HER2 Amplification Research Articles

Concordance between human epidermal growth factor receptor 2 (HER2) status by immunohistochemistry (IHC) and next-generation sequencing (NGS) in tissue and blood samples from gastric and gastroesophageal junction cancers (GC/GEJC).

478 Background: An estimated 18% of patients (pts) with GC/GEJC have HER2-positive (HER2+; IHC 3+ or 2+/in situ hybridization–positive [ISH+]) tumors. Although IHC/ISH are the standard testing methods for determining HER2 positivity, NGS can identify HER2 ( ERBB2 ) amplification alongside other biomarkers. This study assessed the concordance of detecting HER2 amplification by tissue/plasma NGS with IHC/ISH HER2 status in pts with GC/GEJC. Methods: Pts were retrospectively identified in the Tempus database. The primary analysis assessed tissue and plasma NGS HER2 copy number (CN) concordance with a HER2+ (IHC 3+ or 2+/ISH+) or HER2− (IHC 0, 1+, or 2+/ISH−) status; NGS samples were collected in ≤30 days of the IHC sample collection date. HER2 amplification was defined as tissue CN ≥8 or plasma log2(CN) ≥0.5; additional amplifications were captured by manual inspection. A secondary analysis assessed the correlation between paired tissue and plasma HER2 log2(CN) in pts with plasma and tissue NGS samples collected ≤30 days apart. The secondary analysis included pts irrespective of IHC sample collection date. Results: Of 1578 pts with GC/GEJC and a curated IHC score evaluated in the primary analysis, 1507 and 305 pts underwent tissue and plasma NGS testing, respectively. Among pts with tissue and plasma NGS results, 902 (59%) and 201 (66%) had primary tumors of the stomach, respectively; all remaining pts had primary tumors of the cardia.According to IHC/ISH, 229 (15%) and 40 (13%) pts were HER2+ among those with tissue and plasma NGS testing, respectively. Of the pts identified as HER2 amplified by tissue and plasma NGS,95% and 92% were HER2+ by IHC/ISH, respectively. Positive percent agreement (PPA) of HER2 amplification by tissue and plasma NGS with IHC/ISH HER2+ status was 70% and 58%, respectively. Concordance rates by PPA, negative PA (NPA), and overall PA (OPA) are summarized in Table. In the secondary analysis, a positive correlation in NGS HER2 log2(CN) was observed in pts with paired tissue and plasma NGS (mutant variant allele frequency >5%; R=0.85; P<2.2e−16; n=307). Conclusions: Based on PPA, we observed moderate concordance between NGS HER2 amplification detection (plasma or tissue) and standard HER2 IHC assessment. NGS is not sufficient to detect all pts with HER2+ GC/GEJC. Further testing is warranted to determine if NGS testing could complement IHC/ISH in identifying pts who may benefit from HER2-directed therapies. IHC 3+ or 2+/ISH+ (HER2+), n IHC 0, 1+, or 2+/ISH− (HER2−), n Total Total 229 1120 Tissue HER2 amplified, n 160 8 168 Tissue HER2 not amplified, n 69 1112 1181 PPA: 69.9% NPA: 99.3% OPA: 94.3% IHC 3+ or 2+/ISH+ (HER2+), n IHC 0, 1+, or 2+/ISH − (HER2 − ), n Total Total 40 233 Plasma HER2 amplified, n 23 2 25 Plasma HER2 not amplified, n 17 231 248 PPA: 57.5% NPA: 99.1% OPA: 93.0%

A global comparative study on real-world clinical and genomic differences in advanced biliary tract cancer.

628 Background: Biliary tract cancers (BTC) are aggressive malignancies with poor survival outcomes and limited treatment options. This study compared the epidemiological and molecular differences of advanced BTC patients through a global collaboration involving the Mayo Clinic and National Cancer Center Hospital East. Methods: We included patients with advanced BTC who underwent comprehensive tumor molecular profiling who received at least 30 days of systemic therapy. Patients were grouped based on specific alterations. Overall survival (OS) was calculated from the start of first-line therapy until death and compared among subgroups using Cox regression models. Results: 332 (36%) out of 932 patients had at least one actionable mutation : somatic BRCA1/2 mutations N=106, FGFR2 fusions N=61, HER2 amplifications N=65, KRAS G12C/D mutations N=46, IDH1 mutations N=38, and TMB >10 mut/Mb N=16. BRCA1/2 -mutated BTC were more commonly found in older Asian males with intrahepatic tumors and often presented with peritoneal and liver metastases upon initial diagnosis. FGFR2 fusions were more prevalent in younger White females with intrahepatic tumors, while HER2 amplifications were mainly seen in Asian females with gallbladder tumors. KRAS mutations were primarily observed in Asian males, and IDH1 mutations were common in White males with intrahepatic tumors, frequently presenting with liver metastasis. Survival outcomes varied significantly based on the alteration type: patients with FGFR2 fusions had the longest median OS (mOS) at 23.3 months (mo) (95%CI 18.4-34.9), followed by those with IDH1 mutations (19.3 mo; 95% CI 17.4-NE) and TMB >10 mut/Mb (17.8 mo; 95% CI 13.6-NE). BRCA mutations had a mOS of 16 mo (95% CI 14.6-19.8), while HER2 amplifications and KRAS mutations had mOS of 15.5 (95% CI 11.9-20.2) and 11.6 mo (95% CI 10.0-20.9), respectively. Conclusions: These findings underscore the heterogeneity in clinical and genomic characteristics among BTC patients, highlighting the importance of tailored therapeutic approaches. Baseline patient characteristics stratified by mutation status. KRAS G12C/D (N=46) BRCA 1/2 (N=106) IDH1 (N=38) HER2 amplification(N=65) FGFR2 fusion(N=61) TMB>10 mut/Mb(N=16) P-value Median Age, year 67 68 64 66 52 73 <0.001 Female, n (%) 19 (41) 34 (32) 18(47) 34 (52) 45(74) 6 (38) <0.001 Race, n (%) <0.001 Asian 31 (67) 97 (92) 13 (34) 51 (79) 10 (16) 6 (38) White 14 (30) 8 (8) 24 (63) 13 (20) 48 (79) 10 (63) Primary tumor location, n (%) <0.001 Intrahepatic 28 (68) 45 (45) 33 (100) 19 (31) 54(95) 10 (67) Extrahepatic 9 (22) 27(27) 0 (0) 10 (16) 2 (4) 4 (27) Gallbladder 4 (10) 28 (28) 0 (0) 32 (53) 1 (2) 1 (7) Sites of Metastasis at diagnosis, n (%) Abdominal Wall / Peritoneal 9 (24) 15 (19) 4(13) 7 (12) 6 (10) 4 (27) 0.309 Bone 33 (92) 70 (90) 26 (87) 2 (3) 9(15) 0 (0) 0.212 Liver 20 (49) 52 (61) 28 (82) 45 (73) 45 (76) 6 (38) 0.001 Lung 26 (70) 65 (82) 22 (69) 36 (58) 27(45) 11 (69) 0.006

Impact of education on clinical confidence utilizing HER2-targeted therapies in colorectal cancer.

225 Background: Novel targeted therapies entered the treatment landscape in patients with HER2-positive metastatic colorectal cancer (mCRC). To ensure that these therapies are used effectively, clinicians must assess tumor HER2 status and recognize how HER2-directed treatment modalities fit into the current paradigm. To further enable clinicians to integrate HER2-targeted therapies, an educational program was designed in 2023. Methods: A one-hour online, video-based program was designed for clinicians and their teams and hosted on MedLive from February 2023 for one year. Knowledge and competence questions were administered pre-, and immediate post-activity. Additional questions assessed attitudes, barriers, and intended practice changes related to HER-positive mCRC. Results: Over 4,000 participants engaged in the educational activity, 67% of whom were physicians, and 63% noting their specialty as oncology. Approximately 68% identified as treaters seeing 14 patients with CRC per week with 78% of those patients have HER2-positive CRC. All pre-post questions showed significant improvements in knowledge and competence related to HER2 amplification testing methodologies, trial eligibility criteria, identification of patients who would benefit from anti-HER2 targeted regimens, and adverse events associated with anti-HER2 agents. Following the activity, 51% were more likely to incorporate testing for HER2 expression level or amplification prior to the selection of targeted therapy for their patients. The greatest challenges faced when managing patients with HER2-positive mCRC were affordability of therapy for patients (50%), adherence to treatment schedules (25%) and patient anxiety about treatment efficacy (24%). The most common barriers to patient enrollment in clinical trials identified by clinicians were lack of trials at their institutions (35%), lack of trials in their geographic region (21%), and patient lack of interest (20%). Qualitative insights on intended practice changes were shared by learners post-activity. Conclusions: The outcomes of this educational activity demonstrate the effectiveness of education in improving knowledge and confidence from testing to adverse event management to better integrate new regimens into practice. Encouraging practice changes were also seen from the write-in responses from learners. Additional needs were also identified to further solidify HER2 testing as a prerequisite for anti-HER2 targeted therapies, selecting eligible patients, and timely management of adverse events.

Clinical outcomes and molecular characteristics of patients with locoregional ampullary carcinoma.

732 Background: Ampullary carcinoma (AC) is a rare malignancy arising from the ampulla of Vater which has a more favorable prognosis compared to other pancreatic malignancies. Current guidelines favor surgical resection followed by adjuvant therapy for average risk patients (pts), however the type of regimen as well as other perioperative treatment modalities are still being explored. The objective of this retrospective study aims to provide insight into the management of locoregional disease. Methods: Pathology records from Mayo Clinic (AZ, FL, MN) denoting AC between 2010 to 2024 were searched using Mayo Data Explorer and selected for retrospective review. Pt demographics, treatment courses, and next generation sequencing (NGS) data were collected. Statistical analysis was conducted using SAS version 9.04. Results: A total of 137 pts (62% male, n = 85) were identified with median age 66 years old. Histologic subtypes were 54% pancreatobiliary (44/81), 40% intestinal (32/81), 6% mixed (5/81), with 56 pts unknown. Stages at diagnosis were 81% resectable (111/137), 11% locally advanced (15/137), and 8% metastatic (11/137). Nearly all (93%, n = 103/111) pts with localized disease had resection with a 97% R0 resection rate (100/103). Majority (68%, n = 75/111) of resectable pts had perioperative chemotherapy with 5-FU or gemcitabine-based regimens (24%, n = 18/75 neoadjuvant vs. 76%, n = 57/75 adjuvant). Perioperative chemoradiation was given in 17% (19/111) of pts. At median follow up of 30.5 months, recurrence rates were 40% (41/103), primarily to the liver (18/41). Median recurrence free survival (RFS) was 29.3 months (95% CI 24.7 – NE). Median overall survival (OS) has not been reached. The 5-year RFS and OS rates were 0.37 (95% CI 0.25 - 0.55) and 0.69 (95% CI 0.58 - 0.82), respectively. Univariate analysis showed no difference in OS with the addition of neoadjuvant or adjuvant chemotherapy or chemoradiation. Somatic NGS testing showed pathogenic mutations in 90% of pts (63/70): 57% KRAS (36/63; 44% G12D, 19% G12V, 8% G12C, 8% G12D, 19% others); 13% homologous recombination (HR) (8/63); 6% ERRB2 /Her2 amplification (4/63), and 5% mismatch repair (MMR) (3/63). Conclusions: AC has a favorable prognosis in resectable pts. However, high recurrence rates indicate the need for better systemic therapies and improved selection of pts who would benefit from these treatments. Future research should focus on refining perioperative strategies to enhance outcomes and reduce recurrence.

A phase Ib study of immunotherapy with ex-vivo pre-activated and expanded CBNK cells in combination with cetuximab, in patients with colorectal cancer (CRC) with minimal residual disease (MRD): Final report.

176 Background: Colorectal cancer (CRC) is the second most common type of cancer. Even following successful curative treatment, patients often relapse, likely because of undetected micro-metastatic foci. Tumor-informed ctDNA testing has helped to identify those patients before they developed radiographic evidence of disease, introducing the concept of minimal residual disease (MRD) in solid tumors. No standard-of-care treatment options are available for MRD patients. Therefore, we have designed a phase IB trial of the combination of in vitro pre-activated and pre-expanded cord blood natural killer (pre-A+E CBNK) cells with cetuximab. We hypothesized that we could achieve CAR-like efficacy without genetic engineering, by pre-activating NK cells with inflammatory cytokines and by using antibodies to redirect NK cell specificity and antibody-dependent cellular cytotoxicity. The highly immunosuppressive CRC tumor microenvironment is understudied, and novel NK-cell therapy could be highly effective in MRD clearance. Methods: 15 patients with CRC-MRD were enrolled in this trial and completed treatment. All the patients received immunodepleting treatment prior to an infusion of pre-A+E CBNK cells, up to a dose of 1x10^8/kg. Patients were monitored inpatient for 24 hours after treatment for infusion-related reaction, and were then seen for toxicity follow-up in the outpatient clinic twice/week for 4 weeks (DLT window). Samples for correlative analysis were collected at different times. Results: 8/15 patients enrolled were female and 7 were male, age range 26-69. They all had no measurable radiographic evidence of disease and a positive ctDNA Signatera test at baseline. 11/15 were previously treated for metastatic disease; 13 patients had a history of metastases (including 7 liver, 2 lymph nodal, 2 peritoneal). All of them received 5-FU-based chemotherapy. 7/15 were RAS/RAF WT, 4/10 were KRAS mut, 1/10 had BRAF mutation, 1/10 had HER2 amplification. They were all pMMR. No DLT was observed, only 1 patient had G2 CRS requiring tocilizumab. 9/15 patients treated so far had ctDNA clearance at some point. One patient had negative ctDNA at 3 and 6 months, 4/15 were negative at d28 and 7/15 at d14. 4/15 patients have not met the 90-day time point yet. 10/12 patients analyzed up to the primary endpoint had decreased ctDNA over baseline CBNK cells in blood peaked 3 hours post-infusion and persisted at 3 days. Phenotype analysis of CBNK pre-post, donor NK and other immune cells is ongoing. Conclusions: Cetuximab in combination with pre-A+E CBNK is safe and showed promising results for the treatment of MRD-CRC. Clinical trial information: NCT050468 .

Real-world impact of matched targeted therapy on survival in advanced biliary tract cancer: An international collaborative study.

536 Background: The prognosis of advanced BTC remains poor. Although targeted agents have expanded late-line options, many patients (pts) become ineligible for these therapies after progressing on first-line treatment. Herein, we examined the real-world impact of targeted therapy on survival in advanced BTC. Methods: In this international collaborative study between the Mayo Clinic (US) and the National Cancer Center East (Japan), we included pts with advanced BTC with comprehensive tumor molecular profiling who had received at least 30 days of systemic therapy. Pts were categorized into 3 groups: 1) non-actionable, 2) pts with alterations who received matched targeted therapy at any point during their treatment (matched group), and 3) those with mutations who did not receive matched therapy (unmatched group). OS was calculated from the start date of first-line therapy until death and compared among three subgroups using Cox regression models. Results: Of 932 pts, 366 (39.3%) had an actionable alteration, and 135 of these received matched therapy. 566 (61.7%) did not have an actionable alteration (Table). The most common alterations were somatic BRCA1/2 (N=106, 11.4%), HER2 amplification (N=65, 7%), FGFR2 fusion (N=61, 6.5%), KRAS G12C/D (N=46, 4.9%), and IDH1 (N=38, 4.1%). 78.7% of FGFR2 fusions were found in Caucasian pts, whereas BRCA1/2 (97.5%), HER2 (78.5%), and KRAS G12C/D (67.4%) alterations were more prevalent in Asian pts. The median OS was significantly longer in the matched group (21.4 months; 95% CI 18.4-27.9) compared to the unmatched group (14.6 months; 95% CI 12.8-17.6) and the non-actionable group (17.2 months; 95% CI 15.5-19.0). The 36-month OS rate was 33% in the matched group vs. 7% in the unmatched group (p < 0.0001). After adjusting for age, gender, prior surgical resection, primary tumor location, and country of origin, matched targeted therapy remained a strong independent predictor for improved OS (HR 0.61; 95% CI 0.48-0.79). Conclusions: These real-world findings suggest that matched targeted therapies significantly improve survival in advanced BTC, underscoring the need to incorporate them earlier in the treatment course and address barriers to treatment access. The regional prevalence of actionable alterations should also be considered when developing new targeted therapies. Patient baseline characteristics stratified by actionable alterations and targeted therapy. Non-actionable(N=566) Unmatched group(N=231) Matched group(N=135) Site, n (%) Japan 409 (72%) 166 (72%) 50 (37%) US 157 (28%) 65 (28%) 85 (63%) Age at Dx > 65, n (%) 324 (57%) 122 (53%) 54 (40%) Female, n (%) 235 (42%) 92 (40%) 77 (57%) Primary tumor location (category), n (%) Intrahepatic 217 (43%) 115 (55%) 99 (76%) Extrahepatic 150 (30%) 39 (19%) 17 (13%) Gallbladder 142 (28%) 54 (26%) 14 (11%) Prior surgical resection, n (%) 220 (42%) 74 (34%) 48 (37%)

Association between ex vivo pharmacotyping of patient-derived tumor organoids and personalized therapeutic options for patients with biliary tract cancer.

618 Background: Biliary tract cancers (BTC), including cholangiocarcinomas and gallbladder adenocarcinomas, present significant therapeutic challenges due to limited treatment options and poor prognoses. We report findings from the CLIA-certified PARIS assay, evaluating drug sensitivities of patient-derived tumor organoids (PDTOs) to a panel of oncology drugs. Methods: PDTOs were successfully cultured from 27 out of 46 live tumor samples obtained from 43 BTC patients. The majority of patients presented with advanced metastatic disease (60% stage IV, 13% stage III, 10% stage II, 4.4% stage I, 10% unknown) and had exhausted standard therapeutic options. Each PDTO culture underwent testing against an average of 50 drugs, encompassing chemotherapeutic agents and targeted therapies. Results: Comparative analysis of PDTO drug sensitivities with patients' prior treatment histories revealed non-responsiveness to >80% of drugs associated with clinical progression, including gemcitabine, cisplatin, and targeted therapies for FGFR translocations. In contrast, 100% (26/26) of the samples demonstrated significant sensitivity to one or more targeted agents, particularly inhibitors of EGFR, MEK, ERK, mTOR, PI3K, MDM2, BCL2, and BET families. Despite these shared sensitivities, each individual PDTO culture exhibited unique responses to targeted therapies, highlighting the genetic and phenotypic diversity between BTC patients. Comparison of ex vivo drug sensitivities with tumor genomic profiles validated oncogenic drivers such as HER2 amplification, KRAS and PIK3CA pathogenic mutations, correlating with sensitivities to EGFR/HER2 inhibitors, MEK inhibitors, and PI3K inhibitors, respectively. While mutations in KRAS , BRAF , BRCA1 , BRCA2 , ERBB2 , ERBB3 , or MET are present in less than 8% of cases, their role as biomarkers in BTCs requires further validation. PDTOs offer a promising tool to expedite this validation process. The PARIS assay results guided treatment decisions for five patients with metastatic disease, two of whom maintained treatment for over 4 weeks. Notably, two patients showed clinical benefit with everolimus (5 weeks) and dasatinib (11 weeks), experiencing symptom relief and reduced ascites. Intriguingly, 5 out of 7 PDTOs with FGFR alterations exhibited exceptional PARIS test responses to dasatinib, suggesting potential efficacy linked to FGFR activation. Conclusions: In conclusion, our study demonstrates that ex vivo drug testing of PDTO cultures can inform treatment selection and potentially accelerate drug approvals for BTC, leveraging therapies approved for other cancers. Early integration of such assays in patient management, possibly at diagnosis, holds promise for improving outcomes in this challenging disease.

A comprehensive molecular and clinical study of patients with young-onset CRC.

235 Background: Young-onset colorectal cancer (YO-CRC), defined as colorectal cancer diagnosed in individuals under 50 years of age, has emerged as a distinct clinical entity, often presenting at advanced stages. Despite the increasing incidence, molecular and clinical underpinnings of YO-CRC remain underexplored. This study aims to characterize the clinical and molecular features of YO-CRC and to evaluate their impact on OS. Methods: We retrospectively reviewed 110 patients diagnosed with YO-CRCfrom our institution’s molecular database. All patients underwent next-generation sequencing. Demographic, clinical, and molecular data, including age, gender, race, tumor location, cancer stage, and mutation status (KRAS, NRAS, BRAF, POLE, ERBB-2/HER2, microsatellite status), were collected by reviewing electronic medical records. For OS analysis, we focused on patients diagnosed with de novo Stage 4. Cox proportional hazards regression and Kaplan-Meier survival analysis were utilized to assess the association of these factors with OS, with statistical significance determined by a p-value threshold of <0.05. Results: Among 110 patients, N=44 (40%) presented with local disease (stage 1-3), while N=66 (60%) patients presented with de novo metastatic disease at the time of diagnosis. The median age at diagnosis was 44.5 years. The cohort consisted of 64% males and 36% females, with 84% of patients identified as White. Most tumors were left-sided (77%), including the distal colon/sigmoid (44%) and rectum (33%). KRAS and BRAF mutations were present 36% and 5.5%, respectively. ERBB-2/HER2 amplification and microsatellite instability were observed in 4.5% and 6.4% respectively. Tumor mutation burden (TMB) was <10 in 57% of patients, with 14% having TMB >20. CNV analysis revealed that 14% of patients had copy gains, 12% had concurrent gains/losses, and 31% had copy losses. Among the 66 Stage 4 patients, 44% had died by the time of analysis, with a median overall survival (OS) of 43.6 months (95% CI, 28.7 - not reached). KRAS mutation was found to be significantly associated with worse survival outcomes. Cox regression analysis reveals the prognostic significance of KRAS status, with a hazard ratio (HR) of 3.52 (95% CI: 1.59-7.76, P = 0.002 < 0.05), indicating a significantly higher risk of death for KRAS-mutant YO-CRC patients. Conclusions: KRAS mutations are a key prognostic factor in YO-CRC, highlighting the need for therapeutic need to improve outcomes in this high-risk group.

Amivantamab with or without chemotherapy in right-sided metastatic colorectal cancer: Updated results from OrigAMI-1, an open-label, phase 1b/2 study.

197 Background: Amivantamab (ami) is an EGFR-MET bispecific antibody with immune cell-directing activity and is FDA approved in EGFR -mutated advanced non-small cell lung cancer. High MET expression is observed in ~68% of patients (pts) with metastatic colorectal cancer (mCRC). Additionally, MET amplification occurs in up to 23% of EGFR-resistant mCRC and is implicated in driving resistance to EGFR-targeting antibodies (EGFRi). Compared to left (L)-sided disease, right (R)-sided disease is less responsive to EGFRi and associated with poorer outcomes. We present longer follow-up data among pts with R-sided mCRC. Methods: OrigAMI-1 (NCT05379595) is assessing ami as monotherapy and combined with chemotherapy in mCRC. All pts were wild-type for KRAS , NRAS , BRAF , and EGFR ectodomain by central ctDNA testing, without ERBB2 / HER2 amplification. One ami monotherapy cohort (Cohort C) enrolled only pts with R-sided disease (all pts must have 2-3 prior lines; prior EGFRi allowed). The ami plus chemotherapy cohorts (Cohorts D [with FOLFOX] and E [with FOLFIRI) enrolled both L- and R-sided disease (1 prior line max; prior EGFRi use was exclusionary). Primary tumor locations of cecum, ascending colon, hepatic flexure, and transverse colon were considered R-sided. Response was assessed by the investigator per RECIST v1.1. Results: As of 26-Aug-2024, 23 pts with R-sided mCRC received ami monotherapy (median follow-up of 8.1 months [mo]). Median number of prior lines was 2, and 43% had prior EGFRi. There were 7 pts with R-sided disease who received ami plus FOLFOX or FOLFIRI (median follow-up of 6.5 mo); all 7 had received 1 prior line. Among pts receiving ami monotherapy, objective response rate (ORR) was 22% (5/23) and disease control rate (DCR) was 78% (18/23), with 1 achieving a complete response. Median duration of response (DoR) is 7.4 mo; response and treatment are ongoing for 3 of the 5 responders. In pts receiving ami plus FOLFOX or FOLFIRI, ORR was 43% (3/7) and DCR was 86% (6/7). Median DoR is 5.8 mo, with all 3 responders on treatment, of which 2 are ongoing response. Additional details are in the Table. Biomarker data will be presented at the meeting. Safety profile among R-sided disease was consistent with prior reports, with the most common ami-related grade 3+ AEs being rash and hypoalbuminemia (2 pts each). Conclusions: Ami monotherapy or combined with FOLFOX or FOLFIRI demonstrated durable antitumor activity in R-sided mCRC. Clinical trial information: NCT05379595 . Efficacy. Ami monotherapy (n=23) Ami + FOLFOX or FOLFIRI (n=7) Median follow-up, mo (range) 8.1 (0.6–17.5) 6.5 (3.2–8.8) Median number prior lines 2 1 Prior EGFRi, n (%) 10 (43) 0 ORR, % (95% CI) 22 (8–44) 43 (10–82) Median DoR, mo (95% CI) 7.4 (3.7–NE) 5.8 (NE–NE) a DCR, % (95% CI) 78 (56–93) 86 (42–100) Median progression-free survival, mo (95% CI) 3.7 (3.4–5.5) 7.4 (1.8–NE) a All 3 responders remain on treatment; 2 of 3 responders are ongoing response.

Trastuzumab emtansine as second-line therapy in HER2 positive advanced biliary tract cancers: Single arm prospective phase II clinical trial (TAB-3 trial).

584 Background: HER2 overexpression or amplification is a therapeutic target of interest in advanced BTC, especially gallbladder cancers (GBC). Limited studies evaluate the role of T-DM1 in these groups of cancers. Methods: This study was an investigator-initiated, open-label, single-arm, phase II trial in patients (pts) aged 18 years or older with HER2-positive (defined as IHC 3+ or IHC 2+, and FISH positive), advanced BTCs who had previously received systemic therapy (irrespective of prior additional HER2 targeted therapy). Pts received T-DM1 at 3.6mg/kg q 3 -weekly till disease progression (PD), unacceptable toxicities, or patient choice. The primary end-point of the study was an improvement in 3-month progression-free survival (PFS) from 50% (historical cohort) to 70% in the study arm. Secondary endpoints included overall response rates (ORR), disease control rate (DCR), overall survival (OS), and incidence of grade 3 and 4 treatment-related adverse events (TRAE). Results: From July 2023 to July 2024, ‘52’ pts with HER2-positive BTC were screened, and 40 enrolled in the study. A majority of patients had GBC (95%) and most commonly received combination chemotherapy with gemcitabine-cisplatin, with or without nab-Paclitaxel (78%). Six patients (15%) had previously received trastuzumab in combination with chemotherapy. A majority of patients (n=24; 60%) had rapid progression (within 3 months) on prior systemic therapy. With a median follow-up of 7.1 months [95% confidence interval (CI): 5.1-9.1] , 24 patients had disease progression with a 3-month PFS of 51.2% (95% CI: 33.4 - 69) and median PFS of 3.1 months (95% CI: 2.3-3.8); median OS was 7.1 months (95% CI: 5.1-9.1). Partial responses (PR) were seen in 5 patients (12.5 %) and 8 had stable disease (20 %) for a disease control rate of 32.5 %. Patients without rapid disease progression on prior therapy (n=16; 40%) had a 3-month PFS of 70% (95% CI: 44.8 - 95.2) and median PFS of 4.9 months (95% CI: 2.9-6.9). Grade 3 and grade 4 TRAE in the overall cohort were noted in 11 patients (28%), with the commonest being grade 2 fatigue in 6 patients and grade 3 thrombocytopenia in 2 patients. Conclusions: T-DM1 was well tolerated in pre-treated HER2 positive advanced BTCs, but did not statistically improve PFS in comparison to historical data. Patients who did not have rapid progression on prior systemic therapy appeared to have a more favorable survival in comparison to rapid progressors and the role of T-DM1 in such favorable cohorts can be explored in larger studies. Clinical trial information: CTRI/2023/07/055785.

Impact of HER2-positivity on prognosis and targeted therapeutic outcomes in advanced biliary tract cancer.

629 Background: HER2 overexpression and amplification have been identified as a promising druggable target in biliary tract cancer (BTC). This study aims to characterize the clinical and molecular outcomes associated with HER2 positivity in BTC and evaluate the prognostic implications of HER2-targeted therapies. Methods: A retrospective study was conducted involving patients with advanced BTC diagnosed at Yonsei Cancer Center (YCC) and the University of Texas MD Anderson Cancer Center (MDACC) between 2009 and 2023. Patients were classified as HER2-positive either by immunohistochemistry (IHC 3+ or 2+/ISH+) or by next-generation sequencing (NGS, ERBB2 amplification). Overall survival (OS) and palliative first-line progression-free survival (PFS) were analyzed using Kaplan-Meier and Cox regression methods. Results: A total of 389 advanced BTC patients were initially screened (310 from YCC; 79 from MDACC). Among the 310 BTC patients from YCC with available HER2 IHC results, 78 (25.2%) were HER2-positive and 232 (74.8%) were HER2-negative. HER2 positivity was significantly more common in gallbladder cancer (55.1%) compared to its prevalence in intrahepatic or extrahepatic cholangiocarcinoma (22.8%, p<0.001). Of the 201 patients with available NGS data, 186 (92.5%) were ERBB2 non-amplified; among these, 27 (14.9%) were HER2-positive by IHC. For survival analysis, 310 patients were eligible: 239 from YCC (both HER2-positive and negative) and 71 from MDACC (all HER2-positive). Kaplan-Meier survival analysis indicated a trend towards shorter OS in HER2-positive patients compared to HER2-negative patients (median OS, 13.7 vs. 17.1 months, p=0.084, HR=1.246, 95% CI=0.97–1.60) regardless of exposure to HER2-targeted therapy, while first-line PFS on chemotherapy only was significantly shorter in the HER2-positive group (5.1 vs. 7.4 months, p<0.001, HR=1.906, 95% CI=1.46-2.48). Among HER2-positive patients, 56.8% received HER2-targeted therapy in the second line setting. HER2-positive patients who did not receive HER2-targeted therapy had a significantly poorer prognosis compared to HER2-negative patients (median OS 8.1 vs. 17.1 months, HR=2.46, 95% CI=1.73-3.48), whereas those receiving HER2-targeted therapy had comparable OS to HER2-negative patients (median OS 18.2 vs. 17.1 months, HR=0.95, 95% CI=0.71-1.27). Multivariable analysis identified a history of surgical treatment, radiation therapy, HER2 positivity, and HER2-targeted therapy as independent prognostic factors for OS. Conclusions: HER2 positivity is associated with a poorer prognosis in advanced BTC. However, HER2-targeted therapies improve outcomes, suggesting their potential inclusion in standard care for HER2-positive subgroups. Discrepancies between IHC and NGS in HER2 assessment emphasize the need for optimized testing guidelines in BTC.

Prognostic and biological divergence of upper gastrointestinal adenocarcinomas.

350 Background: Esophageal (EAC), gastroesophageal junction (GEJ) or gastric (GAC) adenocarcinoma share similar etiologies and treatment approaches. However, it is not clear if they possess similar or different prognostic and biological characteristics. We aimed to examine overall survival (OS) in associations of the common genomic alterations with these three anatomically closely linked adenocarcinomas. Methods: Eligible patients had recurrent or de novo metastatic GAC, GEJAC or EAC whose tumors had undergone next generation sequencing (NGS) performed from November 2017 to December 2023. We used Cox regression modeling to examine the association between GAC, GEJAC, EAC and OS, adjusting for demographics, performance status, Charlson comorbidity index, receipt of chemotherapy, and HER2 overexpression or amplification (HER2 positive), p53 (mutp53 ) , KRAS (mutKRAS), CDKN2A , PIK3CA co-mutations and MYC amplification. Results: Of 875 total eligible patients, 173 had EAC, 276 had GEJAC and 426 had GAC. GEJAC had substantially better OS than EAC (HR = 0.68, [95% CI, 0.54-0.86]), and modestly better OS than GAC (HR = 0.85, [95% CI, 0.67-1.09]). HER2 positivity was associated with substantially better OS among EAC (HR = 0.62; [95% CI, 0.40-0.96]) and GEJAC (HR = 0.59; [95% CI, 0.38-0.87]) but not among GAC (HR = 0.98; [95% CI, 0.78-1.23]) patients. In addition, p53 gain-of-function versus non-gain-of-function mutations were associated with substantially worse OS among GAC (HR = 1.41; [95% CI, 0.98-2.01]) but not among EAC or GEJAC. MutKRAS was associated with substantially worse OS among EAC (HR = 1.81; [95% CI, 1.10-2.99]) but not among GEJAC or GAC. Surprisingly, MYC amplification was associated with dramatically better OS among EAC (HR = 0.19; [95% CI, 0.08-0.42]) but substantially worse OS among GEJAC (HR = 1.98, 95% CI, 1.14-3.40]) and GAC (HR = 1.75; [95% CI, 1.10-2.80]). Conclusions: GEJAC, EAC and GAC possess substantially different OS that appears differentially associated with HER2 status, mutp53, mutKRAS and MYC amplification. These results suggest distinct prognostic and biological characteristics of these three anatomically closely linked adenocarcinomas that could have important implications in clinical practice and on further investigations on their biological and topological mechanisms.

Acquired gene alterations potentially related to resistance to anti-HER2 therapy in HER2-positive metastatic colorectal cancer (mCRC).

277 Background: While HER2-targeted therapies benefit patients (pts) with advanced HER2-positive mCRC, the development of resistance remains inevitable. This study aims to explore the landscape and frequency of genetic alterations putatively associated with acquired resistance to anti-HER2 therapy in HER2-positive mCRC. Methods: In this retrospective-multicenter international study involving Mayo Clinic, Duke Cancer Center and National Cancer Center Hospital East (Japan), we descriptively compared comprehensive genomic analyses from tissue or blood samples of pts with HER2-positive CRC pre-treatment and post-treatment with HER2-targeting agents. Results: A total of 36 pts were included in this study. 29 (80%) had HER2-positive mCRC as confirmed by immunohistochemistry (IHC) and fluorescent in situ hybridization (ISH), the remaining pts had HER2 amplification detected on blood or tissue . 34/36 (94%) pts were administered their first anti-HER2 agent after progressing on ≥2 prior lines of treatment. The most common anti-HER2 regimen administered was trastuzumab/pertuzumab (50%, 18/36), followed by trastuzumab-tyrosine kinase inhibitors (TKIs) (tucatinib or neratinib) (30%, 11/36) and trastuzumab deruxtecan (14%, 5/36). Putative genomic mechanisms of acquired resistance were detected in 72% (26/36), while the remaining pts lacked new genomic alterations that might explain resistance to anti-HER2 therapy, suggesting alternative escape mechanisms. Acquired alterations in downstream or alternate bypass pathways were identified in 72% (26/36) of pts; most commonly in the RAS/RAF pathway and in EGFR (34.6%, 9/26 each), followed by 27% (7/26) of pts with MET point mutations (mts) and amplification; activating muts in PI3K/AKT and in 27% (7/26), most commonly CDK12 (60%, 4/7) . HER2 receptor alterations were detected in 3 pts (8.3%), all of whom received trastuzumab-tucatinib and included HER2 L755S, HER2 V777L, HER2 D769Y, HER2 G727A and HER2 S310F, which are known to cause resistance to , however, information about tucatinib resistance is not yet available. Other altered pathways included AR, RB, NOTCH and HRD. Two pts lost HER2 expression on blood and tissue NGS post anti-HER2 therapy. Conclusions: The emergence of resistance might be in part related to acquired alterations that constitutively activate signaling pathways parallel or downstream of HER2, bypassing HER2 inhibition. Additional histologic studies, deep mutational scanning, larger populations and correlative analysis are needed to validate these findings.

The prognostic value of HER2 gene dosage and heterogeneity at a single-cell resolution in gastroesophageal adenocarcinoma.

473 Background: HER2 gene dosage (i.e. level of amplification) and heterogeneity are key prognostic variables for HER2+ gastroesophageal adenocarcinoma (GEA), yet no standardized approach to test these in routine clinical use exists. Here we modify the analytical pipeline for HER2 FISH to quantitatively estimate these prognostic variables and correlate them with clinical outcomes. Methods: Patients with metastatic HER2+ GEA who received trastuzumab-based therapy between 2011-2024 at Dana-Farber Cancer Institute and had available pre-treatment tissue for FISH analysis were included. HER2 FISH was conducted in a CLIA approved clinical pathology laboratory. Standard HER2/CEP17 ratio (an average of 50 cells) and individual cell HER2/CEP17 ratio were calculated. Kaplan-Meier method was used to estimate progression free (PFS) on trastuzumab-based therapy and overall survival (OS). Survival outcomes were adjusted for age, gender, tumor site, grade, prior resection, number of metastatic sites, and HER2 IHC using cox proportional hazard regression models. Results: A total of 77 patients were included. Median age was 60 years, 88% were male, and 92% had HER2 3+ tumors. Individual cells with HER2/CEP17 ratio ≥ 2.0 were defined as amplified (same cutoff as current bulk standard) and those with HER2/CEP17 ratio ≥ 4.95 (population median) were labeled highly HER2 amplified providing objective metrics to assess heterogeneity and gene dosage. Using these metrics we defined three subgroups: 1. Heterogenous (Het, n = 15): <75% cells HER2+; 2. Homogenous, low gene dosage (HL, n = 30): >75% cells HER2+, <50% cells with high HER2 amplification; 3. Homogenous, high gene dosage (HH, n = 32): >75% cells HER2+, >50% cells with high HER2 amplification. Cutoffs for both metrics were determined using data distribution patterns. The subgroups were prognostically relevant and PFS and OS were significantly lower for heterogenous tumors and numerically lower for homogenous tumors with low gene dosage after multivariable adjustment (Table). Conclusions: HER2 gene dosage and heterogeneity significantly impact outcomes in HER2+ GEA. More importantly by deconvoluting clinical HER2 FISH data at single cell level, we demonstrate an objective metric with prognostic value that could be adopted for routine clinical use and for patient stratification. Further insights into differing biology of these molecular subtypes are needed. Outcomes of patients by HER2 heterogeneity and gene dosage. Hazards ratio (HR) and p values are adjusted using multivariable cox proportional hazard regression model. PFS OS Patients, No. Median, mo HR (95%CI) P Value Median, mo HR (95%CI) P Value HH 32 16.2 1 16.4 1 HL 30 12.1 1.70 (0.79-3.66) 0.173 15.7 1.71 (0.93-3.14) 0.085 Het 15 6.7 3.12 (1.35-7.2) 0.008 12.4 2.72 (1.28-5.75) 0.009

Changes in HER2 Amplification Status for Breast Cancer Patients After Immunohistochemistry Directed In Situ Hybridization.

The 2018 ASCO/CAP guidelines for HER2 testing for breast cancer implemented the addition of immunohistochemistry (IHC) directed in situ hybridization (ISH) recount to resolve equivocal results. The implementation of an additional 2+ IHC-directed ISH recount adds additional complexity to the testing workflow for an unclear impact on HER2 results. A retrospective review of all equivocal ISH cases (groups 2, 3, and 4) that underwent 2+ IHC-directed ISH, since the 2018 guidelines, which were finalized as either amplified or not amplified, was performed. HER2 group number and final HER2 amplification status frequently changed after IHC guided ISH assessment, which was due to significant changes in HER2/CEP17 ratio and average HER2 signal number per cell. Equivocal groups 2, 3, and 4 samples with a result of HER2 amplified after 2+ IHC-directed ISH counts were closer to the threshold for amplification on the original ISH count, yet their counts also increased significantly after IHC-directed count in comparison to those samples, which were not amplified. Groups 2 and 4 ISH counts significantly increased after IHC directed ISH for HER2/CEP17 ratio and HER2 signal number per cell. This study represents the most extensive examination of efforts to resolve equivocal HER2 ISH results, highlighting a significant shift in therapeutic options after IHC-guided ISH for a subset of breast cancer patients.

Outcomes of Broader Genomic Profiling in Metastatic Colorectal Cancer: A Portuguese Cohort Study

Background: Colorectal cancer (CRC) is the third most diagnosed cancer globally and the second leading cause of cancer-related deaths. Despite advancements, metastatic CRC (mCRC) has a five-year survival rate below 20%. Next-generation sequencing (NGS) is recommended nowadays to guide mCRC treatment; however, its clinical utility when compared with traditional molecular testing in mCRC is debated due to limited survival improvement and cost-effectiveness concerns. Methods: This retrospective study included mCRC patients (≥18 years) treated at a single oncology centre who underwent NGS during treatment planning. Tumour samples were analysed using either a 52-gene Oncomine™ Focus Assay or a 500+-gene Oncomine™ Comprehensive Assay Plus. Variants were classified by clinical significance (ESMO ESCAT) and potential benefit (ESMO-MCBS and OncoKBTM). The Mann–Whitney and Chi square tests were used to compare characteristics of different groups, with significance at p < 0.05. Results: Eighty-six metastatic colorectal cancer (mCRC) patients were analysed, all being MMR proficient. Most cases (73.3%) underwent sequencing at diagnosis of metastatic disease, using primary tumour samples (74.4%) and a focused NGS assay (75.6%). A total of 206 somatic variants were detected in 86.0% of patients, 31.1% of which were classified as clinically significant, predominantly KRAS mutations (76.6%), with G12D and G12V variants as the most frequent. Among 33.7% RAS/BRAF wild-type patients, 65.5% received anti-EGFR therapies. Eleven patients (12.8%) had other actionable variants which were ESCAT level I-II, including four identified as TMB-high, four KRAS G12C, two BRAF V600E, and one HER2 amplification. Four received therapies classified as OncoKbTM level 1–2 and ESMO-MCBS score 4, leading to disease control in three cases. Conclusions: NGS enables the detection of rare variants, supports personalised treatments, and expands therapeutic options. As new drugs emerge and genomic data integration improves, NGS is poised to enhance real-world mCRC management.

HER2 Status in Gastric and Gastroesophageal Carcinomas: Evaluation of Histopathological Fingings, Paired ResectionBiopsy Specimens, and the Effect of Neoadjuvant Therapy: A Single Center Study.

Accurately determining the human epidermal growth factor receptor 2 (HER2) status is crucial in identifying suitable candidates for targeted therapy in gastric cancer, considering the cost and potential side effects of anti-HER2 treatments. This study aimed to assess HER2 overexpression/amplification prevalence in gastric and gastroesophageal cancer patients, its correlation with clinicopathological characteristics, and the consistency of HER2 status between biopsy and radical specimens. We analyzed data from 667 specimens of 600 gastric/gastroesophageal cancer patients at Dokuz Eylül University Faculty of Medicine from 2012 to 2021. The correlation of HER2 expression and amplification status and clinicopathological parameters were assessed. Furthermore, we compared HER2 status concordance between paired biopsy-radical specimens when both endoscopic and radical materials were present. Additionally, we compared HER2 status before and after treatment in patients receiving neoadjuvant chemotherapy. In our study, +3 HER2 immunohistochemistry results were more common in gastroesophageal junction tumors (23%). Human epidermal growth factor receptor 2 immunohistochemistry positivity and HER2 gene amplification were found to be significantly more common in males, people over 65 years of age, and intestinal morphology. Overall concordance of HER2 status between radical and biopsy materials was 95.5%. The high HER2 concordance between the paired biopsy and radical samples holds significant importance in clinical management. This finding is particularly noteworthy since the HER2 assessment can generally be conducted on small/limited biopsy materials in routine practice. Our study is the most extensive series from Türkiye and the Balkans, which will shed light on our country's data by investigating the relationship of HER2 with clinicopathological parameters in gastric/gastroesophageal carcinomas.

Trastuzumab Plus Pertuzumab Versus Cetuximab Plus Irinotecan in Patients With RAS/BRAF Wild-Type, HER2-Positive, Metastatic Colorectal Cancer (S1613): A Randomized Phase II Trial.

ERBB2 overexpression/amplification in RAS/BRAF wild-type (WT) metastatic colorectal cancer (mCRC; human epidermal growth factor receptor 2 [HER2]-positive mCRC) appears to be associated with limited benefit from anti-EGFR antibodies and promising responses to dual-HER2 inhibition; however, comparative efficacy has not been investigated. We conducted a randomized phase II trial to evaluate efficacy and safety of dual-HER2 inhibition against standard-of-care anti-EGFR antibody-based therapy as second/third-line treatment in HER2-positive mCRC. Patients with RAS/BRAF-WT mCRC after central confirmation of HER2 positivity (immunohistochemistry 3+ or 2+ and in situ hybridization amplified [HER2/CEP17 ratio >2.0]) were assigned (1:1) to either trastuzumab plus pertuzumab (TP; trastuzumab 6 mg/kg and pertuzumab 420 mg once every 3 weeks) or cetuximab plus irinotecan (CETIRI; cetuximab 500 mg/m2 and irinotecan 180 mg/m2 once every 2 weeks) until progression or unacceptable toxicity. Crossover to TP was allowed after progression on CETIRI. The primary end point was progression-free survival (PFS). Secondary end points included objective response rate (ORR), overall survival, safety, and HER2 gene copy number (GCN ≥20/<20) as a predictive factor. Between October 2017 and March 2022, 54 participants were assigned to TP (n = 26) and CETIRI (n = 28). Median PFS did not vary significantly by treatment: 4.7 (95% CI, 1.9 to 7.6) and 3.7 (95% CI, 1.6 to 6.7) months in the TP and CETIRI groups, respectively. Efficacy of TP versus CETIRI differed significantly by HER2 GCN (median PFS, GCN ≥20 [9.9 v 2.9 months] and GCN <20 [3.0 v 4.2 months], respectively; P interaction = .003). On TP, ORR was 34.6% (57.1% with GCN ≥20 v 9.1% with GCN <20) with median GCN of 29.7 versus 13.2 for responders and nonresponders, respectively (P = .004). Grade ≥3 adverse events occurred in 23.1% and 46.1% of participants with TP and CETIRI, respectively. TP appears to be a safe and effective cytotoxic chemotherapy-free option for patients with RAS/BRAF-WT, HER2-positive mCRC. Higher levels of HER2 amplification were associated with greater degree of clinical benefit from TP vis-à-vis CETIRI.

Deep learning-based prediction of HER2 status and trastuzumab treatment efficacy of gastric adenocarcinoma based on morphological features

BackgroundFirst-line treatment for advanced gastric adenocarcinoma (GAC) with human epidermal growth factor receptor 2 (HER2) is trastuzumab combined with chemotherapy. In clinical practice, HER2 positivity is identified through immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH), whereas deep learning (DL) can predict HER2 status based on tumor histopathological features. However, it remains uncertain whether these deep learning-derived features can predict the efficacy of anti-HER2 therapy.MethodsWe analyzed a cohort of 300 consecutive surgical specimens and 101 biopsy specimens, all undergoing HER2 testing, along with 41 biopsy specimens receiving trastuzumab-based therapy for HER2-positive GAC.ResultsWe developed a convolutional neural network (CNN) model using surgical specimens that achieved an area under the curve (AUC) value of 0.847 in predicting HER2 amplification, and achieved an AUC of 0.903 in predicting HER2 status specifically in patients with HER2 2 + expression. The model also predicted HER2 status in gastric biopsy specimens, achieving an AUC of 0.723. Furthermore, our classifier was trained using 41 HER2-positive gastric biopsy specimens that had undergone trastuzumab treatment, our model demonstrated an AUC of 0.833 for the (CR + PR) / (SD + PD) subgroup.ConclusionThis work explores an algorithm that utilizes hematoxylin and eosin (H&E) staining to accurately predict HER2 status and assess the response to trastuzumab in GAC, potentially facilitating clinical decision-making.

SLC26A9 promotes the initiation and progression of breast cancer by activating the PI3K/AKT signaling pathway.

SLC26A9 is a member of the Slc26a family of multifunctional anion transporters that function as Cl- channels in the stomach. We reported for the first time that SLC26A9 is involved in gastric tumorigenesis. However, the role of SLC26A9 in breast cancer has not yet been investigated. We first demonstrated that the upregulation of SLC26A9 is associated with the clinicopathological progression and poor prognosis of patients with breast cancer and is positively correlated with HER2 amplification. SLC26A9 alters the proliferation, migration, and invasion potential of breast cancer cells by regulating the PI3K/AKT signaling pathway. SLC26A9 acts as an oncogene in the development of breast cancer. These findings provide valuable insights for the development of future diagnostic and therapeutic strategies for BC.