61%) were enrolled. The DCR was 54% (80% confidence interval [CI] 44-63; P = 0.12), with a partial response rate of 3.6%. Median progression-free survival (PFS) was 2.4 months (95% CI 2.1-3.7). In the circulating tumor DNA analysis, patients without any alterations of the six genes (n = 20) demonstrated higher DCR (75% vs. 39%; P = 0.02) and longer PFS (median 4.7 vs. 2.1 months; P < 0.01) than those with any gene alterations (n = 33). The most common grade 3/4 hematologic adverse event was neutropenia (55%). No treatment-related deaths occurred. CONCLUSIONS: FTD/TPI plus cetuximab rechallenge did not demonstrate clinically meaningful efficacy in all mCRC patients, but might be beneficial for the molecularly selected population.

Abstract Background Uterine angioleiomyoma is benign tumor that composed of smooth muscle cells and thick-walled vessels. It is a very rare condition reported to present as lower abdominal mass, accompanied by dysmenorrhea and hypermenorrhea. However, its clinical presentation is not known. Case presentation We report the case of a 44-year-old Japanese woman who developed severe anemia with disseminated intravascular coagulation without obvious external bleeding. The patient had a huge abdominal mass of over 20 cm in size, which was thought to be a uterine tumor. She received daily blood transfusions and her condition improved rapidly after she underwent hysterectomy. Pathological examination of the tumor revealed spindle-shaped cells with little atypia and mitosis, and numerous large vessels with smooth muscle and thrombus in the vessels. Conclusions Uterine angioleiomyoma was identified as the cause of the coagulation abnormality. CCND2 and AR gene amplification was detected in the tumor. Uterine tumors that present with coagulopathy despite a clinical course suggestive of benign disease should undergo differential diagnosis for uterine angioleiomyoma.

Vestibular schwannoma (VS) is the most common tumor of the cerebellopontine angle. Despite the increasing diagnosis of sporadic VS over the past decade, the use of traditional microsurgeries to treat VS has decreased. This is likely a result of the adoption of serial imaging as the most common initial evaluation and treatment strategy, especially for small-sized VS. However, the pathobiology of VSs remains unclear, and elucidating the genetic information of tumor tissue may reveal novel insights. The present study performed a comprehensive genomic analysis of all exons in the key tumor suppressor and oncogenes from 10 small (<15 mm) sporadic VS samples. The evaluations identified NF2, SYNE1, IRS2, APC, CIC, SDHC, BRAF, NUMA1, EXT2, HRAS, BCL11B, MAGI1, RNF123, NLRP1, ASXL1, ADAMTS20, TAF1L, XPC, DDB2 and ETS1 as mutated genes. The current study could not draw any new conclusions about the relationship between VS-related hearing loss and gene mutations; however, it did reveal that NF2 was the most frequently mutated gene in small sporadic VS.

The development of BCR::ABL1 tyrosine kinase inhibitors (TKIs), such as dasatinib, has dramatically improved survival in cases of chronic myeloid leukaemia (CML). However, the development of resistance to BCR::ABL1 TKIs is a clinical problem. BCR::ABL1 TKI resistance is known to have BCR::ABL1-dependent or BCR::ABL1-independent mechanisms, but the mechanism of BCR::ABL1 independence is not well understood. In the present study, we investigated the mechanism of BCR::ABL1-independent dasatinib resistance. The expression and activation level of genes or proteins were evaluated using array CGH, real time PCR, or western blot analysis. Gene expression was modulated using siRNA-mediated knockdown. Cell survival was assessed by using trypan blue dye method. We found that dasatinib-resistant K562/DR and KU812/DR cells did not harbour a BCR::ABL1 mutation but had elevated expression and/or activation of MOS, TPL2 and ERK1/2. In addition, MOS siRNA, TPL2 siRNA and trametinib resensitized dasatinib-resistant cells to dasatinib. Moreover, expression levels of MOS in dasatinib non-responder patients with CML were higher than those in dasatinib responders, and the expression of TPL2 tended to increase in dasatinib non-responder patients compared with that in responder patients. Our results indicate that activation of ERK1/2 by elevated MOS and TPL2 expression is involved in dasatinib resistance, and inhibition of these proteins overcomes dasatinib resistance. Therefore, MOS, TPL2 and ERK1/2 inhibitors may be therapeutically useful for treating BCR::ABL1-independent dasatinib-resistant CML.

Our previous real-world studies raised concerns that sequential biomarker testing may lead to increased time to treatment when compared with simultaneous single biomarker testing. The Oncomine Dx target test (ODxTT), a next-generation sequencing-based multiplex biomarker panel test approved in Japan in 2019, is expected to improve time to treatment due to changes in testing methods. This retrospective observational study examined data claims for reimbursement submitted for patients with lung cancer in Japan between June 1, 2019, and March 31, 2020. To evaluate the change in testing prevalence over time and associated improvements in time to treatment, descriptive statistics were used to characterize biomarker testing patterns and rates and evaluate the time to treatment in the time following the approval of ODxTT considering transitions over time during the evaluation period. EGFR and programmed death ligand 1 (PD-L1) were the most tested biomarkers in overall single and simultaneous single testing in the 6177 patients in this study. Individual single biomarker testing gradually decreased over time, except testing for PD-L1, which remained constant. The use of ODxTT gradually increased in this period. Time to treatment decreased from 29 to 22 days with ODxTT, in contrast to single biomarker tests (median 21-23 days overall). These results indicate that biomarker testing frequency changed in Japanese clinical practice during the study and that the use of ODxTT has increased over time, which potentially contributed to the shortening of time to treatment.

Anaplastic lymphoma kinase (ALK)-positive lung cancer is a rare cancer that occurs in approximately 5% of non-small-cell lung cancer (NSCLCs) patients. Despite the excellent efficacy of ALK-tyrosine kinase inhibitor in ALK-positive NSCLCs, most patients experience resistance. We conducted a phase II study to investigate the combination of alectinib with bevacizumab in ALK-positive NSCLC patients after failure of alectinib. In this study, ALK-positive nonsquamous NSCLC patients previously treated with alectinib received bevacizumab 15 mg/kg on day 1 every 3 weeks and alectinib 600 mg/day until disease progression. The primary endpoints were progression-free survival (PFS) and the safety of alectinib and bevacizumab. The secondary endpoints included overall survival (OS) and correlation of circulating tumor DNA and plasma proteins with PFS. Of the 12 patients treated, the median PFS was 3.1 months (95% CI 1.2-16.1), and the median OS was 24.1 months (95% CI 8.3-not estimable). The EML4-ALK fusion gene in circulating tumor DNA was significantly correlated with shorter PFS (1.2 months vs. 11.4 months, HR 5.2, p = 0.0153). Two patients experienced grade 3 adverse events; however, none of the patients required dose reduction. Although the primary endpoint was not met, alectinib combined with bevacizumab showed clinical efficacy in ALK-positive patients.

Despite the success of cancer immunotherapies such as programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) inhibitors, patients often develop resistance. New combination therapies with PD-1/PD-L1 inhibitors are needed to overcome this issue. Bezafibrate, a ligand of peroxisome proliferator-activated receptor-γ coactivator 1α/peroxisome proliferator-activated receptor complexes, has shown a synergistic antitumor effect with PD-1 blockade in mice that is mediated by activation of mitochondria in T cells. We have therefore now performed a phase 1 trial (UMIN000017854) of bezafibrate with nivolumab in previously treated patients with advanced non-small cell lung cancer. The primary end point was the percentage of patients who experience dose-limiting toxicity, and this combination regimen was found to be well tolerated. Preplanned comprehensive analysis of plasma metabolites and gene expression in peripheral cytotoxic T cells indicated that bezafibrate promoted T cell function through up-regulation of mitochondrial metabolism including fatty acid oxidation and may thereby have prolonged the duration of response. This combination strategy targeting T cell metabolism thus has the potential to maintain antitumor activity of immune checkpoint inhibitors and warrants further validation.

Abstract Background We hypothesized that the high-dose opioid requirement in patients carrying the rs4680-GG variant in the COMT gene encoding catechol-O-methyltransferase would be greater for patients taking morphine than for those taking oxycodone, thus providing a much-needed biomarker to inform opioid selection for cancer pain. Methods A randomized, multicenter, open-label trial was conducted at a Japanese hospital’s palliative care service. Patients with cancer pain treated with regular doses of nonsteroidal anti-inflammatory drugs or acetaminophen were enrolled and randomized (1:1) into morphine (group M) and oxycodone (group O) groups. The minimum standard dose of immediate-release (IR) oral opioids was repeatedly administered by palliative care physicians to achieve pain-reduction goals (Pain reduction ≥ 33% from baseline and up to ≤ 3 on a numerical rating scale). The primary endpoint was the proportion of subjects requiring high-dose opioids on day 0 with the GG genotype. Results Of 140 participants who developed cancer-related pain among 378 subjects registered and pre-screened for the genotype, 139 were evaluated in the current study. Among patients carrying a COMT rs4680-GG genotype, 48.3% required high-dose opioids in group M, compared with the 20.0% in group O (95% CI, 3.7%-50.8%; P = .029). Of those with the non-GG genotype, 41.5% treated with morphine and 23.1% with oxycodone required high-dose opioids (95% CI, 3.3%-38.3%; P = 0.098). Conclusion Using the COMT rs4680 genotype alone is not recommended for selecting between morphine and oxycodone for pain relief.

OBJECTIVE: Immune checkpoint inhibitors (ICIs) have become a key therapeutic modality for advanced non-small cell lung cancer (NSCLC), but most patients experience primary or acquired resistance to these drugs. We here explored the mechanisms underlying both types of ICI resistance by analysis of the tumor immune microenvironment (TME). MATERIALS AND METHODS: Four patients who experienced a long-term response to ICI treatment (progression-free survival [PFS] of ≥12 months) followed by disease progression, after which a rebiopsy was immediately performed (cohort-A), as well as four patients who experienced early tumor progression during ICI treatment (PFS of <9 weeks, cohort-B) were enrolled in this retrospective study. The pretreatment TME was evaluated by 16- or 17-color multiplex immunohistochemistry (mIHC)-based spatial profiling at the single-cell level for both cohorts. In cohort-A, changes in the TME after disease progression during ICI treatment were also investigated by mIHC analysis and transcriptomic analysis. RESULTS: Pretreatment tumor tissue from cohort-B manifested poor infiltration of tumor-reactive CD8+ T cells characterized by CD39 and CD103 expression or by programmed cell death-1 expression, implicating insufficient recognition of tumor cells by CD8+ T cells as a mechanism of primary ICI resistance. Analysis of the paired tumor specimens from cohort-A revealed various changes in the TME associated with acquired ICI resistance, including substantial infiltration of myeloid-derived suppressor cells and M2-type tumor-associated macrophages without a marked decline in the number of tumor-reactive CD8+ T cells; a decrease in the number of tumor-reactive CD8+ T cells; and an apparent decrease in neoantigen presentation by tumor cells. CONCLUSION: The presence of intratumoral tumor-reactive CD8+ T cells may be a prerequisite for a long-term response to ICI treatment in advanced NSCLC, but it is not sufficient for cancer cell eradication. Various TME profiles are associated with acquired ICI resistance, suggesting that patient-specific strategies to overcome such resistance may be necessary.

During tumorigenesis, certain tissues are colonized by mutant clones with oncogenic driver mutations as precancer lesions. These mutations can facilitate clonal expansion and may contribute to malignant transformation. The molecular features of low-grade non-muscle invasive bladder cancer (NMIBC) and high-grade bladder cancer are so distinct that they are thought to follow different evolutionary tumorigenesis pathways. Although NMIBC accounts for most bladder tumors, the somatic mutation patterns in "precancer" urothelium of patients with NMIBC remain unclear. Here, we analyzed specimens of normal urothelium and bladder tumors from patients with low-grade and high-grade NMIBC and investigated the genomic evolution of the cancer. Somatic mutations were analyzed using 50 oncogene-targeted sequences and droplet digital polymerase chain reaction for TERT promoter mutations. Somatic mutations in TERT promoter, FGFR3, and CDKN2A were characteristically identified in the normal urothelium of patients with NMIBC. These mutations, consistently identified in both tumor and normal specimens, likely affect clonal expansion during the malignant transformation of NMIBC. Though larger samples and comprehensive study are warranted to confirm our results, the difference in mutational landscape of the precancerous urothelium of patients with bladder cancer could offer deeper understandings of genomic evolution in bladder tumorigenesis.

Introduction: Despite a considerable benefit of adding immune checkpoint inhibitors (ICIs) to platinum-based chemotherapy for patients with extensive-stage SCLC (ES-SCLC), a durable response to ICIs occurs in only a small minority of such patients. Methods: A total of 135 patients with ES-SCLC treated with chemotherapy either alone (chemo-cohort, n = 71) or together with an ICI (ICI combo-cohort, n = 64) was included in this retrospective study. Tumors were classified pathologically as inflamed or noninflamed on the basis of programmed death-ligand 1 expression and CD8+ tumor-infiltrating lymphocyte density. Immune-related gene expression profiling was performed, and predicted neoantigen load was determined by whole-exome sequencing. Results: Among patients in the ICI combo-cohort, median progression-free survival was 10.8 and 5.1 months for those with inflamed (n = 7) or noninflamed (n = 56) tumors, respectively (log-rank test p = 0.002; hazard ratio of 0.26). Among the 89 patients with immune-related gene expression profiling data available, inflamed tumors had a higher T cell-inflamed GEP score than did noninflamed tumors (-0.18 versus -0.58, p < 0.001). The 12-month progression-free survival rate was 16.1% and 0% for patients in the ICI combo-cohort harboring tumors with a high (n = 26) or low (n = 18) frameshift neoantigen load, respectively. A high-frameshift neoantigen load was associated with up-regulation of gene signatures related to antigen presentation and costimulatory signaling. A durable clinical benefit of ICI therapy was observed only in patients with inflamed tumors and a high-frameshift neoantigen load. Conclusions: Expression of programmed death-ligand 1, CD8+ T cell infiltration, and a high-frameshift neoantigen load are associated with clinical benefit of ICI therapy in ES-SCLC. Clinical trial registration: UMIN000041056.

Advances in cancer genome care over the past few years have included the development of gene panel testing for various biomarkers. This article summarizes issues and provides recommendations related to analytical performance evaluations for new oncology gene panels. The scope of these recommendations includes comprehensive genomic profiling assays related to gene panel testing that uses histological or serum specimens to detect gene mutations. As a research project of the Japan Agency for Medical Research and Development Research on Regulatory Science of Pharmaceuticals and Medical Devices, we convened the working group committee that consisted of more than 30 experts from academia, industry, and government. We have discussed the points that should be considered to allow maximal simplification of assessments using clinical specimens in evaluating accuracy and limit of detection in equivalence and analytical performance for three years. We provide recommendations specific to each type of gene mutation as well as to reference standards or specimens used for evaluations. In addition, in order to facilitate the discussion on the analytical performance of gene panel tests by multidisciplinary tumor boards of hospitals, the present recommendations also describe the items that companies are expected to provide information on in their packaging inserts and reports, and the items that are expected to be discussed by multidisciplinary tumor boards. Our working group document will be important for participants in multidisciplinary tumor boards including medical oncologists and genome scientists, and developers of gene panels not only in Japan but also in other countries.

Abstract Recent studies suggested that aggregates of mutant p53 proteins may propagate and impair normal p53 functioning in recipient cells. Our previous study showed that cancer cell-derived p53 aggregates that cells internalized interfered with p53-dependent apoptosis in recipient cells. However, involvement of p53 aggregate propagation in cancer pathology has not been fully elucidated. Here, we screened patients with high-grade serous ovarian carcinoma, which is characterized by an extremely high frequency of TP53 gene mutations, to show that patients with cytoplasmic p53 deposits have a poor prognosis compared with patients with complete p53 absence or strong nuclear p53 positivity. Cytoplasmic p53 in the patients with poor prognosis consisted of protein aggregates, which suggests that p53 aggregates are oncogenic drivers. Indeed, an inhibitor of p53 aggregation restored cellular apoptosis, a proper p53 function, in p53 aggregate-bearing patient-derived tumor organoids. In cell-based assays, endogenous and exogenous mutant p53 aggregates hindered chemotherapeutic activity of cisplatin, which depends on normal p53 functions. This inhibition was reduced by blocking p53 aggregation or internalization of p53 aggregates. Our study, thus indicates the involvement of p53 aggregate transmission in poor prognosis and in chemotherapy resistance in cancers.

BACKGROUND: The Biocartis Idylla™ platform is a fully automated, real-time PCR-based diagnostic system. The Idylla™ KRAS and NRAS-BRAF Mutation Tests have been developed for the qualitative detection of mutations in KRAS, NRAS and BRAF genes, facilitating the genomic profiling of patients with colorectal cancer. The aim of the present study was to evaluate clinical performances of these tests in Japan. METHODS: The RAS and BRAF mutation statuses of 253 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues were analyzed using the Investigational Use Only Idylla™ KRAS Mutation Test and the Idylla™ NRAS-BRAF Mutation Test and an in vitro diagnostics (IVD) kit (MEBGEN RASKET™-B kit). RESULTS: The success rate for obtaining a valid mutational data without retest of the Idylla tests was 97.6% (247/253): 111 KRAS mutations (43.8%), 9 NRAS mutations (3.6%), and 36 BRAF V600E mutations (14.2%) were detected using the Idylla tests. Compared with the MEBGEN RASKET-B results, the positive concordance rate was 97.4%, the negative concordance rate was 95.7%, and the overall concordance rate was 95.3% (κ = 0.919, 95% CI 0.871-0.967). The average turnaround time to Idylla™ KRAS and NRAS-BRAF Mutation Test was 5.6 working days (range: 3-11 days). CONCLUSION: This result demonstrates a high concordance between the Idylla™ KRAS and NRAS-BRAF Mutation Tests and an existing IVD kit. In this manner, the Idylla™ mutation tests were validated for the detection of clinically significant KRAS, NRAS, and BRAF mutations in FFPE samples from colorectal cancer patients.

INTRODUCTION: Alternation of osimertinib and afatinib is a potential approach to overcome osimertinib resistance and to allow complementation of drug efficacy without compromising safety in patients with epidermal growth factor receptor gene (EGFR)-mutated non-small cell lung cancer (NSCLC). METHODS: Treatment-naive patients with stage IV NSCLC harboring an activating EGFR mutation (L858R or exon-19 deletion) were enrolled. Alternating cycles of osimertinib at 80 mg/day for 8 weeks followed by afatinib at 20 mg/day for 8 weeks were administered. The primary end point was 12-month progression-free survival (PFS) probability. RESULTS: Forty-six patients were enrolled and treated with study therapy. The 12-month PFS probability was 70.2% (60% confidence interval [CI], 63.9-75.6%; 95% CI, 54.2-81.5%), which did not meet the primary end point. After a median follow-up time of 25.7 months, the median PFS was 21.3 months (95% CI, 16.3 months-not reached). The overall response rate was 69.6% (95% CI, 54.2-82.3%). The most common treatment-related adverse events (any grade or grade ≥ 3, respectively) were diarrhea (73.9%, 4.3%), rash acneiform (63.0%, 2.2%), and paronychia (52.2%, 0%). Five cases of pneumonitis, two of grade 2 and thres of grade 3, were apparent, all of which developed during osimertinib treatment. Exploratory evaluation of circulating tumor DNA suggested that coexisting TP53 mutations did not influence PFS for the alternating therapy. CONCLUSIONS: Alternating therapy with osimertinib and afatinib for treatment-naive patients with EGFR- mutated advanced NSCLC did not meet its primary end point, despite the encouraging efficacy and safety profile of this treatment strategy.

Introduction: Ramucirumab (RAM) plus erlotinib was found to have superior progression-free survival (PFS) versus placebo plus erlotinib in untreated EGFR-mutated metastatic NSCLC in the global phase 3 RELAY study. RELAY+ was an open-label, two-period, single-arm, exploratory study of RAM plus gefitinib (GEF; period 1) and RAM plus osimertinib (period 2) in East Asia (NCT02411448). Methods: Period 1 evaluated RAM (10 mg/kg) plus GEF (250 mg/d) in patients with untreated EGFR-mutated metastatic NSCLC. Period 2 evaluated RAM plus osimertinib (80 mg/d) in patients with disease progression who acquired T790M mutation in period 1. Exploratory end points included 1-year PFS rate (primary), other efficacy parameters, safety, and biomarker analyses of plasma (baseline, on-treatment, follow-up) using next-generation sequencing. Results: From December 2017 to August 2018, a total of 82 patients were enrolled and started treatment (period 1, RAM + GEF). The 1-year PFS rate was 62.9% (95% confidence interval: 50.3-73.1). Treatment-emergent adverse events of grade three or higher were reported with RAM plus GEF in 60 of 82 patients (73.2%; five patients [6.1%] grade four). There were two deaths owing to adverse events that occurred (acute cardiac failure, congestive cardiac failure). T790M rate at disease progression in plasma was 81.0% (13 of 16 patients). Conclusions: RELAY+ was found to have a favorable benefit-risk profile for RAM plus GEF in first-line treatment of East Asian patients with EGFR-mutated NSCLC.

The mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol-3 kinase (PI3K)/AKT pathways are dysregulated in various human cancers, including pancreatic ductal adenocarcinoma (PDAC), which has a very poor prognosis due to its lack of efficient therapies. We have previously identified ACAGT-007a (GT-7), an anti-cancer compound that kills ERK-active melanoma cells by inducing ERK-dependent apoptosis. Here, we investigated the apoptosis-inducing effect of GT-7 on three PDAC cell lines and its relevance with the MAPK/ERK and PI3K/AKT signaling pathways. GT-7 induced apoptosis in PDAC cells with different KRAS mutations (MIA-Pa-Ca-2 (KRAS G12C), T3M4 (KRAS Q61H), and PANC-1 (KRAS G12D)), being T3M4 most susceptible, followed by MIA-Pa-Ca-2, and PANC-1 was most resistant to apoptosis induction by GT-7. GT-7 stimulated ERK phosphorylation in the three PDAC cells, but only T3M4 displayed ERK-activation-dependent apoptosis. Furthermore, GT-7 induced a marked down-regulation of AKT phosphorylation after a transient peak in T3M4, whereas PANC-1 displayed the strongest and most sustained AKT activation, followed by MIA-Pa-Ca-2, suggesting that sustained AKT phosphorylation as a determinant for the resistance to GT-7-mediated apoptosis. Consistently, a PI3K inhibitor, Wortmannin, abolished AKT phosphorylation and enhanced GT-7-mediated apoptosis in T3M4 and MIA-Pa-Ca-2, but not in PANC-1, which showed residual AKT phosphorylation. This is the first report that ERK stimulation alone or in combination with AKT signaling inhibition can effectively induce apoptosis in PDAC and provides a rationale for a novel concurrent targeting of the PI3K/AKT and ERK pathways.

BACKGROUND/PURPOSE OF THE STUDY: Tumor heterogeneity based on copy number variations is associated with the evolution of cancer and its clinical grade. Clonal composition (CC) represents the number of clones based on the distribution of B-allele frequency (BAF) obtained from a genome-wide single nucleotide polymorphism (SNP) array. A higher CC number represents a high degree of heterogeneity. We hypothesized and evaluated that the CC number in hepatocellular carcinoma (HCC) tissues might be associated with the clinical outcomes of patients. METHODS: Somatic mutation, whole transcriptome, and CC number based on copy number variations of 36 frozen tissue samples of operably resected HCC tissues were analyzed by targeted deep sequencing, transcriptome analysis, and SNP array. RESULTS: The samples were classified into the heterogeneous tumors as poly-CC (n = 26) and the homogeneous tumors as mono-CC (n = 8). The patients with poly-CC had a higher rate of early recurrence and a significantly shorter recurrence-free survival period than the mono-CC patients (7.0 months vs. not reached, p = 0.0084). No differences in pathogenic non-synonymous mutations, such as TP53, were observed between the two groups when targeted deep sequencing was applied. A transcriptome analysis showed that cell cycle-related pathways were enriched in the poly-CC tumors, compared to the mono-CC tumors. Poly-CC HCC is highly proliferative and has a high risk of early recurrence. CONCLUSION: CC is a possible candidate biomarker for predicting the risk of early postoperative recurrence and warrants further investigation.

BACKGROUND: Cancer of unknown primary (CUP) has a poor prognosis. Given the recent approval of immune checkpoint inhibitors for several cancer types, we carried out a multicenter phase II study to assess the efficacy of nivolumab for patients with CUP. PATIENTS AND METHODS: Patients with CUP who were previously treated with at least one line of systemic chemotherapy constituted the principal study population. Previously untreated patients with CUP were also enrolled for exploratory analysis. Nivolumab (240 mg/body) was administered every 2 weeks for up to 52 cycles. The primary endpoint was objective response rate in previously treated patients as determined by blinded independent central review according to RECIST version 1.1. RESULTS: Fifty-six patients with CUP were enrolled in the trial. For the 45 previously treated patients, objective response rate was 22.2% [95% confidence interval (CI), 11.2% to 37.1%], with a median progression-free survival and overall survival of 4.0 months (95% CI, 1.9-5.8 months) and 15.9 months (95% CI, 8.4-21.5 months), respectively. Similar clinical benefits were also observed in the 11 previously untreated patients. Better clinical efficacy of nivolumab was apparent for tumors with a higher programmed death-ligand 1 expression level, for those with a higher tumor mutation burden, and for microsatellite instability-high tumors. In contrast, no differences in efficacy were apparent between tumor subgroups based on estimated tissue of origin. Adverse events were consistent with the known safety profile of nivolumab. No treatment-related death was observed. CONCLUSIONS: Our results demonstrate a clinical benefit of nivolumab for patients with CUP, suggesting that nivolumab is a potential additional therapeutic option for CUP.

To implement precision oncology, analytical validity as well as clinical validity and utility are important. However, proficiency testing (PT) to assess validity has not yet been systematically performed in Japan. To investigate the quality of next-generation sequencing (NGS) platforms and cancer genome testing prevalent in laboratories, we performed pilot PT using patient samples. We prepared genomic DNA from the cancer tissue and peripheral blood of 5 cancer patients and distributed these to 15 laboratories. Most participating laboratories successfully identified the pathogenic variants, except for two closely located KRAS variants and 25 bp delins in EGFR. Conversely, the EGFR L858R variant was successfully identified, and the allele frequency was similar for all the laboratories. A high DNA integrity number led to excellent depth and reliable NGS results. By conducting this pilot study using patient samples, we were able to obtain a glimpse of the current status of cancer genome testing at participating laboratories. To enhance domestic cancer genome testing, it is important to conduct local PT and to involve the parties concerned as organizers and participants.

Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows great clinical activity in non-small cell lung cancer (NSCLC) patients with EGFR mutations regardless of T790M mutation at first-line chemotherapy. Previous studies demonstrated that there are few patients with initial resistance to osimertinib. Here, we describe a case to report the efficacy of afatinib in an EGFR-mutated NSCLC patient with early progression to first-line osimertinib treatment. A 68-year-old Japanese male was diagnosed with stage IVB lung adenocarcinoma with the EGFR L858R mutation in exon 21. Two months after the start of osimertinib, his tumor progressed at the initial response evaluation. Because he refused to receive cytotoxic chemotherapy, afatinib treatment was initiated. He was administered afatinib, and the tumor shrank. After five months of afatinib treatment, nevertheless the primary tumor was not enlarged, he experienced disease progression with leptomeningeal metastasis and passed away. To elucidate the resistance mechanisms of osimertinib in this patient, we performed next-generation sequencing (NGS) on tumor samples from pleural effusions after osimertinib failure. NGS revealed no specific gene mutations causing resistance to osimertinib except for the EGFR L858R mutation; however, his tumor had a relatively high tumor mutational burden. Afatinib is considered an option for EGFR-mutated patients with early progression to osimertinib.

PURPOSE: Although the efficacy of programmed cell death-1 (PD-1) blockade is generally poor for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR) gene, EGFR tyrosine kinase inhibitors (TKIs) may improve the tumor immune microenvironment. We performed a randomized study to assess whether nivolumab improves outcome compared with chemotherapy in such patients previously treated with EGFR-TKIs. PATIENTS AND METHODS: Patients with EGFR-mutated NSCLC who acquired EGFR-TKI resistance not due to a secondary T790M mutation of EGFR were randomized 1:1 to nivolumab (n=52) or carboplatin-pemetrexed (n=50). The primary endpoint was progression-free survival (PFS). RESULTS: Median PFS and 1-year PFS probability were 1.7 months and 9.6% for nivolumab versus 5.6 months and 14.0% for carboplatin-pemetrexed [log-rank P < 001; hazard ratio (HR) of 1.92, with a 60% confidence interval (CI) of 1.61-2.29]. Overall survival was 20.7 and 19.9 months [HR = 0.88 (95% CI, 0.53-1.47)], and response rate was 9.6% and 36.0% for nivolumab and carboplatin-pemetrexed, respectively. No subgroup including patients with a high tumor mutation burden showed a substantially longer PFS with nivolumab than with carboplatin-pemetrexed. The T cell-inflamed gene expression profile score (0.11 versus -0.17, P = 0.036) and expression of genes related to cytotoxic T lymphocytes or their recruitment were higher in tumors that showed a benefit from nivolumab. CONCLUSIONS: Nivolumab did not confer a longer PFS compared with carboplatin-pemetrexed in the study patients. Gene expression profiling identified some cases with a favorable tumor immune microenvironment that was associated with nivolumab efficacy.

PURPOSE: EGFR-tyrosine kinase inhibitor (TKI) is a standard first-line therapy for activated EGFR-mutated non-small cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance. Clinical trials of the HER3-targeting antibody-drug conjugate patritumab deruxtecan (HER3-DXd) demonstrated its anticancer activity in EGFR-mutated NSCLC; however, the mechanisms that regulate HER3 expression are unknown. This study was conducted with the aim to clarify the mechanisms underlying HER3 regulation in EGFR-mutated NSCLC tumors and explored the strategy for enhancing the anticancer activity of HER3-DXd in EGFR-mutated NSCLC. EXPERIMENTAL DESIGN: Paired tumor samples were obtained from 48 patients with EGFR-mutated NSCLC treated with EGFR-TKI(s). HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pretreatment to post-EGFR-TKI resistance acquisition. The anticancer efficacy of HER3-DXd and osimertinib was evaluated in EGFR-mutated NSCLC cells. RESULTS: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared with paired pretreatment samples. RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation, especially in tumors from patients who received continuous EGFR-TKI therapy. An in vitro study also showed that EGFR-TKI increased HER3 expression, repressed AKT phosphorylation in multiple EGFR-mutated cancers, and enhanced the anticancer activity of HER3-DXd. CONCLUSIONS: Our findings help clarify the mechanisms of HER3 regulation in EGFR-mutated NSCLC tumors and highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC.

BACKGROUND: Tumour burden (TB) is implicated in resistance to programmed cell death-1/PD-L1 inhibitor (immune checkpoint inhibitor [ICI]) therapy. However, whether TB contributes to such resistance in non-small-cell lung cancer (NSCLC) has remained unknown. METHODS: A total of 260 treatment-naïve patients with advanced NSCLC who started ICI monotherapy (ICI cohort), platinum-doublet therapy (Chemo cohort) or ICI and platinum-doublet therapy (ICI+Chemo cohort) as first-line treatment were consecutively included. TB was estimated on the basis of the sum of the diameters of measurable target lesions as per Response Evaluation Criteria in Solid Tumours. Progression-free survival (PFS) in the ICI cohort was evaluated as per TB as a preplanned primary objective, with the analysis based on propensity score-weighted survival curves and estimation of restricted mean survival time (RMST). The Chemo cohort served as a control to determine whether TB is predictive of ICI treatment outcomes. The ICI+Chemo cohort was exploratory. The relation of TB to tumour immune status was assessed by immune-related gene expression profiling (irGEP) of pretreatment tumour tissue. RESULTS: In the ICI cohort, patients with a low TB showed a significantly longer PFS than did those with a high TB (median, 17.9 vs 4.3 months; weighted hazard ratio, 0.32 [95% confidence interval, 0.19-0.53]). No such difference was apparent in the other cohorts. A significant difference in overall survival was also observed only in the ICI cohort. RMST-based analysis confirmed these results. The irGEP analysis implicated M2-type macrophages, angiogenesis and transforming growth factor-β as well as protumourigenic signalling pathways in ICI resistance conferred by a high TB. CONCLUSION: A high TB was associated with a poor outcome of ICI therapy for advanced NSCLC as a result of immunosuppressive phenotypes. Development of combination or novel treatment strategies for such disease is thus warranted.

We previously examined the utility of rituximab-bendamustine (RB) in patients with follicular lymphoma (FL) exhibiting less than optimal responses to 2 cycles of the R-CHOP chemotherapy regimen. The aim of this study was to identify molecular biomarkers that can predict prognosis in RB-treated patients in the context of the prospective cohort. We first analyzed the mutational status of 410 genes in diagnostic tumor specimens by target capture and Sanger sequencing. CREBBP, KMT2D, MEF2B, BCL2, EZH2, and CARD11 were recurrently mutated as reported before, however none was predictive for progression-free survival (PFS) in the RB-treated patients (n = 34). A gene expression analysis by nCounter including 800 genes associated with carcinogenesis and/or the immune response showed that expression levels of CD8+ T-cell markers and half of the genes regulating Th1 and Th2 responses were significantly lower in progression of disease within the 24-mo (POD24) group (n = 8) than in the no POD24 group (n = 31). Collectively, we selected 10 genes (TBX21, CXCR3, CCR4, CD8A, CD8B, GZMM, FLT3LG, CD3E, EOMES, GZMK), and generated an immune infiltration score (IIS) for predicting PFS using principal component analysis, which dichotomized the RB-treated patients into immune IIShigh (n = 19) and IISlow (n = 20) groups. The 3-y PFS rate was significantly lower in the IISlow group than in the IIShigh group (50.0% [95% CI: 27.1-69.2%] vs. 84.2% [95% CI: 58.7-94.6%], P = .0237). Furthermore, the IIS was correlates with absolute lymphocyte counts at diagnosis (r = 0.460, P = .00355). These results suggest that the T-cell-associated immune markers could be useful to predict prognosis in RB-treated FL patients. (UMIN:000 013 795, jRCT:051 180 181).

Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in "Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)" published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be ≥30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.

The clinical utility of systemic therapy and genomic profiling in non-squamous-cell head and neck cancer (NSCHNC) has not been fully elucidated. This phase II trial evaluated the efficacy and safety of docetaxel and cisplatin combination in the first-line setting. Eligibility criteria were recurrent and/or metastatic NSCHNC; progressive disease within the last 6 months; no prior systemic therapy; and ECOG performance status of 0-1. Patients received docetaxel (75 mg/m2 on day 1) and cisplatin (75 mg/m2 on day 1), repeated every 21 days for 6 cycles. The primary endpoint was confirmed objective response rate (ORR). The secondary endpoints included progression-free survival (PFS), overall survival (OS), and adverse events. Next-generation sequencing (NGS) was performed using the Ion AmpliSeq Cancer Hotspot Panel v2. Twenty-three patients were enrolled from November 2012 to October 2016, of whom 8 were male. Median age was 57 years. Ninety-six percent of cases were metastatic. Among 22 evaluable patients, confirmed ORR was 45% (95% confidential interval 24-68%). With a median follow-up period of 18.8 months, median PFS and OS were 6.7 and 20.1 months, respectively. Grade 3/4 adverse events included febrile neutropenia (39%) and anemia (22%). No treatment-related deaths were observed. NGS analysis revealed potential treatment targets, including ERBB2, KIT, and ALK. The docetaxel and cisplatin combination regimen can be considered a new treatment option in recurrent and/or metastatic NSCHNC, although primary prophylaxis for febrile neutropenia should be considered. Diverse genomic alterations may lead novel treatment options.This trial was registered with the UMIN Clinical Trials Registry as UMIN000008333 on [September 1st, 2012].

BACKGROUND: MET exon 14 skipping mutation is a driver mutation in lung cancer and is highly enriched in pulmonary pleomorphic carcinomas (PPCs). Whether there is intratumor or intertumor heterogeneity in MET exon 14 skipping status or in co-occurring genetic alterations in lung cancers driven by MET exon 14 skipping is unknown. METHODS: We analyzed tumor specimens obtained from 23 PPC patients (10 autopsied and 13 surgically resected). MET exon 14 skipping was detected by RT-PCR. For patients with MET exon 14 skipping mutation, further analyses were performed. Genomic DNA (gDNA) was extracted from various histological components for each patient who underwent surgical resection (to assess intratumor heterogeneity). In autopsied patients, gDNA and total RNA were extracted from all metastatic lesions (to assess intertumor heterogeneity). RESULTS: MET exon 14 skipping mutation was detected in 4 patients (4/23, 17.4%): two surgically resected and two autopsied patients. We found no intratumor or intertumor heterogeneity in MET exon 14 skipping mutation status in these patients. We observed intratumor and intertumor heterogeneity in the copy number variations and/or mutational status of cancer-related genes; some of these differences may have an impact on MET tyrosine kinase inhibitor (TKI) efficacy. CONCLUSION: In our exploratory analysis of four cases, we observed that MET exon 14 skipping mutations are distributed homogeneously throughout histological components and between metastatic lesions. Our results also suggest that there is marked intertumor and intratumor heterogeneity in co-occurring genetic alterations, and therapeutic implications of such heterogeneity should be evaluated in future studies.

Introduction: Although treatment with osimertinib confers survival benefits in patients with lung cancer with the EGFR T790M mutation, the mechanism of acquired resistance to osimertinib remains poorly understood. We conducted a prospective observational study to identify the mechanism on the basis of repeated tissue biopsies. Methods: Patients with EGFR-mutated advanced lung cancer with a T790M mutation detected on a tissue biopsy underwent a rebiopsy after developing acquired resistance to osimertinib. Nucleic acids extracted from the biopsy samples were subjected to targeted resequencing (Oncomine Comprehensive Assay), and circulating cell-free DNA (ccfDNA) was analyzed by CAncer Personalized Profiling by deep Sequencing (AVENIO ctDNA Surveillance Kit). Results: Between November 2016 and March 2020, a total of 87 patients were screened. Among them, 44 developed acquired resistance. Of these, 19 samples from rebiopsies and 12 from preosimertinib biopsies were able to be analyzed by an Oncomine Comprehensive Assay. A ccfDNA analysis was performed in 16 patients. Regarding the mechanisms of acquired resistance, structural change in EGFR, namely, C797S, G796S, or L792V, was the most frequent alteration, being observed in 57.9% of the cases. MET gain was observed in 31.6% of the cases, and gains in cell cycle genes were observed in 26.3% of the cases. In addition, we identified GAS6 gain and an ATM mutation in a patient with small-cell transformation and a BRAF V600E mutation in a patient with oligoprogressive disease. Conclusions: A repeated tissue biopsy and a ccfDNA analysis were useful in analyzing the mechanisms underlying acquired resistance. A long treatment history of EGFR TKIs may result in a high percentage of EGFR structural change.

Introduction: The phase 3 RELAY global study (NCT02411448) revealed significant improvement in progression-free survival (PFS) with ramucirumab plus erlotinib (RAM + ERL) compared with placebo plus ERL (PL + ERL) in untreated EGFR-mutated metastatic NSCLC (hazard ratio [HR] = 0.59 [95% confidence interval (CI): 0.46-0.76, p < 0.0001]). This prespecified analysis evaluates efficacy, safety, and postprogression EGFR T790M rates of RELAY patients enrolled in Japan. Methods: Patients were randomized (1:1) to oral ERL (150 mg/d) plus intravenous RAM (10 mg/kg) or PL every 2 weeks. End points included PFS (primary), safety (secondary), and biomarker analyses (exploratory). Plasma samples collected at baseline and poststudy treatment discontinuation were evaluated for EGFR T790M mutations by next-generation sequencing. Results: The Japanese subset included 211 of 449 (47.0%) RELAY patients (RAM + ERL, n = 106; PL + ERL, n = 105). Median PFS was 19.4 versus 11.2 months for RAM + ERL versus PL + ERL treatment (HR = 0.610 [0.431-0.864]) in the Japanese intent-to-treat population, 16.6 versus 12.5 months (HR = 0.701 [0.424-1.159]) in the EGFR exon 19 deletion subgroup, and 19.4 versus 10.9 months (HR = 0.514 [0.317-0.835]) in the EGFR exon 21 L858R subgroup, respectively. Adverse events of grade 3 or above with RAM + ERL included hypertension (24.8%, all grade 3) and dermatitis acneiform (23.8%). Postprogression treatment-emergent T790M rates were similar between arms (RAM + ERL: 47%, 9 of 19 patients; PL + ERL: 50%, 20 of 40 patients). Conclusions: Clinically meaningful efficacy was observed with RAM + ERL versus PL + ERL in the RELAY Japanese subset, with no new safety concerns. Postprogression T790M rates were similar across treatment arms, indicating the addition of RAM did not affect the ERL-associated EGFR T790M rates at disease progression.

Immune checkpoint inhibitors (ICIs) have become the standard of care for several cancers. However, ICI therapy has also been associated with various immune-related adverse events (irAEs). Clinical manifestations of immune-related colitis resemble those of inflammatory bowel diseases such as ulcerative colitis (UC). The composition of the bowel microflora is thought to influence the development of inflammatory bowel disease and irAE colitis. We profiled the gene expressions and microbe compositions of colonic mucosa from patients with solid cancers receiving anti-PD-L1 antibody treatment; we then compared the expression profiles associated with irAE colitis with those associated with UC. The pathway enrichment analysis revealed functional similarities between inflamed regions of irAE colitis and UC. The common enriched pathways included leukocyte extravasation and immune responses, whereas non-inflamed mucosa from patients with irAE colitis was distinct from patients with UC and was characterized by the recruitment of immune cells. A similarity between the microbiota profiles was also identified. A decreased abundance of Bacteroides species was observed in inflamed regions from both irAE colitis and UC based on a microbiota composition analysis of 16S rDNA sequencing. Pathways associated with molecule transport systems, including fatty acids, were enriched in inflamed and non-inflamed irAE colitis and inflamed UC, similar to Piphillin-inferred KEGG pathways. While UC is characterized by local regions of inflammation, ICI treatment extends to non-inflammatory regions of the colonial mucosa where immune cells are reconstituted. This analysis of the similarity and heterogeneity of irAE colitis and UC provides important information for the management of irAE colitis.

We developed a prodrug type of curcumin, curcumin monoglucuronide (CMG), whose intravenous/intraperitoneal injection achieves a high serum concentration of free-form curcumin. Although curcumin has been reported to alter the gut microbiota and immune responses, it is unclear whether the altered microbiota could be associated with inflammation in immune-mediated diseases, such as multiple sclerosis (MS). We aimed to determine whether CMG administration could affect the gut microbiota at three anatomical sites (feces, ileal contents, and the ileal mucosa), leading to suppression of inflammation in the central nervous system (CNS) in an autoimmune model for MS, experimental autoimmune encephalomyelitis (EAE). We injected EAE mice with CMG, harvested the brains and spinal cords for histological analyses, and conducted microbiome analyses using 16S rRNA sequencing. CMG administration modulated EAE clinically and histologically, and altered overall microbiota compositions in feces and ileal contents, but not the ileal mucosa. Principal component analysis (PCA) of the microbiome showed that principal component (PC) 1 values in ileal contents, but not in feces, correlated with the clinical and histological EAE scores. On the other hand, when we analyzed the individual bacteria of the microbiota, the EAE scores correlated with significant increases in the relative abundance of two bacterial species at each anatomical site: Ruminococcus bromii and Blautia (Ruminococcus) gnavus in feces, Turicibacter sp. and Alistipes finegoldii in ileal contents, and Burkholderia spp. and Azoarcus spp. in the ileal mucosa. Therefore, CMG administration could alter the gut microbiota at the three different sites differentially in not only the overall gut microbiome compositions but also the abundance of individual bacteria, each of which was associated with modulation of neuroinflammation.

BACKGROUND: Concomitant ulcerative colitis (UC) and idiopathic thrombocytopenic purpura (ITP) is a rare phenomenon. The management of UC with ITP can be challenging, since a decreased platelet count augments UC. CASE SUMMARY: A 24-year-old man with UC and steroid-resistant ITP experienced UC flare. Although continuous infusion of cyclosporine was initiated, UC did not improve. The administration of tofacitinib subsequently led to the induction of remission. The patient has maintained remission of UC and ITP for over one year on tofacitinib treatment. Whole transcriptomic sequencing was performed for inflamed rectal mucosae obtained before and after the initiation of Janus kinase (JAK) inhibitor, suggesting that distinct molecular signatures seemed to be regulated by JAK inhibitors and other conventional therapies including tumor necrosis factor lockers. CONCLUSION: Tofacitinib should be considered in refractory cases of UC with ITP.

Myelodysplastic/myeloproliferative neoplasm (MDS/MPN) with ring sideroblasts and thrombocytosis (MDS/MPN with RS-T), which exhibits both an increased number of marrow ring sideroblasts and thrombocytosis, is a rare disorder classified as one of the newly established forms of MDS/MPN in the WHO 2016 classification. A 77-year-old female with marked thrombocytosis of 1,024×109/L was tentatively diagnosed with essential thrombocythemia in 2011, and the thrombocytosis was controlled using hydroxycarbamide and low-dose busulfan. In 2016, the leukocyte count increased to a peak value of 68.8×109/L (86.6% mature neutrophils) during platelet-reduction therapy. Bone marrow aspirate exhibited hypercellularity with ring sideroblasts comprising 41.5% erythroblasts without excess myeloblasts. Cytogenetic examination demonstrated the JAK2 V617F mutation and chromosomal abnormality of 46,XX,del(20)(q1?). Furthermore, dysplastic features of erythroid and granuloid precursors, as well as many large atypical megakaryocytes, were observed. Further genetic examinations revealed the SF3B1 K700E mutation, but not amplification of the JAK2 gene or pathogenic mutations in the 13 other genes examined. A diagnosis of MDS/MPN with RS-T was established and hyperleukocytosis was controlled using a higher dose of hydroxycarbamide. Although the patient maintained a stable disease state, she became RBC transfusion-dependent. Hyperleukocytosis, regardless of chemotherapy, is rare and may be novel in this disorder.

PURPOSE: Although gene expression profiling is a promising diagnostic technique to determine the tissue of origin for patients with cancer of unknown primary site (CUP), no clinical trial has evaluated yet site-specific therapy directed by this approach compared with empirical chemotherapy. We therefore performed a randomized study to assess whether such site-specific therapy improves outcome compared with empirical chemotherapy in previously untreated patients with CUP. PATIENTS AND METHODS: Comprehensive gene expression profiling was performed by microarray analysis, and an established algorithm was applied to predict tumor origin. Patients with CUP were randomly assigned (1:1) to receive standard site-specific therapy or empirical paclitaxel and carboplatin (PC). The primary end point was 1-year survival rate. RESULTS: One hundred thirty patients were randomly assigned and had sufficient biopsy tissue for molecular analysis. Efficacy analysis was performed for 50 and 51 patients in the site-specific therapy and empirical PC arms, respectively. Cancer types most commonly predicted were pancreatic (21%), gastric (21%), and lymphoma (20%). The 1-year survival rate was 44.0% and 54.

非小細胞肺癌の細胞株や患者由来検体を実験材料とし、上皮成長因子受容体(EGFR)チロシンキナーゼ阻害薬(TKI)がMHCクラスIの発現に及ぼす影響について調査した。EGFR遺伝子にT790M二次的突然変異などの変異がある細胞株を適切なEGFR-TKIで処理するとMHCクラスIのmRNA/蛋白質発現は亢進した。細胞外シグナル調節キナーゼ(ERK)キナーゼであるMEKの阻害薬で処理した場合もMHCクラスI発現が亢進したことから、EGFR活性化に応答してみられるMHCクラスI発現の下方制御はMEK-ERK経路が媒介していることが示唆された。EGFR-TKI治療を施行したEGFR変異非小細胞肺癌患者から得た検体の免疫組織化学解析からも、疾患進行後においては、リン酸化EGFRやリン酸化ERKの下方制御が、MHC-Iの上方制御、CD8+浸潤T細胞の増加、およびPD-1リガンド1発現亢進と関連していることが明らかになった。これらの結果から、非小細胞癌においてEGFRが変異活性化するとMEK-ERK経路を通じてMHCクラスI発現が阻害され、免疫療法に不応となるのに寄与することが示唆された。

Carnitine deficiency is reportedly associated with increased pain sensation in diabetes mellitus and fibromyalgia, but the association between serum carnitine concentration and cancer pain has not been fully elucidated. We investigated the incidence of carnitine deficiency in patients with cancer pain, and examined the effect of the patients' demographic and clinical characteristics on pain intensity and carnitine deficiency. The serum carnitine concentration was measured in 50 patients with cancer pain receiving non-steroidal anti-inflammatory drugs, but not opioids. Multivariate regression analysis was used to determine the association of carnitine concentration, pain intensity, age and gender with hemoglobin and C-reactive protein (CRP) concentrations. Carnitine deficiency was detected in 9 of the patients (18.0%) and found to be significantly correlated with an elevated CRP concentration (P=0.039). In conclusion, although there does not appear to be an association between carnitine deficiency and cancer pain, it may be affected by inflammation or infection.

肝癌根治切除後再発肝細胞癌(HCC)に対してソラフェニブを投与した45例から得られた初回肝切除時の腫瘍部および非腫瘍部のホルマリン固定パラフィン包埋組織を用いた。効果判定は、奏効(CR)が6例、部分奏効(PR)が4例、安定(SD)が13例、進行(PD)が22例であった。FGFRの全てのエクソンをスクリーニングし、FGFR2変異2例(4.4%)、FGFR3変異2例(4.4%)、FGFR1変異およびFGFR4変異が各1例(2.2%)であった。FGFRの変異状態とソラフェニブの奏効との関連性を分析し、変異状態と臨床的変数との間には有意な関連はなかった。腫瘍サンプルのコピー数変化を解析した。FGF19のコピー数の増加は、CR2例(33.3%)で検出した。FGF3、FGF4、FGFR1、FGFR2、FGFR3、FGFR4については、コピー数の増加は検出しなかった。ソラフェニブに奏効しなかった35例の腫瘍検体を解析し、FGF19のコピー数の増加は2例(5.7%)で検出した。FGF19の遺伝子の増幅は、HCC患者におけるソラフェニブの有効性を予測できるバイオマーカーであると考えられた。

Castration-resistant prostate cancer is an incurable heterogeneous disease that is characterized by a complex multistep process involving different cellular and biochemical changes brought on by genetic and epigenetic alterations. These changes lead to the activation or overexpression of key survival pathways that also serve as potential therapeutic targets. Despite promising preclinical results, molecular targeted therapies aimed at such signaling pathways have so far been dismal. In the present study, we used a PTEN-deficient mouse model of prostate cancer to show that plasticity in castration-resistant tumors promotes therapeutic escape. Unlike castration-naïve tumors which depend on androgen receptor and PI3K/AKT signal activation for growth and survival, castration-resistant tumors undergo phenotypic plasticity leading to increased intratumoral heterogeneity. These tumors attain highly heterogeneous phenotypes that are characterized by cancer cells relying on alternate signal transduction pathways for growth and survival, such as mitogen-activated protein kinase and janus kinase/signal transducer and activator of transcription, and losing their dependence on PI3K signaling. These features thus enabled castration-resistant tumors to become insensitive to the therapeutic effects of PI3K/AKT targeted therapy. Overall, our findings provide evidence that androgen deprivation drives phenotypic plasticity in prostate cancer cells and implicate it as a crucial contributor to therapeutic resistance in castration-resistant prostate cancer. Therefore, incorporating intratumoral heterogeneity in a dynamic tumor model as a part of preclinical efficacy determination could improve prediction for response and provide better rationale for the development of more effective therapies.

The heparan sulfate sulfotransferase gene family catalyzes the transfer of sulfate groups to heparan sulfate and regulates various growth factor-receptor signaling pathways. However, the involvement of this gene family in cancer biology has not been elucidated. It was demonstrated that the heparan sulfate D-glucosaminyl 6-O-sulfotransferase-2 (HS6ST2) gene is overexpressed in colorectal cancer (CRC) and its clinical significance in patients with CRC was investigated. The mRNA levels of HS6ST2 in clinical CRC samples and various cancer cell lines were assessed using a microarray analysis and quantitative RT-PCR, respectively. An immunohistochemical (IHC) analysis of the HS6ST2 protein was performed using 102 surgical specimens of CRC. The correlations between the HS6ST2 expression status and clinicopathological characteristics were then evaluated. HS6ST2 mRNA was significantly overexpressed by 37-fold in CRC samples compared to paired colonic mucosa. High levels of HS6ST2 mRNA expression were also observed in colorectal, esophageal and lung cancer cell lines. The IHC analysis demonstrated that HS6ST2 was expressed in the cytoplasmic region of CRC cells, but not in normal colonic mucosal cells. Positive staining for HS6ST2 was detected in 40 patients (39.2%). There was no significant association between the clinicopathological characteristics and HS6ST2 expression. However, positive staining for HS6ST2 was associated with a poor survival (P=0.074, log-rank test). In conclusion, HS6ST2 was found to be overexpressed in CRC and its expression tended to be a poor prognostic factor, although the correlation was not significant. These findings indicate that HS6ST2 may be a novel cancer-related marker that may provide insight into the glycobiology of CRC.

OTS11101 is a novel peptide vaccine that acts as an angiogenesis inhibitor by inducing cytotoxic T lymphocyte (CTL) cells that specifically target vascular endothelial cells expressing vascular endothelial growth factor (VEGF) receptor 1. We conducted a phase I study to evaluate the safety, tolerability, maximum tolerated dose, and pharmacodynamic biomarker status of this vaccine. Nine patients with advanced solid tumors received 1.0, 2.0, or 3.0 mg of OTS11101 subcutaneously, once a week in a 28-day cycle. Three patients experienced grade 1 injection site reactions, which were the most frequent adverse events. Grade 2 proteinuria and hypertension each occurred in one patient. As other toxicities were generally mild, the maximum tolerated dose was not reached. Furthermore, we explored the induction of specific activated CTLs, and biomarkers related to angiogenesis. A pharmacodynamics study revealed that induction of specific CTLs was observed for a dose of 2.0 and 3.0 mg. The serum concentrations of soluble VEGF receptor 1 and 2 after vaccination increased significantly compared with baseline. A microarray was performed to give a comprehensive analysis of gene expression, suggesting that OTS11101 vaccination resulted in T cell activation in a clinical setting. In conclusion, OTS11101 was well tolerated in patients up to 3.0 mg once weekly and our biomarker analysis suggested that this anti-angiogenesis vaccine is biologically active.

Precise and objective measurements of tumor response have yet to be standardized in the mouse orthotopic bladder cancer model. In this study, we used image analysis and green fluorescent protein (GFP) to objectively measure tumor size in response to chemotherapy. KU-7 human bladder cancer cells transfected with GFP were intravesically inoculated into 8-week-old female nude mice. Fourteen days after tumor cell inoculation, the mice were assigned into a control (PBS) group or a doxorubicin (conc. 1.0 mg/ml) treatment group and received a single instillation of treatment. Fourteen days after treatment, the bladders were surgically exposed and fluorescent images were captured and later analyzed using image analysis. Bladders were processed for histological examination. Tumor incidence determined by GFP expression and histology was 100 and 80%, respectively, in the doxorubicin treatment group. A 9-fold (histology) vs. 12-fold (GFP expression) difference in tumor regression measured by tumor area (P<0.05) and a 5-fold (histology) vs. 9-fold (GFP expression) difference in tumor regression measured by the percent of tumor area in the bladder (P<0.001) were observed in the doxorubicin treatment group. Our findings suggest that using image analysis provides a precise, sensitive and objective means to measure tumor growth and treatment response in the mouse orthotopic bladder cancer model in lieu of histological methods. Consequently, the number of mice required in an experiment can be reduced since tissue samples are not needed for histology, thus making tissue samples readily available for additional assays in both a labor-effective and cost-effective manner.

L858R point mutation in exon 21 of the EGFR gene, accounting for approximately 40% of non-small cell lung cancer (NSCLC)-associated EGFR mutations, has been known to hyper-respond to gefitinib, a selective EGFR tyrosine kinase inhibitor. From this view point, it is important to detect EGFR mutations. Immunohistochemistry (IHC) is commonly used to analyze the molecular status of several clinical specimens. We have developed specific antibodies recognizing the mutant EGFR (L858R) protein and characterized the antibodies by ELISA, Western blot, immunocytochemistry, and immunohistochemistry. Using any of these evaluation methods, we found an antibody, AbyD02889, which could detect the EGFR (L858R) protein with specificity. AbyD02889 may be a useful tool to detect the EGFR (L858R) mutation of NSCLC in the clinical situation. IHC using the mutant-specific anti-EGFR antibody will be a powerful assay to predict or select subpopulation sensitive to EGFR-TKI.

Recent progress in the field of molecular biology has been expected to contribute to progress in the field of clinical medicine. Personalized medicine could be achieved by pharmacogenomics. Prospective clinical studies using biomarkers are considered to be important. Investigators should plan the study design and carefully perform such studies.

Cisplatin (CDDP) is an effective anticancer agent that is widely used in the treatment of testicular, ovarian, bladder and lung cancers. However the development of resistance to CDDP by tumor cells is a major obstacle to treatment. We reported that decreased accumulation of CDDP was observed in CDDP-resistant cell lines from NSCLC, and a good correlation was found between the amount of intracellular platinum and the sensitivity of lung cancer cell lines to CDDP. In the present study, to investigate the CDDP resistance mechanism, several platinum compounds were exposed to those cell lines, and we measured the cellular platinum using inductively coupled plasma mass spectrometry (ICP-MS). Furthermore, the proportion of intact CDDP in total platinum was also determined by LC-ICP-MS for pharmacokinetic study of CDDP.