HER2-negative Patients Research Articles

Abstract 4915: Identification and validation of novel prognostic genetic markers in HER2-negative advanced gastric cancer (AGC) by artificial intelligence (AI) deep learning and machine learning algorithm

Abstract Introduction: Despite advances in anticancer treatments, there has been urgent need to identify novel prognostic markers for HER2-negative AGC. We propose AI based deep learning and machine learning pipeline to explore genetic markers that predict patient response to chemotherapy in HER2-negative AGC. Methods: We retrospectively assessed 179 stage IV HER2-negative AGC patients who have been treated with first-line chemotherapy in Yonsei Cancer Center, Korea between 2015 and 2021. In-house targeted sequencing panel data (CancerSCANTM and CancerMaster) were used to identify candidate prognostic markers. DeepSurv was mainly used to analyze the progression-free survival (PFS), which is a deep learning algorithm that can investigate prognostic roles. The SHapley Additive exPlanations (SHAP) method was applied to open the deep learning models’ black-box and calculate the importance ranking of gene features. Machine learning models-random survival forest, elastic-net, and lasso-were also investigated to identify potentially meaningful gene variants. To rank the gene features, we calculated variable importance (VIMP) for random survival forest, and penalized regression coefficients for elastic-net and lasso. All deep learning and machine learning models were trained using 5-folds cross-validation in 1,000 iterations of re-sampling bootstrapping with hyperparameter tuning by grid search. To classify responder or non-responder related genetic markers to chemotherapy, we used the signs of averaged penalize coefficients obtained from elastic-net and lasso. Based on the median linear predictor with candidate gene features, patients were classified into high-risk and low-risk group. Results: A total of 7,824 common variants were analyzed and 20 prognostic genetic markers were identified based on the top average values of SHAP, VIMP, and penalized regression coefficients. The top 10 non-responder genetic markers are included as follows: PARP4(p.S873N), CHUK(p.V268I), BARD1(359_365del), CREBBP(p.Y1125F), BARD1(p.P24S), PHLPP2(p.R1312Q), FAT3(p.Q3375R), CASP5(p.T106A), PHLPP2(p.I544V), and PTCH2(p.T988M). Otherwise, the top 10 responder genetic markers are defined as follows: IL7R(p.I66T), NOTCH4(L16delinsLL), FGFR3, BCL2A1(p.G82D), ZNF217(p.T548I), BRCA1(p.K1183R), FANCA(p.A412V), ADGRA2, CDKN2B, and FANCM(p.S175F). The high-risk group had worse PFS compared with low-risk group (median PFS 4.1 vs. 9.1 months; P-value <0.0001). Conclusion: This study showed the potential of AI deep learning and machine learning pipeline that employs an innovate prognostic genetic markers for HER2-negative AGC. Current ongoing analyses of larger cohort with more comprehensive clinical data will be presented. Citation Format: Sejung Park, Seok-Jae Heo, Choong-Kun Lee, Yaeji Lee, Woo Sun Kwon, Jingmin Che, Minseok Kim, Hyun Cheol Chung, Sun Young Rha. Identification and validation of novel prognostic genetic markers in HER2-negative advanced gastric cancer (AGC) by artificial intelligence (AI) deep learning and machine learning algorithm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4915.

Abstract 2269: Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer

Abstract PIK3CA mutations occur in ~15% of triple-negative breast cancers (TNBCs) and aberrantly activate the PI3K-AKT pathway, resulting in uncontrolled cell proliferation. Alpelisib is approved for hormone receptor-positive (HR+), HER2-negative BC patients with a PIK3CA mutation; however, not all PIK3CA-mutated (MUT), HR+ patients respond to alpelisib and others acquire resistance following exposure, suggesting adaptive mechanisms of PI3K activation. It has been proven that in TNBC, there exists a cohort of mutated genes which activate PI3K-AKT signaling. We hypothesize that testing alpelisib in combination with repurposed approved drugs, targeting alternative mechanisms of PI3K-AKT pathway activation, may be used to treat a cohort of TNBC patients. TNBC patient genetic data from the MSK (N=168), METABRIC (N=209), and TCGA (N=171) datasets were analyzed using cBioPortal to identify frequently occurring mutations. Differential expression analyses based on mutational status were performed on CCLE cell line (N=705) microarray and TCGA BC patient (N=981) RNA-sequencing data using R. Dysregulated genes were functionally analyzed using DAVID Ontology and those associated with poorer survival in TNBC were identified using KM-Plotter. STRING database was used to investigate protein interactions. The antiproliferative effects of targeted drugs tested alone and in combination in a panel of 6 TNBC cell lines were determined using acid phosphatase assays. Reverse-phase protein array analysis was carried out on protein lysates from cells following 24 hour treatment with drugs used alone and in combination.TP53 is the most frequently mutated gene in TNBC and mutations were associated with significant genetic dysregulation. A comprehensive analysis of upregulated genes in TP53-MUT TNBC led to the identification of 5 potential therapeutic targets - MET, S100A9, LCN2, CA9, TACSTD2, and KLK5 - based on their association with poorer survival in TNBC, protein interactions with TP53 and PIK3CA, and availability of targeted drugs. MET was selected for in vitro analysis based on its strong correlation with PI3K-AKT signaling. The presence of a TP53 mutation was associated with decreased antiproliferative effects in TNBC cell lines treated with alpelisib. Treatment with alpelisib in combination with a MET inhibitor, crizotinib, demonstrated greater antiproliferative effects in TP53-MUT/PIK3CA-MUT cell lines when compared to TP53 or PIK3CA MUT only models.TP53 mutations are associated with genetic dysregulation in TNBC and may confer resistance to alpelisib. Targeting upregulated proteins in a cohort of TP53-MUT/PIK3CA-MUT patients may improve sensitivity to alpelisib via dual inhibition of PI3K-AKT pathway activation. Protein analysis will uncover the mechanisms of synergy observed between alpelisib and crizotinib, and the therapeutic potential of the combination will be experimentally investigated in vivo. Citation Format: Grace Colley, Alexander Eustace, Dalal AlSultan. Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2269.

Abstract 2013: Extracellular vesicles transfer miR-181a to confer PARP inhibitor resistance and induce STING degradation in BRCA-mutated triple-negative breast and ovarian cancers cells

Abstract Background: Patients with BRCA-mutated breast cancer (BC), including triple-negative BC (TNBC), and ovarian cancer (OvCa) are treated with poly (ADP-ribose) polymerase inhibitors (PARPi). BC and OvCa tumors develop similar mechanisms of resistance to platinum-based agents and PARPi. This study aimed to identify microRNAs (miRs) inducing PARPi resistance in cancer cells that can be transferred by extracellular vesicles (EV). Methods: Olaparib-resistant (OlaR) cell lines were analyzed for 2,083 miRs using HTG miRNA Whole Transcriptomic Analysis (WTA) and NGS. Functional assays were performed in BRCA1-mutated TNBC and OvCa cell lines. In-silico analysis were performed using TCGA, GTEx, CCLE, GDSC, and GEO databases. Results: The miR-181 family (including miR-181a, p=0.001) was significantly upregulated in OlaR TNBC cell lines. High miR-181a levels occurred in tumor tissues of TNBC patients (p=0.001). miR-181a binds to the stimulator of interferon genes (STING) mRNA to promote STING mRNA degradation in OvCa. We hypothesized that miR-181a downregulates STING and downstream proinflammatory cytokines, to mediate PARPi resistance. miR-181a-overexpression induced olaparib resistance and STING downregulation in BRCA1-mutated TNBC cell lines. EV isolated from OlaR TNBC cell lines horizontally transferred miR-181a to non-resistant parental cell lines, which conferred PARPi-resistance and decreased STING. In clinical settings, STING levels were positively correlated with interferon gamma (IFNG) response scores (p=0.01). Low-IFNG response scores in HER2-negative BC patients showed a worse response to neoadjuvant treatment (p=0.001). Consistently, OlaR TNBC cell lines showed resistance to platinum-based drugs. OvCa cell lines resistant to cisplatin showed resistance to olaparib; but miR-181a knockout significantly improved olaparib sensitivity (p=0.001). Conclusions: High levels of miR-181a decrease STING pathway, correlate with IFNG scores, drive PARPi and platinum-based drug resistance. Horizontal miR-181a EVs transfer can facilitate drug resistance in cells. The miR-181a-STING axis can be used for predicting PARPi responses in TNBC and OvCa tumors. Citation Format: Matias A. Bustos, Takamichi Yokoe, Yoshiaki Shoji, Yuta Kobayashi, Shodai Mizuno, Tomohiro Murakami, Sreeja C. Sekhar, Matthew Knarr, Steven A. Vasilev, Analisa DiFeo, Ronny Drapkin, Dave S. Hoon. Extracellular vesicles transfer miR-181a to confer PARP inhibitor resistance and induce STING degradation in BRCA-mutated triple-negative breast and ovarian cancers cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2013.

Use of axillary ultrasound to guide breast cancer management in the genomic assay era.

Chemotherapy is conventionally offered to non-stage IV breast cancer patients with metastatic nodes. However, the RxPONDER trial showed that chemotherapy can be omitted in selected patients with 1-3 metastatic nodes if the 21-gene assay recurrence score is ≤25. We aimed to investigate if axillary ultrasound can identify this group of patients with limited nodal burden so that they can undergo upfront surgery followed by gene assay testing, to potentially avoid chemotherapy. T1-3, node positive, hormone receptor-positive and HER2-negative breast cancer patients ≥50 years old with axillary lymph node dissection (ALND) were reviewed from 2 centres. Patients with neoadjuvant chemotherapy and bilateral cancers were excluded. Number of ultrasound-detected abnormal axillary nodes, demographic and histological parameters were correlated with the number of metastatic nodes found on ALND. 138 patients were included, 59 (42.8%) and 79 (57.2%) patients had 1-3 and >3 metastatic nodes on ALND respectively. On logistic regression and ROC analysis, the number of ultrasound-detected abnormal nodes was significant (p <0.001) for predicting limited nodal burden (ROC AUC = 0.7135). Probabilities of <4 metastatic nodes with ultrasound cut-offs of 5, 6 and 8 abnormal nodes were 0.057, 0.026 and 0.005 respectively, with 100% specificity. A cut-off of ≤5 ultrasound-detected abnormal nodes can distinguish between patients with limited versus high nodal burden, with high specificity. Hence, incorporating the number of abnormal ultrasound-detected nodes into clinical practice may prove useful in guiding between upfront surgery and gene assay testing or neoadjuvant chemotherapy in this group of patients.

Association between Homologous Recombination Repair Defect Status and Long-Term Prognosis of Early HER2-Low Breast Cancer: A Retrospective Cohort Study.

As a newly identified subtype of HER2-negative tumors associated with a less favorable prognosis, it remains crucial to evaluate potential prognostic and predictive factors, particularly non-invasive biomarkers, for individuals with human epidermal growth factor 2 (HER2) low early-stage breast cancer (EBC). Multiple investigations have highlighted that HER2-negative patients with EBC exhibiting high homologous recombination deficiency (HRD) scores display lower rates of pathological complete response (PCR) to neoadjuvant chemotherapy (NAC). Nevertheless, no study to date has explored the correlation between HRD and the long-term prognosis in HER2-low patients with EBC. This retrospective observational study focuses on primary EBC sourced from The Cancer Genome Atlas dataset (TCGA). It reveals the gene mutation landscape in EBC with low HER2 expression and elucidates the tumor immune landscape across different HRD states. Utilizing bioinformatics analysis and Cox proportional models, along with the Kaplan-Meier method, the study assesses the correlation between HRD status and disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI). Subgroup analyses were conducted to identify potential variations in the association between HRD and prognosis. In the patients with HER2-low breast cancer, patients with homologous recombination related genes (HRRGs) defects had an HRD score about twice that of those without related genes mutations, and were at higher risk of acquiring ARID1A, ATM, and BRCA2 mutations. We also found that most immune cell abundances were significantly higher in EBC tumors with high HRD than in EBC tumors with low HRD or HRD-medium, particularly plasma B-cell abundance, CD8 T-cell abundance, and M1 macrophages. In addition, these tumors with HRD-high also appear to have significantly higher tumor immune scores and lower interstitial scores. Then, we analyzed the relationship between different HRD status and prognosis. There was statistical significance (P = .036 and P = .046, respectively) in DSS and PFI between the HRD-low and HRD-high groups, and patients with HRD-high EBC showed relatively poor survival outcomes. A medium HRD score (hazard ratio, HR = 2.15, 95% CI: 1.04-4.41, P = .038) was a significant risk factor for PFI. Hormone receptor positivity is an important factor in obtaining medium-high HRD score and poor prognosis. Higher HRD scores were associated with poorer PFI outcomes, particularly in people with HR+/HER2-low. Varied HRD states exhibited distinctions in HRRGs and the tumor immune landscape. These insights have the potential to assist clinicians in promptly identifying high-risk groups and tailoring personalized treatments for patients with HER2-low EBC, aiming to enhance long-term outcomes.

Comparison of efficacy and tolerability of single agent and double agent chemotherapy regimens in first-line treatment of elderly patients with her-2 negative metastatic gastric cancer

Aims: Chemotherapy remains a cornerstone in treating metastatic gastric cancer (GC), yet the management of elderly patients, who often face distinct challenges, lacks comprehensive guidelines. The aim of this study was to compare the efficacy and side effects of single-agent and double-agent chemotherapy regimens in first-line treatment of elderly patients with HER-2 negative metastatic GC. Methods: We retrospectively evaluated HER-2 negative metastatic GC patients aged 80 years and older who were treated at Van Yüzüncü Yıl University Medical Faculty Dursun Odabaşı Medical Center Oncology Clinic between 2010 and 2023. Demographic characteristics, treatment regimens and responses, prognostic factors, grade 3-4 toxicity, progression-free survival (PFS), and overall survival (OS) were analyzed. Results: The mean age of 56 patients was 82.6 ± 2.3 years and 24 (42.9%) of them were women. Single-agent chemotherapy was administered to 33 (58.9%) patients, while 23 (41.1%) received double-agent chemotherapy. The median OS was 5 months (95% CI, 2.9 to 7.1) in the single-agent group and 10 months (95% CI, 4.2 to 15.8) in the double-agent group (p = 0.237), although there was a numerical difference, it was not statistically significant. Median PFS was longer with double-agent chemotherapy, but not statistically significant (6 months vs. 4 months, p = 0.668). No statistically significant difference was found in the side effect rates of patients receiving single and double-agent chemotherapy. Conclusion: In our study, despite the absence of statistical significance in the survival rates among patients receiving double chemotherapeutic agents, their survival was twice as long as that of individuals receiving a single agent. Furthermore, no significant differences in terms of side effects were observed. These findings suggest that, even in individuals aged 80 years and older, a preference for double-agent chemotherapy should be considered when feasible.

Abstract A026: An alternative to HER2 IHC 0/1+/2+ status to predict which clinically HER2-negative patients will respond to anti-HER2 therapies: A rationale for the likely superiority of quantitative HER2 pathway RPPA measurements

Abstract Immunohistochemistry (IHC) may not accurately measure HER2 levels and does not reflect HER2 activity/phosphorylation in HER2-negative patients in the HER2 LOW (IHC 1+/2+) and ULTRA-LOW (IHC 0) range [PMID: 35595825). Yet, accurate quantification of HER2 may be key for predicting response to antibody drug conjugates (ADC) such as fam-trastuzumab-deruxtecan-nxki (T-DxD). We showed previously that response to the receptor tyrosine kinase inhibitor (RTKI) neratinib and the ADC ado-trastuzumab emtansine (TDM-1) can be predicted by phospho(p) HER2 Y1248 and pEGFR Y1173, but not total HER2, in TN and HER2+ pts, respectively (PMID: 34741023; 32914002). We now utilize I-SPY2 TRIAL data to explore further relationships between HER2 IHC, Reverse Phase Protein Array (RPPA)-based quantitative HER2 measurement and a HER2 Activation Response Predictive Signature (HARPS) as candidate response predictors for HER2low-targeted agents. Tumors from 395 HER2-negative patients (HR+=200, TN=195) from 8 arms of the I-SPY2 TRIAL had HER2 IHC/FISH data, along with RPPA-based HER2, pHER2 Y1248 and pEGFR Y1173 data from Laser Capture Microdissection (LCM)-enriched tumor epithelium. Of these tumors, 37% (145/395) were HER2 IHC 0, 42% (168/395) were IHC 1+ and 21% (82/395) were IHC 2+. RPPA-based HARPS was categorized as HARPS+ (pHER2-high/pEGFR-high) or HARPS- (pHER2-low and/or pEGFR-low) using previously established combined cut points. Association between quantitative protein values and HER2 IHC was assessed using ANOVA/Tukey tests. Association between endpoint quartiles and IHC was assessed using chi-squared/posthoc tests. RPPA quantitative HER2 total protein levels were associated with HER2 IHC (Tukey p&amp;lt;1E-09), however, the median and ranges of HER2 expression highly overlapped between HER2 IHC 0, 1+ and 2+ (e.g. HER2=0.44 [0.27 - 0.6] in IHC 0 vs 0.69 [0.42-1.0] in IHC 2+). A significant number (34%) of HER2 IHC 0 tumors expressed HER2 above the median level in the HER2 LOW population. While pHER2 and HARPS status were not associated with HER2 IHC class, HARPS associated with total HER2 overall (chi-squared p=8E-05) and in TNBC (chi-squared p=2.4E-06). We found that 42% (81/195) of TNBC were HARPS+, as were 44% (40/89) of HER2 ULTRA-LOW and 40% (41/104) of HER2 LOW tumors. Our results reveal general disconcordance between IHC and RPPA quantitative HER2, with a third of HER2 IHC 0 tumors expressing HER2 and thus, potentially responsive to ADCs such as T-DxD or TDM1. The high proportion of HER2 ULTRA-LOW TNBC tumors classified HARPS+ by our CLIA-based assay identifies tumors predicted to respond to HER2-directed TKI therapies, representing a cohort with actionable HER2 biology missed by IHC. As HER2Low targeted therapies enter ISPY 2.2, we will test the hypothesis that quantitative HER2 is most predictive for HER2 ADCs, and HARPS is most predictive for RTKIs. Citation Format: Julia Wulfkuhle, Denise M. Wolf, Angela DeMichele, Christina Yau, Laura van ‘t Veer, Hope S. Rugo, Lajos Pusztai, Lamorna Brown-Swigart, Gillian L. Hirst, Rosa I. Gallagher, Amy L. Delson, Alexander D. Borowsky, Laura J. Esserman, Paula R. Pohlmann, Emanuel F. Petricoin. An alternative to HER2 IHC 0/1+/2+ status to predict which clinically HER2-negative patients will respond to anti-HER2 therapies: A rationale for the likely superiority of quantitative HER2 pathway RPPA measurements [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A026.

Risk Factors of Response to Neoadjuvant Chemotherapy in Patients with Luminal (HER2 Negative) Breast Cancer: Roc Curve and Logistic Regression Model Results

Background: Neoadjuvant chemotherapy (NAC) is less effective for luminal human epidermal growth factor receptor 2 (HER2) negative breast cancer (BC) patients and generally shows a low pathological complete response (pCR) after NAC compared to HER2 positive and triple negative breast cancer (TNBC). This study aimed to determine the factors associated with histopathologic response following NAC in luminal (HER2 negative) BC. Methods: This is a cross-sectional study conducted on 255 estrogen (ER) positive and HER2 negative BC patients after NAC between January 2018 and July 2023. Demographic and clinicopathological characteristics of the patients were collected for the statistical analysis. Chi-Square tests were used in the qualitative comparisons between study groups. Receiver Operating Characteristic (ROC) analysis was used for the diagnostic performance of Ki-67 expression and ER in determining the pCR rates. Using the Youden index, optimum cut points were determined. Also, multivariate logistic regression analysis was applied to determine the independent variables associated with the dependent variable (pCR). Results: After NAC, pCR was achieved in the breast in 35 (14%) patients, in the axilla in 44 (17%) patients, and in both the breast and axilla in 18 (7%) patients. Ki-67 expression was the only common variable associated with the breast, axilla and both the breast and axilla pCR. The most appropriate Ki-67 expression cut-off value for determining the breast and axilla complete response was found to be 40%. ER positivity level was only associated with pCR in the breast and the cut-off value was found to be 85%. Conclusion: The results of this study raise the possibility of patients with luminal (HER2 negative) BC with Ki-67 expression higher than 40% benefiting from chemotherapy, as they showed increased pCR rates.

Trastuzumab and first-line taxane chemotherapy in metastatic breast cancer patients with a HER2-negative tumor and HER2-positive circulating tumor cells: a phase II trial

PurposeHER2 overexpressing circulating tumor cells (CTCs) are observed in up to 25% of HER2-negative metastatic breast cancer patients. Since targeted anti-HER2 therapy has drastically improved clinical outcomes of patients with HER2-positive breast cancer, we hypothesized that patients with HER2 overexpressing CTCs might benefit from the addition of trastuzumab to chemotherapy.MethodsIn this single-arm, phase II trial, patients with HER2-positive CTCs received trastuzumab as addition to first-line treatment with taxane chemotherapy. Patients with detectable CTCs but without HER2 overexpression that received taxane chemotherapy only, were used as control group. The primary outcome measure was progression-free rate at 6 months (PFR6), with a target of 80%. In November 2022, the study was terminated early due to slow patient accrual.Results63 patients were screened, of which eight patients had HER2-positive CTCs and were treated with trastuzumab. The median number of CTCs was 15 per 7.5 ml of blood (range 1–131) in patients with HER2-positive CTCs, compared to median 5 (range 1–1047) in the control group. PFR6 was 50% in the trastuzumab group and 54% in the taxane monotherapy group, with no significant difference in median PFS (8 versus 9 months, p = 0.51).ConclusionNo clinical benefit of trastuzumab was observed, although this study was performed in a limited number of patients. Additionally, we observed a strong correlation between the number of evaluable CTCs and the presence of HER2-positive CTCs. We argue that randomized studies investigating agents that are proven to be solely effective in the HER2-positive patient group in patients with HER2-positive CTCs and HER2-negative tissue are currently infeasible. Several factors contribute to this impracticality, including the need for more stringent thresholds, and the rapidly evolving landscape of cancer treatments.

Exploratory analysis of biomarkers of response to durvalumab in advanced HER2-negative oesophago-gastric adenocarcinoma within a phase 2 clinical trial.

384 Background: Advanced oesophago-gastric cancers have showcased varied responses to immunotherapy based on biomarkers such as tumour mutational burden, microsatellite instability, and PD-L1 expression. However, these markers may not fully capture tumour heterogeneity and evolutionary dynamics. Hence, understanding the intricate relationship between immune selection, specifically immune dN/dS,1 and tumour evolution is vital for devising personalized therapeutic strategies. Methods: In our phase 2 open label, multicentre, randomised PLATFORM trial (NCT02678182), patients received maintenance durvalumab post first-line chemotherapy. A survival advantage was not seen with maintenance durvalumab compared to surveillance and exploratory analysis of PD-L1 expression was not associated with improved survival outcomes.2 However, a subset of patients treated with durvalumab experienced durable responses and some experienced incremental radiological responses. A pilot substudy on a selected cohort (n=24) of these patients was designed to explore the potential of immunogenomics evolutionary-based metrics, notably dN/dS, as predictive biomarkers. DNA and RNA from FFPE baseline tumour samples underwent analysis, examining genomic factors including tumour mutational burden, indel signatures, PDL1 status, and immune evasion mechanisms. Furthermore, the evolutionary metric, immune dN/dS, which has implications in tumour immunoediting and its response to immunotherapies, was scrutinized. Results: A dominant indel mutational signature emerged from the analysis. The majority of patients displayed a significantly higher number of indel variants, compared to non-synonymous and synonymous single-nucleotide variants. A recurring observation was dN/dS values consistently above 1, indicating positive selection for non-synonymous somatic variants. Additionally, hypermutation in driver genes, notably TP53 and XIRP2, was detected. One patient, with a unique dN/dS &lt; 1, showcased survival beyond four years without liver metastases. Immune dN/dS &gt; 1 and no evidence of liver metastases displayed the best prognosis after treatment in contrast with those that have immune dN/dS &lt; 1 with or without liver metastases. Conclusions: Immune dN/dS emerges as a promising tool to decode tumour evolutionary mechanisms, providing profound insights into the dynamics of tumour evolution in advanced HER2 negative oesophago-gastric cancer patients receiving durvalumab immunotherapy. This pilot study underscores the potential of this novel immunogenomic approach in improving patient stratification and tailoring therapeutic strategies in cancer immunotherapy. 1. Zapata et al, Nature Genetics, 2023. 2. Fong et al, J Clin Oncol, 2021. Clinical trial information: NCT02678182 .

Preliminary results of surufatinib (Suru) plus standard chemotherapy (ChT) as second line (2L) treatment of advanced gastric cancer (aGC): A single-arm, phase 2 clinical trial.

353 Background: Limited options are available for aGC patients who have failed first line (1L) systemic treatment, and single-agent ChT with paclitaxel (Pac) or irinotecan (Iri) remains the mainstay. Recent studies have suggested improved efficacy with the combination of ChT and antiangiogenic agents in the 2L setting. Suru is a tyrosine kinase inhibitor targeting VEGFR1-3, FGFR1 and CSF-1R. This trial is aimed to evaluate the efficacy and safety of Suru plus Pac or Iri as 2L treatment for aGC. Methods: This single-arm, single center, phase 2 trial was designed to enroll up to 35 histologically confirmed HER2-negative aGC patients with evaluable disease who failed 1L ChT with or without immunotherapy (IO). Eligible patients were administered 21-day cycles of Suru (250mg, po, qd) plus Pac (150mg/m2, iv, d1) or Iri (125mg/m2, iv, d1, d8) per investigator choice for 6 cycles, followed by Suru (250mg, po, qd) maintenance if no disease progression (PD), until intolerable toxicity, PD, or death. The primary endpoint was ORR (per RECIST v1.1), and secondary endpoints included DCR, PFS, OS, and safety. Results: As of Sep 11, 2023, 15 patients were enrolled, and 6 patients remained on treatment. The median follow-up time was 5.77 (95% CI: 3.37~10.13) months. Among all 15 patients, the median age was 66 (range: 33-76) years, 14 (93.3%) were male, and 14 (93.3%) had an ECOG PS of 1. Ten (66.7%) patients had received prior anti-PD-1 antibodies, and all 15 patients received Pac as the standard ChT. All 15 patients were evaluable for tumor response, with 5 PR, 9 SD, and 1 PD as their best overall response (BOR). The ORR and DCR were 33.3% and 93.3%, respectively. The median PFS was 5.23 months, while the OS data was immature yet. The ORR, DCR and PFS seemed comparable between patients with and without prior IO. In terms of safety, the combined treatment was generally well tolerated, treatment-related Grade 3/4 adverse events included neutropenia (6, 40%), leukopenia (1, 6.7%), lymphopenia (1, 6.7%), and proteinuria (1, 6.7%). There were no treatment-related serious adverse events or treatment-related deaths. Conclusions: These results suggested promising anti-tumor activity and a manageable safety profile of Suru plus standard ChT in 2L treatment of aGC. The trial is still ongoing, and more data will be disclosed in the future. Clinical trial information: ChiCTR2200063336 . [Table: see text]

A phase II trial of CapeOX plus nivolumab for early relapsed HER2-negative gastric cancer (JACCRO GC-11: FirSTAR trial).

TPS423 Background: Fluoropyrimidine-based post-operative chemotherapy is one of the standard adjuvant therapies for curatively resected gastric cancer (GC).1,2 The current standard first-line chemotherapy consists of fluoropyrimidine and oxaliplatin combined with nivolumab in advanced HER2-negative GC patients including cases with a recurrence &gt; 6 months after completion of post-operative chemotherapy, based on the CheckMate 649 and ATTRACTION-4 trials. However, in GC patients with early recurrence during or ≤ 6 months after completion of post-operative 5-FU based chemotherapy, optimal treatments remain to be established. A previous phase II study showed that capecitabine plus cisplatin is an active regimen for early relapsed GC after post-operative S-1 monotherapy.3 This trial aims to investigate the efficacy of capecitabine plus oxaliplatin (CapeOX) plus nivolumab for patients with early relapsed HER2-negative GC. Methods: This is a multicenter single-arm phase II trial. The main eligibility criteria include: histologically confirmed HER2-negative adenocarcinoma of stomach or esophagogastric junction; radiologically diagnosed recurrence during or ≤ 6 months after post-operative chemotherapy with S-1 or S-1 plus docetaxel conducted for stage II/III GC after curative resection; age ≥ 18 years; ECOG performance status 0-1; having measurable lesions according to RECIST ver. 1.1; and adequate oral intake and organ functions. Enrolled patients will receive 21-day cycles of nivolumab with CapeOX at the following dose until disease progression or unacceptable toxicities: capecitabine, 1000 mg/m2 twice per day on day 1-14; oxaliplatin, 130 mg/m2 on day 1; and nivolumab, 360 mg on day 1. The primary endpoint is objective response rate (ORR), and the secondary endpoints include overall survival, progression-free survival, disease control rate, duration of response, and safety. PD-L1 combined positive score will be tested for the association with efficacy. We assume null ORR of 20% and alternative ORR of 32%. With two-sided alpha level of 10% and 80% power, sample size would be calculated as 85. Thus, a total of 92 patients are planned for enrollment within a 2.5-year accrual period. Enrollment opened in March 2023. Clinical trial information: jRCTs031220572. 1. N Engl J Med. 2007;357:1810-20. 2. J Clin Oncol. 2019;37:1296-1304. 3. Gastric Cancer. 2018;21:811-8. Clinical trial information: jRCTs031220572 .

Efficacy and safety of nivolumab and CapeOX in patients with previously untreated FGFR2-positive, PD-L1-positive advanced gastric cancer: A single-arm, multicenter, phase 2 study NIVOFGFR2.

303 Background: Nivolumab in combination with chemotherapy has been approved in the first-line treatment of PD-L1-positive metastatic gastric cancer (GC). Fibroblast growth factor receptor 2 (FGFR2) is overexpressed in 30% of patients with GC and is a potential new target for targeted therapy with monoclonal antibodies1 or allosteric extracellular inhibitors.2 The aim of the NIVOFGFR2 study was to evaluate the preliminary efficacy of nivolumab in metastatic GC co-expressing PD-L1 and FGFR2. Methods: In this single-arm, multi-center, phase 2 study, eligible patients had metastatic untreated HER2-negative gastric adenocarcinoma with centrally confirmed expression of PD-L1 (CPS≥5; DAKO 28-8) and FGFR2 (moderate (2+) and strong (3+) membranous staining in more than 1% of tumor cells; Abсam EPR24075-418). Patients received nivolumab 360 mg with CapeOX (capecitabine and oxaliplatin) every 3 weeks. The primary endpoint was 1-year progression-free survival (PFS). Secondary endpoints included median PFS and overall survival (OS), objective response rate (ORR) and Grade ≥3 adverse events rate. Results: Seventy-four HER2-negative patients were screened and23 (31%) patients were enrolled (median age 61 years, men 74%, Caucasian 91%, ECOG PS 1-2 78%, organs with ≥2 metastases 78%; CPS (5-9) 22%, (≥10) 78%). 1-year PFS rate was 30,4%. The median PFS was 6.2 months (95% CI 4.4-7.6). The ORR was 21.7% with 1 complete response. With a median follow-up of 11.8 months at data cutoff, the median OS was not reached. Grade ≥3 treatment-related adverse events were reported in 9 (39.1%) patients. Conclusions: An interim analysis demonstrates modest efficacy and an acceptable safety profile of nivolumab in combination with chemotherapy in patients with FGFR2-positive, PD-L1-positive metastatic GC. Further follow-up is ongoing. 1. Wainberg, Lancet Oncol. 2022. 2. Tsimafeyeu, Invest New Drugs. 2023. Clinical trial information: NCT05859477 .

First-in-human validation of enzymolysis clearance strategy for decreasing renal radioactivity using modified [68Ga]Ga-HER2 Affibody.

Enzymolysis clearance strategy, characterized by releasing the non-reabsorbable radioactive fragment under the specific cleavage of enzymes, is confirmed to be a safe and effective way to reduce the renal radioactivity accumulation in mice. However, the effectiveness of this strategy in humans remains unknown. Human epidermal growth factor receptor 2 (HER2) is overexpressed in various types of tumors, and radiolabeled HER2 Affibody is believed to be an attractive tool for HER2-targeted theranostics. However, its wide application is limited by the high and persistent renal uptake. In this study, we intend to validate the effectiveness of enzymolysis clearance strategy in reducing renal accumulation by using a modified HER2 Affibody. A new HER2 Affibody ligand, NOTA-MVK-ZHER2:2891, containing a cleavable Met-Val-Lys (MVK) linker was synthesized and labeled with 68Ga. The microPET imaging study was performed in SKOV-3 tumor mice to assess the uptakes of the control ligand and the MVK one in tumors and kidneys. Seven healthy volunteers were included for biodistribution and dosimetric studies with both the control and MVK ligands performed 1week apart. Urine and blood samples from healthy volunteers were collected for in vivo metabolism study of the two ligands. Four HER2-positive and two HER2-negative patients were recruited for [68Ga]Ga-NOTA-MVK-ZHER2:2891 PET/CT imaging at 2 and 4h post-injection (p.i.). [68Ga]Ga-NOTA-MVK-ZHER2:2891 was stable both in PBS and in mouse serum. MicroPET images showed that the tumor uptake of [68Ga]Ga-NOTA-MVK-ZHER2:2891 was comparable to that of [68Ga]Ga-NOTA-ZHER2:2891 at all the time points, while the kidney uptake was significantly reduced 40min p.i. (P < 0.05). The biodistribution study in healthy volunteers showed that the kidney uptake of MVK ligand was significantly lower than that of the control ligand at 1h p.i. (P < 0.05), with the SUVmean of 34.3 and 45.8, respectively, while the uptakes of the two ligands in the other organs showed negligible difference. The effective doses of the MVK ligand and the control one were 26.1 and 28.7 µSv/MBq, respectively. The enzymolysis fragment of [68Ga]Ga-NOTA-Met-OH was observed in the urine samples of healthy volunteers injected with the MVK ligand, indicating that the enzymolysis clearance strategy worked in humans. The PET/CT study of patients showed that the range of SUVmax of HER2-positive lesions was 9.4-21, while that of HER2-negative lesions was 2.7-6.2, which suggested that the MVK modification did not affect the ability of ZHER2:2891 structure to bind with HER2. We for the first time demonstrated that enzymolysis clearance strategy can effectively reduce renal radioactivity accumulation in humans. This strategy is expected to decrease renal radiation dose of peptide and small protein-based radiotracers, especially in the field of radionuclide therapy.

Intratumoral heterogeneity of Ki67 proliferation index outperforms conventional immunohistochemistry prognostic factors in estrogen receptor-positive HER2-negative breast cancer.

In breast cancer (BC), pathologists visually score ER, PR, HER2, and Ki67 biomarkers to assess tumor properties and predict patient outcomes. This does not systematically account for intratumoral heterogeneity (ITH) which has been reported to provide prognostic value. This study utilized digital image analysis (DIA) and computational pathology methods to investigate the prognostic value of ITH indicators in ER-positive (ER+) HER2-negative (HER2-) BC patients. Whole slide images (WSIs) of surgically excised specimens stained for ER, PR, Ki67, and HER2 from 254 patients were used. DIA with tumor tissue segmentation and detection of biomarker-positive cells was performed. The DIA-generated data were subsampled by a hexagonal grid to compute Haralick's texture indicators for ER, PR, and Ki67. Cox regression analyses were performed to assess the prognostic significance of the immunohistochemistry (IHC) and ITH indicators in the context of clinicopathologic variables. In multivariable analysis, the ITH of Ki67-positive cells, measured by Haralick's texture entropy, emerged as an independent predictor of worse BC-specific survival (BCSS) (hazard ratio (HR) = 2.64, p-value = 0.0049), along with lymph node involvement (HR = 2.26, p-value = 0.0195). Remarkably, the entropy representing the spatial disarrangement of tumor proliferation outperformed the proliferation rate per se established either by pathology reports or DIA. We conclude that the Ki67 entropy indicator enables a more comprehensive risk assessment with regard to BCSS, especially in cases with borderline Ki67 proliferation rates. The study further demonstrates the benefits of high-capacity DIA-generated data for quantifying the essentially subvisual ITH properties.

The Magee 3 Equation Predicts Favorable Pathologic Response to Neoadjuvant Endocrine Therapy in Breast Cancer Patients.

Breast cancer (BC) remains a significant health care challenge, and treatment approaches continue to evolve. Among these, neoadjuvant endocrine therapy (NET) has gained prominence, particularly for postmenopausal, hormone-receptor positive, HER2-negative (HR+/HER2-) BC patients. Despite this, a significant gap exists in identifying patients who stand to benefit from NET. The objective of this study was to assess whether Magee equations (MEs) could serve as predictors of response to NET. This retrospective study included adult patients with invasive BC who underwent NET followed by curative surgery. Assessment of sociodemographic, clinical, and tumor-related variables was conducted. The ME1, ME2, ME3, and ME mean were analyzed to explore their predictive role for NET response. Receiver operating characteristic (ROC) curves were employed, along with the determination of optimal cutoff points. Logistic regression models were utilized to identify the most significant predictors of pathological response. Among the 75 female participants, the mean age was 69.4 years, with the majority being postmenopausal (n = 72, 96%) and having an ECOG-PS of 0/1 (n = 63, 84%). Most patients were classified as luminal A (n = 41, 54.7%). ME3 emerged as a promising predictor, boasting an AUC of 0.734, with sensitivity of 90.62% and specificity of 57.50% when the threshold was ≤ 19.97. In univariate analysis, clinical staging (p = 0.002), molecular subtype (p = 0.001), and ME3 (continuous = 0.001, original 3-tier: p = 0.013, new 2-tier: <0.001) categories exhibited significant associations with pathological response. In the multivariate model, clinical staging and new 2-tier ME3 (<20 vs. ≥20) were included as significant variables. Patients with ME3 < 20 have a higher likelihood of presenting a pathological response, offering a cost-effective alternative tool to Oncotype DX. Larger future studies with a prospective design are awaited to confirm our findings.

Prognostic factors in non-metastatic HER2 'low' and HER2 'negative' breast cancer: single institute experience.

Comparison of prognosis and survival in human epidermal growth factor receptor 2 (HER2)-low and HER2-negative patients with early stage or locally advanced, hormone receptor-positive breast cancer. In aretrospective single center study, the patients with early stage or locally advanced stage, hormone receptor (HR)-positive [estrogen receptor (ER) ≥ 1% and/or progesterone receptor (PR) ≥ 1%] and HER2 negative or HER2 low invasive breast cancer diagnosis were included. Atotal of 444 patients were included in the study. Patients were divided into two groups: HER2 negative and HER2 low. There were 235 (53%) patients in the HER2 negative group and 209 (47%) patients in the HER2 low group. The HER2 low group had significantly longer 5‑year disease-free survival (DFS) than the HER2 negative group. The patients with lower Ki67 (< 20%) also had a longer 5‑year DFS. Nonmetastatic HR+/HER2 low breast cancer patients had better DFS than HR+/HER2 negative ones. The Ki67 level and HER2 low status were independent prognostic factors. Randomized clinical trials are needed in early stage HER2 low breast cancer patients.

Evolution and prognostic significance of HER-2 conversion from primary to residual disease in HER-2 negative patients with breast cancer after neoadjuvant chemotherapy.

This study aimed to analyze HER-2 zero or HER-2 low conversion in HER-2 negative patients after neoadjuvant chemotherapy (NAC) and evaluate its prognostic significance. HER-2 negative patients with breast cancer with residual disease after NAC and paired pre- and post-therapeutic HER-2 testing results were analyzed retrospectively. HER-2 low, defined as immunohistochemistry (IHC) scores of 1+ or 2+/in situ hybridization (ISH), were not amplified. HER-2 zero is defined as an IHC score of 0. A total of 571 patients were enrolled, including primary HER-2 zero (n=201, 35.2%) and HER-2 low (n=370, 64.8%). The overall HER-2 change rate was 32.4%. Multivariable logistic regression showed that patients with hormone receptor-positive status before NAC was significantly associated with the conversion of HER-2 zero to low (OR=3.436, P < 0.0001). The median follow-up time was 50.0 months. In patients who are primary HER-2 zero, HER-2 zero to low was significantly associated with better disease-free survival (DFS) than constant HER-2 zero (HR=0.49, P=0.01) after adjustment (4-year DFS 80.1% vs 55.7%, Log-rank P=0.033). Subgroup analysis revealed that among patients who are primary HER-2 zero with hormone receptor-positive, HER-2 zero to low had a significantly better DFS than constant HER-2 zero (Log-rank P=0.037). In contrast, patients with hormone receptor-negative status did not. In conclusion, almost one-third of patients who are HER-2 negative underwent HER-2 zero or HER-2 low conversion after NAC. HER-2 zero to low conversion was associated with better DFS in patients who are HER-2 zero. These results provide a valuable reference for the potential application of anti-HER-2 ADC in an adjuvant setting for patients with residual disease after NAC.

Androgen Receptor Influenced Recurrence Score Correlation in Hormone Positive and HER2 Negative Breast Cancer Indian Patients: A Comparative Approach

Breast cancer (BC) recurrence is a major concern for both patients and healthcare providers. Accurately predicting the risk of BC recurrence can help guide treatment decisions and improve patient outcomes for a disease-free survival. There are several approaches and models that have been developed to predict BC recurrence risk. These include derived clinical assays such as genetic profiling (Oncotye Dx, MammaPrint, CanAssist and others), and algorithm derived open access tools such as Magee equations (ME), CTS5 Calculator and Predict Breast cancer. All the clinical assays are well accepted, but affordability and feasibility remain the challenge due to a noteworthy price tag of USD 3000. As per The American Society of Clinical Oncology (ASCO) updates, open access tools are possible substitutes but the availability of limited information on their applicability is a concern. These tools take into consideration the histopathologic parameters and immunohistochemistry (IHC) biomarkers data for estrogen receptor/progesterone (ER/PR), human epidermal growth factor receptor 2 (HER2), and Ki67. The current study focuses on the application of these tools in a subset of 55 Indian BC patients considering the influence of the androgen receptor (AR) IHC expression profile. AR is a potent target and a close interacting neighbor protein to ER and available literature also suggests their crosstalk expression in BC clinical models. Our comparative recurrence scores (RSs) predictive data showed a statistically significant AR expression correlation with average ME scores. No significance was noted across different prediction tools. The findings are suggestive that ME predictive scores are more relevant and informative compared to other online tools and with an additional AR IHC expression analysis the recurrence prediction might prove to be beneficial and feasible to many deprived BC patients.

HER2 becomes a novel survival biomarker for gastric cancer patients: a pooled analysis.

Although anti-HER2 therapies have been widely used against gastric carcinoma, the prognostic significance of HER2 overexpression remains unclear. Previous studies failed to provide convincible evidence due to inconsistent HER2 evaluation criteria and heterogeneous clinical characteristics. To figure out the prognostic significance of HER2 expression in gastric cancer, we rigorously designed and conducted this study. Meta-analysis. Record retrieval was performed by searching PubMed, Web of Science, Cochrane Library, Embase, ASCO, and ESMO meeting libraries from inception to November 2022. Cohort studies investigating overall survival comparison between HER2-positive and HER2-negative gastric cancer patients were included. Both resectable and advanced cases were separately collected while HER2 evaluation standards should be consistent across eligible studies. Newcastle-Ottawa Scale was used for quality assessment. Overall survival was the only endpoint and effect size was presented by hazard ratio (HR) with its 95% confidence interval. The pooled calculation was conducted on Review Manager 5.4. Thirty studies were eligible, including 9945 patients. Eligible studies were mostly high quality (n = 31). Regarding resectable cases (n = 22), HER2-positive groups had significantly worse prognosis than HER2-negative counterparts (HR 1.56, 95%CI 1.32-1.85, p < 0.00001). For HER2-positive patients with advanced gastric cancer (n = 10), HER2 overexpression was also an unfavorable survival indicator (HR 1.70, 95%CI 1.23-2.35, p = 0.001). Potential heterogeneous studies had been eliminated while outcomes remained stable by sensitivity analysis. Subgroup analysis suggested HER2-positive patients had a poorer prognosis in both East Asian (resectable: HR 1.56; advanced: HR 1.32) and non-East Asian countries (HR 1.58; HR 3.27). As a novel survival biomarker in gastric cancer, HER2 overexpression indicates unfavorable prognosis among both resectable and advanced patients, irrespective of East Asian or non-East Asian populations. PROSPERO (CRD42020168051).