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.

PURPOSE: We aimed to compare the efficacy and safety of docetaxel plus ramucirumab and atezolizumab as second-line treatment for programmed death-ligand 1 (PD-L1)-negative or low advanced non-small-cell-lung cancer (NSCLC) after platinum-based chemotherapy. PATIENTS AND METHODS: This multicenter randomized phase III study enrolled patients with advanced NSCLC who had progressed during or after first-line platinum-based chemotherapy. Patients were allocated randomly (1:1) to receive atezolizumab (arm A) or docetaxel plus ramucirumab (arm B) every 3 weeks. The primary endpoint was overall survival (OS). RESULTS: This study was activated in April 2018 and closed in March 2020 due to slow accrual. Seventy eligible patients were enrolled from 26 institutions, including 36 patients in arm A and 34 in arm B. The median OS (median follow-up, 24.2 months) were 17.1 and 15.8 months (HR = 1.508, 95 % confidence interval (CI), 0.86-2.65; P = 0.23), respectively. The 2-year OS rates were 42.8 % (95 % CI, 26.2 %-58.4 %) and 19.4 % (95 %CI, 7.5-35.3), the objective response rates (ORRs) were 5.6 % and 35.3 % (P = 0.002), and the median progression-free survival (PFS) were 1.5 and 5.5 months (P = 0.005), respectively. The crossover rates were 55.6 % and 64.7 %, and the median times from randomization to progression or death post-crossover were 12.9 and 9.1 months. Grade ≥ 3 toxicities included neutropenia (2.8 %/17.6 %), thrombocytopenia (2.8 %/8.8 %), anorexia (2.8 %/5.9 %), febrile neutropenia (0 %/5.9 %), and hypertension (2.8 %/8.8 %). CONCLUSIONS: OS was similar in both arms, but docetaxel plus ramucirumab resulted in favorable ORR and PFS. The 2-year OS rates suggested that atezolizumab might enhance the efficacy of post-study cytotoxic chemotherapy; however, interpretation of the data was limited by the small sample size.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are standard therapies for EGFR-mutated non-small-cell lung cancer (NSCLC); however, their efficacy is inconsistent. Secondary mutations in the EGFR or other genes that lead to resistance have been identified, but resistance mechanisms have not been fully identified. Chromosomal instability (CIN) is a hallmark of cancer and results in genetic diversity. In this study, we demonstrated by transcriptomic analysis that CIN activates the cGAS-STING signaling pathway, which leads to EGFR-TKI refractoriness in a subset of EGFR-mutated NSCLC patients. Furthermore, EGFR-mutated H1975dnMCAK cells, which frequently underwent chromosomal mis-segregation, demonstrated refractoriness to the EGFR-TKI osimertinib compared to control cells. Second, H1975dnMCAK cells exhibited activation of cGAS-STING signaling and its downstream signaling, including tumor-promoting cytokine IL-6. Finally, chromosomally unstable EGFR-mutated NSCLC exhibited enhanced epithelial-mesenchymal transition (EMT). Blockade of cGAS-STING-TBK1 signaling reversed EMT, resulting in restored susceptibility to EGFR-TKIs in vitro and in vivo. These results suggest that CIN may lead to the activation of cGAS-STING signaling in some EGFR-mutated NSCLC, resulting in EMT-associated EGFR-TKI resistance.

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.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with limited treatment options and poor prognosis. Recent advances in cancer genomic analysis enable the identification of actionable gene alterations, opening new opportunities for personalized therapy. Among these, homologous recombination DNA repair (HRR) gene alterations are associated with distinct biological behavior, favorable prognosis, and increased sensitivity to platinum-based chemotherapy. However, the prognostic impact of coexisting mutations in key driver genes-KRAS, TP53, CDKN2A, and SMAD4-within HRR-altered PDAC remains poorly understood. METHODS: We retrospectively analyzed PDAC patients who underwent genomic profiling testing with FoundationOne® CDx between June 2019 and December 2021 through the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database. We compared the prevalence and prognostic significance of key gene alterations between HRR-altered and HRR-wild-type (WT) tumors. RESULTS: Of 2,381 PDAC patients, 274 (11.5%) harbored HRR alterations. These patients showed significantly longer overall survival (OS) than those with HRR-WT tumors (HR = 0.66, p = 0.002). The frequencies of KRAS, TP53, and CDKN2A mutations were less frequent in HRR-altered tumors. TP53 mutation was independently associated with poorer OS across both HRR subgroups, while CDKN2A alteration was a poor prognostic factor in HRR-WT tumors. Interestingly, SMAD4 alteration was linked to improved survival in the HRR-altered group. CONCLUSION: HRR-altered PDAC has a distinct genomic profile and is associated with a favorable prognosis. Our findings demonstrate that coexisting alterations are significant prognostic factors in both HRR-altered and HRR-wild-type tumors. These results highlight the clinical relevance of incorporating comprehensive genomic profiling into routine care to stratify patient prognosis better and inform individualized treatment strategies in PDAC.

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.

BACKGROUND: Amplification of the programmed cell death-ligand 1 gene (CD274) is highly prevalent and associated with a high response rate to immune checkpoint inhibitors (ICIs) in lymphomas, and is also a potential biomarker for ICI treatment of solid tumors. However, the efficacy of ICIs for solid tumors with CD274 amplification identified by comprehensive genomic profiling (CGP) has been unclear. We here examined ICI efficacy for solid tumors with CD274 amplification identified by CGP in a national database. METHODS: We retrospectively analyzed data from the Center for Cancer Genomics and Advanced Therapeutics database containing 60,155 CGP test results for individuals with solid tumors. Only clinical data from patients treated with ICIs alone (not those undergoing concomitant therapy with molecularly targeted or cytotoxic chemotherapeutic agents) were evaluated. We matched 48 patients in the CD274 amplification-positive group with 170 patients in the CD274 amplification-negative group in a 1:4 ratio based on tumor type, histology, treatment, and age. Overall survival (OS), time to next treatment (TTNT), and response rate were evaluated as treatment outcomes in the two groups. RESULTS: OS was similar in the CD274-amplified and matched CD274-non-amplified groups (median of 22.1 vs 26.3 months, respectively; HR of 0.92 with a 95% CI of 0.55 to 1.54; p=0.075). TTNT tended to be longer in the CD274-amplified group than in the matched CD274-non-amplified group (median of 16.5 vs 14.0 months; HR of 0.63 with a 95% CI of 0.37 to 1.08; p=0.091). The objective response rate was 33.3% and 18.4% (difference of 14.9%, with a 95% CI of -0.2% to 31.6%), and the disease control rate was 63.9% and 41.1% (difference of 22.8%, with a 95% CI of 5.1% to 40.4%), in the CD274-amplified and matched CD274-non-amplified groups, respectively. CONCLUSIONS: The number of patients with solid tumors positive for CD274 amplification in this analysis is the largest to date, and our results suggest that such gene amplification may be associated with the outcome of ICI treatment in such individuals. CD274 amplification identified by CGP may therefore be a predictor of ICI efficacy for solid tumors. TRIAL REGISTRATION NUMBER: UMIN000029779.

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.

High-grade fetal lung adenocarcinoma (H-FLAC) is a rare tumor, with little known of its response to chemotherapy with or without an immune checkpoint inhibitor or of its molecular profile. We report the first case of a 56-year-old man with stage IV H-FLAC who was successfully treated with carboplatin plus nab-paclitaxel in combination with atezolizumab. In addition, the tumor was found to be positive for amplification of the human epidermal growth factor receptor 2 gene.

BACKGROUND: Neoadjuvant docetaxel plus cisplatin and 5-FU (NAC-DCF) and adjuvant nivolumab monotherapy are the standard care for locally advanced resectable esophageal squamous cell carcinoma (ESCC). However, no effective biomarkers have been found in perioperative setting. We investigated how programmed death-ligand 1 (PD-L1) changes before and after NAC-DCF and how it relates to the therapeutic effect of NAC-DCF in resectable ESCC. METHODS: PD-L1 expression in paired diagnostic biopsy and surgically resected tissues from ESCC patients who underwent surgical resection after receiving two or three NAC-DCF cycles was evaluated. PD-L1 positivity was defined as a combined positive score (CPS) of 10% ≤ . Gene expression analysis was conducted using samples before NAC-DCF. RESULTS: Sixty-six paired samples from 33 patients were included in PD-L1 expression analysis, and 33 Pre-NAC samples acquired by diagnostic biopsy were included in gene expression analysis. Pretreatment, 3 (9%), 13 (39%), and 17 (52%) patients harbored tumors with CPS ranges of < 1%, 1%-10%, and 10% ≤ , respectively. After NAC-DCF, 5 (15%), 15 (45%), and 13 (39%) tumors presented CPS ranges of < 1%, 1%-10%, and 10% ≤ , respectively. The concordance rate between Pre-and Post-NAC-DCF samples was 45%. Patients with PD-L1-negative tumors both before and after NAC-DCF (n = 9) had shorter survival and different gene expression profile characterized by upregulation in WNT signaling or neutrophils. CONCLUSIONS: A substantial PD-L1 expression alteration was observed, resulting in low concordance rate before and after NAC-DCF. Tumors persistently lacking PD-L1 had distinct gene expression profile with worse clinical outcomes, raising the need for further investigation.

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.

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.

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.

Epidermal growth factor receptor (EGFR) is one of the anticancer drug targets for certain malignancies, including nonsmall cell lung cancer (NSCLC), colorectal cancer (CRC), and head and neck squamous cell carcinoma. However, the grave issue of drug resistance through diverse mechanisms persists, including secondary EGFR-mutation and its downstream RAS/RAF mutation. Since the discovery of the role of human epidermal growth factor receptor 2 (HER2) and HER3 in drug resistance, HER2- or HER3-targeting treatment strategies using monoclonal antibodies have been intensively examined and have demonstrated impressive responsiveness and limitations. Finally, an innovative targeted therapy called antibody drug conjugates (ADC) has provided a solution to overcome this resistance. Specifically, a new cleavable linker-payload system enables stable drug delivery to cancer cells, causing selective destruction. HER2-targeting ADC trastuzumab deruxtecan demonstrated promising responsiveness in patients with HER2-positive CRC, in a phase 2 clinical trial (objective response rate = 45.3%). Furthermore, HER3-targeting patritumab deruxtecan, another ADC, exhibited impressive tumor shrinkage in pretreated patients with EGFR-mutated NSCLC, in a phase 1 clinical trial. This manuscript presents an overview of the accumulated evidence on HER2- and HER3-targeting therapy, especially ADCs, and discussion of remaining issues for further improving these treatments in cancers resistant to EGFR inhibitors.

BACKGROUND: Predictive markers for the clinical outcomes of second-line treatment in patients with metastatic colorectal cancer (mCRC) remain unclear. OBJECTIVE: This retrospective biomarker study was conducted to explore predictive markers for patients with KRAS exon 2 wild-type mCRC who were treated with FOLFIRI plus panitumumab (Pani) or bevacizumab (Bev) in the WJOG6210G trial. PATIENTS AND METHODS: The associations of early tumor shrinkage (ETS), tumor location, and VEGF-D with progression-free survival (PFS) and overall survival (OS) were analyzed using a Cox proportional hazards model. Spearman's correlation coefficient was used to analyze the association of depth of response (DpR) with PFS and OS. Serum VEGF-D levels were measured in samples collected before treatment using magnetic bead panel Milliplex xMAP kits. RESULTS: In total, 101 patients (Pani, n = 49; Bev, n = 52) were enrolled in this study. Patients with ETS had longer PFS (Pani: hazard ratio (HR) 0.40, P = 0.009; Bev: HR 0.078, P = 0.0002) and OS (Pani: HR 0.49, P = 0.044; Bev: HR 0.35, P = 0.048) than patients without ETS. The DpR was moderately correlated with PFS and OS in Pani (rs = 0.75, P < 0.001; rs = 0.60, P < 0.001) and Bev groups (rs = 0.68, P < 0.001; rs = 0.44, P = 0.002). No significant differences were observed in PFS and OS between the two treatment groups even if in left-sided tumors. No significant interaction between VEGF-D levels and treatment was observed in PFS and OS. CONCLUSIONS: ETS and DpR serve as surrogate markers of PFS and OS in the second-line treatment with FOLFIRI plus targeted agent for mCRC.

Immunotherapy targeting programmed cell death-1 (PD-1) induces durable antitumor efficacy in many types of cancer. However, such clinical benefit is limited because of the insufficient reinvigoration of antitumor immunity with the drug alone; therefore, rational therapeutic combinations are required to improve its efficacy. In our preclinical study, we evaluated the antitumor effect of U3-1402, a human epidermal growth factor receptor 3-targeting (HER3-targeting) antibody-drug conjugate, and its potential synergism with PD-1 inhibition. Using a syngeneic mouse tumor model that is refractory to anti-PD-1 therapy, we found that treatment with U3-1402 exhibited an obvious antitumor effect via direct lysis of tumor cells. Disruption of tumor cells by U3-1402 enhanced the infiltration of innate and adaptive immune cells. Chemotherapy with exatecan derivative (Dxd, the drug payload of U3-1402) revealed that the enhanced antitumor immunity produced by U3-1402 was associated with the induction of alarmins, including high-mobility group box-1 (HMGB-1), via tumor-specific cytotoxicity. Notably, U3-1402 significantly sensitized the tumor to PD-1 blockade, as a combination of U3-1402 and the PD-1 inhibitor significantly enhanced antitumor immunity. Further, clinical analyses indicated that tumor-specific HER3 expression was frequently observed in patients with PD-1 inhibitor-resistant solid tumors. Overall, U3-1402 is a promising candidate as a partner of immunotherapy for such patients.

OBJECTIVES: The T790M secondary mutation of epidermal growth factor receptor gene (EGFR) is the most common mechanism of acquired resistance to first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). We investigated the association between gene mutation profile in EGFR mutation-positive non-small cell lung cancer (NSCLC) before EGFR-TKI treatment and T790M status after EGFR-TKI treatment. MATERIALS AND METHODS: A total of 57 EGFR mutation-positive NSCLC patients who had undergone a repeat biopsy (tissue or liquid) after failure of treatment with a first- or second-generation EGFR-TKI and who had sufficient tumor tissue available from before treatment for genetic analysis was enrolled. The gene mutation profile of tumor tissue obtained before EGFR-TKI treatment was evaluated by next-generation sequencing with a comprehensive cancer gene panel (409 genes). The number of potentially damaging nonsynonymous mutations was predicted with PolyPhen-2 software. RESULTS: Progression-free survival during EGFR-TKI treatment did not differ significantly between patients who developed T790M-mediated resistance and those who developed T790M-independent resistance. The predicted number of damaging nonsynonymous mutations in pretreatment tumor tissue was significantly lower in patients who developed T790M-mediated resistance than in those with T790M-independent resistance (P =  0.049). CONCLUSIONS: Coexisting mutations in tumor tissue before EGFR-TKI treatment may contribute to the emergence of cell clones responsible for development of T790M-dependent or T790M-independent TKI resistance in patients with EGFR-mutated NSCLC. Multiplex genomic testing of pretreatment tumor tissue may thus provide a means of identifying patients likely to develop T790M-mediated TKI resistance and therefore inform treatment selection.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are standard therapy for EGFR-mutant non-small cell lung cancer (NSCLC). Preclinically, HER3 ligand heregulin induces resistance to EGFR-TKIs, whereas the pan-human EGFR family inhibitor afatinib remains effective. Here, we examined whether soluble heregulin levels have clinical implications for EGFR-mutant NSCLC treated with EGFR-TKIs. Soluble heregulin was immunologically measured in plasma from EGFR-mutant NSCLC patients. Cutoff values were determined by 1-year PFS ROC curve. The relationship between soluble heregulin and PFS following EGFR-TKI therapy was analyzed by Cox proportional hazards model. Seventy-three patients were enrolled: 44 were treated with 1st-generation and 29 with 2nd-generation EGFR-TKIs. Soluble heregulin levels varied (range: 274-7,138 pg/mL, median: 739 pg/mL). Among patients treated with 1st-generation EGFR-TKIs, those with high heregulin (n = 20, >800 pg/mL) had a tendency for shorter PFS than those with low heregulin (n = 24, <800 pg/mL), with median PFS of 322 and 671 days, respectively. Cox proportional hazards model also indicated a trend toward resistance against 1st-generation EGFR-TKIs (HR: 1.825, 95% CI: 0.865-4.318) but not against 2nd-generation EGFR-TKIs. Soluble heregulin potentially correlates with resistance to EGFR-TKIs but not 2nd-generation EGFR-TKIs in patients with EGFR-mutant NSCLC.

Therapies targeted to human epidermal growth factor receptor 2 (HER2) have proven effective against tumors positive for HER2 amplification, but there is an unmet clinical need for the treatment of tumors that express HER2 protein in the absence of HER2 amplification. [fam-] trastuzumab deruxtecan (DS-8201a) is a novel antibody-drug conjugate composed of the anti-HER2 antibody and the topoisomerase I inhibitor, an exatecan derivative. It has shown efficacy against tumors that express HER2 and is currently under evaluation in clinical trials. We here show that the antitumor activity of [fam-] trastuzumab deruxtecan is dependent on the expression level of HER2 protein in colorectal cancer (CRC) cell lines negative for HER2 amplification. We established isogenic CRC cell lines that express various levels of HER2 protein in the absence of HER2 amplification, and we found that cells that express HER2 at a high level were sensitive to [fam-] trastuzumab deruxtecan but not to conventional HER2-targeted therapies. Furthermore, [fam-] trastuzumab deruxtecan manifested a bystander killing effect both in vitro and in vivo, with cells essentially negative for HER2 expression also being killed in the presence of HER2-expressing cells, an effect that has the potential to overcome heterogeneity of HER2 expression in CRC tumors. Our results thus suggest that [fam-] trastuzumab deruxtecan warrants further study as a potential treatment for CRC tumors that express HER2 protein in the absence of HER2 amplification.

BACKGROUND: Non-small cell lung cancer (NSCLC) positive for activating mutations of the epidermal growth factor receptor (EGFR) gene is initially sensitive to EGFR-tyrosine kinase inhibitors (TKIs) but eventually develops resistance to these drugs. Upregulation of the receptor tyrosine kinase AXL in tumor tissue has been detected in about one-fifth of NSCLC patients with acquired resistance to EGFR-TKIs. However, the clinical relevance of the levels of AXL and its ligand GAS6 in plasma remains unknown. METHODS: Tumor tissue and plasma specimens were collected from 25 EGFR-mutated NSCLC patients before EGFR-TKI treatment or after treatment failure. The levels of AXL and of GAS6 mRNA in tumor tissue were evaluated by immunohistochemistry and chromogenic in situ hybridization, respectively. The plasma concentrations of AXL and GAS6 were measured with enzyme-linked immunosorbent assays. RESULTS: AXL expression was detected in three of 12 (25%) and nine of 19 (47%) tumor specimens obtained before and after EGFR-TKI treatment, respectively. All tumor specimens assayed were positive for GAS6 mRNA. The median values for the plasma AXL concentration before and after EGFR TKI treatment were 1 635 and 1 460 pg/mL, respectively, and those for the plasma GAS6 concentration were 4 615 and 6 390 pg./mL, respectively. There was no significant correlation between the plasma levels of AXL or GAS6 and the corresponding expression levels in tumor tissue. CONCLUSION: Plasma concentrations of AXL and GAS6 do not reflect tumor expression levels, and their measurement is thus not a viable alternative to direct analysis of tumor tissue in EGFR-mutated NSCLC.

The anti-epidermal growth factor receptor (EGFR) antibody cetuximab is standard therapy for head and neck squamous cell carcinoma (HNSCC). However, most HNSCC tumors are resistant to it and require alternative treatments. Here, we explored the mechanism of cetuximab resistance and evaluated its clinical relevance in HNSCC. An unbiased comprehensive transcriptome analysis was performed on cetuximab-resistant HNSCC FaDuCR cells. The causative resistance genome was knocked down with siRNA, cell signaling was immunologically analyzed, and drug efficacy was evaluated in vitro and in vivo. The mRNA in situ hybridization (ISH) of the causative genome was performed using 28 excised HNSCC tumors and its relationship with cetuximab efficacy was analyzed. FaDuCR cells were resistant to cetuximab, whereas parental FaDu cells were susceptible to it. FaDuCR cells expressed consistently higher levels of phosphorylated Akt than FaDu cells despite cetuximab exposure. A comprehensive transcriptome analysis revealed that the HER3-ligand heregulin was upregulated in FaDuCR cells compared to FaDu cells. Heregulin knockdown in FaDuCR cells repressed HER3 and Akt phosphorylation and recovered cetuximab anticancer efficacy. In contrast, pan-HER family tyrosine kinase inhibitors such as afatinib decreased HER3 and Akt phosphorylation in FaDuCR cells and inhibited FaDuCR tumor growth. Two of the 28 HNSCC tumor samples presented aberrant heregulin expression comparable to that of FaDuCR cells and were resistant to cetuximab therapy. In HNSCC, heregulin-mediated HER3-Akt activation causes resistance to cetuximab but not to second-generation EGFR-tyrosine kinase inhibitors. Subpopulations with aberrant heregulin-expressing HNSCC might be resistant to cetuximab.

Medical oncologists are challenged to personalize medicine with scientific evidence, drug approvals, and treatment guidelines based on sequencing of clinical samples using next generation sequencer (NGS). Knowledge-based curation systems have the potential to help address this challenge. We report here the results of examining the level of evidence regarding treatment approval and clinical trials between recommendations made by Watson for Genomics (WfG), QIAGEN Clinical Insight Interpret (QCII), and Oncomine knowledge-based reporter (OKR). The tumor samples obtained from the solid cancer patients between May to June 2018 at Kindai University Hospital. The formalin-fixed paraffin-embedded tumor samples (n = 31) were sequenced using Oncomine Comprehensive Assay v3. Variants including copy number alteration and gene fusions identified by the Ion reporter software were used commonly on three curation systems. Curation process of data were provided for 25 solid cancers using three curation systems independently. Concordance and distribution of curated evidence levels of variants were analyzed. As a result of sequencing analysis, nonsynonymous mutation (n = 58), gene fusion (n = 2) or copy number variants (n = 12) were detected in 25 cases, and subsequently subjected to knowledge-based curation systems (WfG, OKR, and QCII). The number of curated information in any systems was 51/72 variants. Concordance of evidence levels was 65.3% between WfG and OKR, 56.9% between WfG and QCII, and 66.7% between OKR and QCII. WfG provided great number of clinical trials for the variants. The annotation of resistance information was also observed. Larger differences were observed in clinical trial matching which could be due to differences in the filtering process among three curation systems. This study demonstrates knowledge-based curation systems (WfG, OKR, and QCII) could be helpful tool for solid cancer treatment decision making. Difference in non-concordant evidence levels was observed between three curation systems, especially in the information of clinical trials. This point will be improved by standardized filtering procedure and enriched database of clinical trials in Japan.

HER2-targeted therapy, especially the anti-HER2 antibody trastuzumab, is standard for HER2-positive breast cancer; however, its efficacy is limited in a subpopulation of patients. HER3 ligand (heregulin)-dependent HER2-HER3 interactions play a critical role in the evasion of apoptosis and are therefore a target for oncotherapy to treat HER2-positive breast cancer. The anti-HER2 antibody pertuzumab and anti-HER3 antibody patritumab both target this heregulin-HER3-HER2 complex in different ways. This study examined the anticancer efficacy of dual HER2 and HER3 blockade in trastuzumab-resistant HER2-positive breast cancer. HER2-positive SKBR3 or BT474 cells overexpressing heregulin (SKBR3-HRG, BT474-HRG) were used to evaluate the efficacy of trastuzumab, pertuzumab, and patritumab in vitro by performing cell viability, immunoblotting, and clonogenic assays. The effects of these agents were then evaluated in vivo using BT474-HRG and an intrinsic heregulin-expressing and HER2-positive JIMT-1 xenograft models. SKBR3-HRG and BT474-HRG cells lost sensitivity to trastuzumab, which was accompanied by Akt activation. Unexpectedly, trastuzumab in combination with pertuzumab or patritumab also showed limited efficacy toward these cells. In contrast, trastuzumab/pertuzumab/patritumab triple treatment demonstrated potent anticancer efficacy, concomitant with strong repression of Akt. Finally, in heregulin-expressing BT474-HRG and JIMT-1 xenograft models, the addition of pertuzumab and patritumab to trastuzumab also enhanced antitumor efficacy leading to tumor regression. The current study found that triple blockade of HER2 and HER3 using trastuzumab, pertuzumab, and patritumab could overcome resistance to trastuzumab therapy in heregulin-expressing and HER2-positive breast cancer, which could be exploited clinically.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‑TKIs) are efficacious drugs for non‑small cell lung cancers (NSCLCs) with EGFR‑activating mutations. Afatinib, a second‑generation EGFR‑TKI and osimertinib, a third‑generation EGFR‑TKI, are both standard therapies for patients with these types of cancer. Each drug possesses distinct binding sites for the tyrosine kinase domain of EGFR. The present study examined the efficacy of single and combination TKI therapy using in vitro growth inhibition assays of Ba/F3 cells with an EGFR‑activating Del19 mutation. Afatinib or osimertinib treatment alone markedly inhibited cell proliferation in Ba/F3 cells, although drug‑resistant cells eventually appeared with secondary EGFR mutations (either T790M or C797S, respectively) as determined by direct sequencing. Notably a combination of afatinib and osimertinib eradicated Ba/F3 cells with no development of resistance. We also evaluated the efficacy of afatinib, osimertinib, and a combination of the two, using drug‑resistant cells with T790M or C797S mutations. Osimertinib was effective for treating Ba/F3 cells with the T790M mutation, whereas afatinib was moderately effective against C797S Ba/F3 cells. However, subsequent treatment, even when both drugs were used in combination, could not completely eradicate the Ba/F3 population and doubly resistant cells with a variety of triple mutations were generated, including Del19/T790M/C797S. In conclusion, an initial treatment with a combination of osimertinib and afatinib is potentially more effective for eradicating mutant EGFR‑dependent cells than sequential drug use. This should be tested in future clinical trials to establish whether such a combination would be effective for the treatment of NSCLC.

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。

EGFR tyrosine kinase inhibitors (TKIs) are standard therapy for EGFR-mutant non-small cell lung cancer (NSCLC); however, these tumours eventually acquire chemoresistance. U3-1402 is an anti-HER3 antibody-drug conjugate with a novel topoisomerase I inhibitor, DXd. In the current study, we evaluated the anticancer efficacy of U3-1402 in EGFR-mutant NSCLC cells with acquired resistance to EGFR-TKIs. HCC827GR5 and PC9AZDR7 are EGFR-TKI-resistant clones for gefitinib and osimertinib, respectively. U3-1402 alone or in combination with the EGFR-TKI erlotinib demonstrated potent anticancer efficacy in HCC827GR5 cells using an in vitro growth inhibition assay and in vivo xenograft mouse model. U3-1402 induced apoptosis in HCC827GR5 cells accompanying phosphorylation of histone H2A.X, a marker of DNA damage, but did not block HER3/PI3K/AKT signalling. Further, we found using flow cytometry that the cell surface HER3 expression level in HCC827GR5 cells was twice that found in HCC827 cells, indicating internalization of U3-1402 was increased in resistant cells. In addition, administration of U3-1402 notably repressed growth of EGFR-TKI osimertinib-resistant PC9AZDR7 xenograft tumours, and that PC9AZDR7 cells expressed five times greater cell surface HER3 than PC9 cells. Furthermore, using immunofluorescent microscopy, HER3 was observed predominantly in the nucleus of PC9 cells, but was localized in the cytoplasm of PC9AZDR7 cells. This finding indicates that altered trafficking of the HER3-U3-1402 complex may accelerate linker payload cleavage by cytoplasmic lysosomal enzymes, resulting in DNA damage. Our results indicate that administration of U3-1402 alone or in combination with an EGFR-TKI may have potential as a novel therapy for EGFR-TKI-resistant EGFR-mutant NSCLC.

The efficacy of programmed cell death-1 (PD-1) blockade in patients with non-small cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutations has been found to be limited, but the underlying mechanisms for this poor response have remained obscure. Given that the recognition by T cells of tumor antigens presented by major histocompatibility complex class I (MHC-I) molecules is essential for an antitumor immune response, we examined the effects of EGFR tyrosine kinase inhibitors (TKIs) on MHC-I expression in NSCLC cell lines. Appropriate EGFR-TKIs increased MHC-I expression at the mRNA and cell surface protein levels in NSCLC cells positive for EGFR mutations including those with the T790M secondary mutation. Trametinib, an inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK, also increased MHC-I expression, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor buparlisib did not, suggesting that the MEK-ERK pathway mediates the down-regulation of MHC-I expression in response to EGFR activation. Immunohistochemical analysis of EGFR-mutated NSCLC specimens obtained before and after EGFR-TKI treatment also revealed down-regulation of phosphorylated forms of EGFR and ERK in association with up-regulation of MHC-I, an increased number of infiltrating CD8+ T cells, and increased PD-1 ligand 1 expression after such treatment. Our results thus suggest that mutational activation of EGFR inhibits MHC-I expression through the MEK-ERK pathway in NSCLC and thereby contributes to the poor response of such tumors to immunotherapy. Further studies are warranted to evaluate the relation between EGFR-MEK-ERK signaling in and the immune response to EGFR-mutated NSCLC. .

survivin はアポトーシス阻害蛋白の一つで, 癌細胞に特異的に発現している. またp53は癌抑制遺伝子であり, DNAの障害に伴い活性化され, 抗癌剤の感受性やアポトーシスに関与する. 我々はp53による survivin の発現調節の有無を探るため, 肺癌細胞株を用いアドリアマイシン暴露後の survivin 発現量の変動を観察した. その結果, 変異型p53を有する細胞株とは対照的に, 野生型p53を有する細胞株ではp53の発現誘導に伴い survivin の発現は顕著に減少した. さらに small interfering (si) RNAを利用し, 野生型p53を有するA549肺癌細胞株のp53発現を抑制したところ, survivin 発現は有意に増加した. 次に survivin 阻害の癌細胞にもたらす影響を調べるために, 変異型p53を有する肺癌細胞株PC9の survivin 発現をsiRNAにより抑制した. その結果, 細胞増殖は有意に抑制され, さらにアドリアマイシンに対する感受性の増強と, 強いアポトーシスの誘導がみられた. 以上により, 我々は survivin 発現はp53の活性化によって抑制され, siRNAによる survivin の阻害は変異型p53を有する癌細胞の増殖を抑制し, またアドリアマイシンに対する感受性を高め, アポトーシスの惹起を促すことを確認した.

KRAS阻害剤ソトラシブ（AMG510）の耐性機序の解明、さらに耐性克服治療の開発につなげることが目的。 研究方法としてKRAS変異陽性でソトラシブ感受性肺癌細胞株H23及び同耐性株H23ARC10を用いて耐性の原因遺伝子の探索を行う。すでに網羅的な遺伝子解析によって耐性株でMET遺伝子増幅の出現を確認。さらに同耐性株に対しMET阻害剤の有効性をマウスモデルで確認。これらの研究成果はすでに論文発表をおこなった（Suzuki S, Yonesaka K, et al. Clin Cancer Research2021)。MET遺伝子増幅はSOS1、SHP2を介し、変異型及び野生型RASシグナルの増強をもたらした。このためSOS1、SHP2阻害剤とソトラシブの併用にも有効性を認めた。 現在あらたに樹立したソトラシブ耐性株H2122ARについて耐性機序の解明と耐性克服治療の検討を行っている。すなわち同耐性株について次世代シークエンサーによる遺伝子解析、マイクロアレイによる発現解析を実施。同定された遺伝子の異常に関連した阻害剤の投与試験を行っている。またその遺伝子について機能解析も行い、耐性のメカニズムを確認中である。同時にマウスモデル試験も開始しており、今後の耐性克服治療の臨床応用を目指した実験を行っている。またKRAS変異陽性患者由来の血液、腫瘍組織を収集しておりこれらについての遺伝子解析も予定している。