HER2 Copy Number Research Articles

Anti-cancer effect of afatinib, dual inhibitor of HER2 and EGFR, on novel mutation HER2 E401G in models of patient-derived cancer

BackgroundPrecision medicine with gene panel testing based on next-generation sequencing for patients with cancer is being used increasingly in clinical practice. HER2, which encodes the human epidermal growth factor receptor 2 (HER2), is a potentially important driver gene. However, therapeutic strategies aimed at mutations in the HER2 extracellular domain have not been clarified. We therefore investigated the effect of EGFR co-targeted therapy with HER2 on patient-derived cancer models with the HER2 extracellular domain mutation E401G, based on our previous findings that this mutation has an epidermal growth factor receptor (EGFR)-mediated activation mechanism.MethodsWe generated a xenograft (PDX) and a cancer tissue-originated spheroid (CTOS) from a patient’s cancer containing an amplified HER2 E401G mutation. With these platforms, we compared the efficacy of afatinib, a tyrosine kinase inhibitor having anti-HER2 and anti-EGFR activity, with two other therapeutic options: lapatinib, which has similar properties but weaker EGFR inhibition, and trastuzumab plus pertuzumab, for which evidence exists of treatment efficacy against cancers with wild-type HER2 amplification. Similar experiments were also performed with H2170, a cell line with wild-type HER2 amplification, to contrast the characteristics of these drug’s efficacies against HER2 E401G.ResultsWe confirmed that PDX and CTOS retained morphological and immunohistochemical characteristics and HER2 gene profiles of the original tumor. In both PDX and CTOS, afatinib reduced tumor size more than lapatinib or trastuzumab plus pertuzumab. In addition, afatinib treatment resulted in a statistically significant reduction in HER2 copy number at the end of treatment. On the other hand, in H2170 xenografts with wild-type HER2 amplification, trastuzumab plus pertuzumab was most effective.ConclusionsAfatinib, a dual inhibitor of HER2 and EGFR, showed a promising effect on cancers with amplified HER2 E401G, which have an EGFR-mediated activation mechanism. Analysis of the activation mechanisms of mutations and development of therapeutic strategies based on those mechanisms are critical in precision medicine for cancer patients.

Molecular Basis of HER2-Targeted Therapy for HER2-Positive Colorectal Cancer.

Human epidermal growth factor receptor 2 (HER2) amplification has emerged as a biomarker in colorectal cancer (CRC), occurring in 1-4% of metastatic CRC (mCRC). In addition to conventional methods, such as immunohistochemistry and fluorescence in situ hybridization, next-generation sequencing-based tissue or circulating tumor DNA analysis has recently been used to identify HER2 amplification and assess HER2 overexpression. Prospective clinical trials have demonstrated the efficacy of HER2-targeted therapies in HER2-positive mCRC. The TRIUMPH study, a phase II study of dual HER2 antibodies, i.e., pertuzumab plus trastuzumab, demonstrated promising efficacy for patients with HER2-positive mCRC confirmed by tissue-and/or blood-based techniques, which led to the regulatory approval of this combination therapy in Japan. The mechanisms associated with efficacy and resistance have also been explored in translational studies that incorporate liquid biopsy in prospective trials. In particular, HER2 copy number and co-alterations have repeatedly been reported as biomarkers related to efficacy. To improve the therapeutic efficacy of the current strategy, many clinical trials with various HER2-targeted agents are ongoing. This review discusses the molecular basis of HER2-targeted therapeutic strategies for patients with HER2-positive mCRC.

Establishment and evaluation of digital PCR methods for HER2 copy number variation in breast cancer

Accurate measurement of human epidermal growth factor receptor 2 (HER2) copy number variation (CNV) is very important for guiding the tumor target therapy in breast cancer. Digital PCR (dPCR) is a sensitive and an absolute quantitative method, which can be used to detect HER2 CNV. Three HER2 exon-specific digital PCR assays along with three new reference genes assays (homo sapiens ribonuclease P RNA component H1 (RPPH1), glucose-6-phosphate isomerase (GPI), and chromosome 1 open reading frame 43 (C1ORF43), on different chromosomes) were established and validated by using standard reference material, 8 different cell lines and 110 clinical Formalin-fixed and paraffin-embedded (FFPE) samples. DPCR can achieve precise quantification of HER2 CNV by calculating the ratio of HER2/reference gene. The positive and negative coincidence rates were 98% (53/54) and 95% (53/56), respectively, compared with fluorescence in situ hybridization (FISH) diagnostic result 110 of FFPE samples. The common reference gene CEP17 used for FISH diagnostic was not suitable as single reference gene for HER2 CNV measurements by dPCR. The best practice of HER2 CNV determination by dPCR is to conduct the three duplex assays of H1 (HER2 exon 4) with the proposed three new reference genes, with a positive cut-off value of H1/RPPH1 ≥ 2.0 or H1/averaged reference gene ≥ 2.0. The proposed dPCR method in our study can accurately provide absolute copy number of HER2 and reference gene on an alternative chromosome, thus avoiding false negative caused by polysomy of chromosome 17. The improved molecular typing and diagnosis of breast cancer will better guide clinical medication.

HER2 Overexpression and Cytogenetical Patterns in Canine Mammary Carcinomas.

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that promotes tumor cell growth and is implicated in the pathogenesis of human breast cancer. The role of HER2 in canine mammary carcinomas (CMCs) is not clear. Therefore, this study aimed to examine the protein expression and cytogenetic changes of HER2 and their correlation with other clinical-pathological parameters in CMC. We retrospectively selected 112 CMCs. HER2, ER, and Ki67 were assessed by immunohistochemistry. HER2 antibody validation was investigated by immunoblot on mammary tumor cell lines. Fluorescence in situ hybridization (FISH) was performed with probes for HER2 and CRYBA1 (control gene present on CFA9). HER2 protein overexpression was detected in 15 carcinomas (13.5%). A total of 90 carcinomas were considered technically adequate by FISH, and 8 out of 90 CMC (10%) were HER2 amplified, 3 of which showed a cluster-type pattern. HER2 overexpression was correlated with an increased number of HER2 gene copies (p = 0.01; R = 0.24) and overall survival (p = 0.03), but no correlation with ER, Ki67, grade, metastases, and tumor-specific survival was found. Surprisingly, co-amplification or polysomy was identified in three tumors, characterized by an increased copy number of both HER2 and CRYBA1. A morphological translocation-fusion pattern was recognized in 20 carcinomas (22%), with a co-localized signal of HER2 and CRYBA1. HER2 is not associated with clinical-pathological parameters of increased malignancy in canine mammary tumors, but it is suitable for studying different amplification patterns.

167P The prognostic importance of high HER2 copy numbers and high HER2/CEP17 ratio in trastuzumab-treated early breast cancer

We aim to investigate the prognostic significance of HER2/CEP17 ratio, HER2 copy numbers, and tumor infiltrating lymphocytes (TILs) among early HER2+ breast cancer patients treated with HER2-targeted therapy.

1090P HER2 copy number variation in non-small cell lung cancer (NSCLC)

1090P HER2 copy number variation in non-small cell lung cancer (NSCLC)

1206P HER2 copy number variation (CNV), HER2 expression and primary resistance mechanisms in patients (pts) with HER2-positive metastatic gastric or gastroesophageal junction cancer (mGC/GEJC) receiving first-line chemotherapy (CT) + trastuzumab (T) +/- pertuzumab (P) in the JACOB trial

In JACOB trial, P added to T-CT did not significantly improve overall survival (OS) in pts with HER2+ mGC/GEJC despite a 3.3 mos increase vs placebo. We previously showed a negative prognostic role of AMNESIA panel, including KRAS/PIK3CA/MET mutations and KRAS/EGFR/MET amplifications (Pietrantonio et al, CCR 2018), whereas higher HER2 CNV may be associated with better outcomes on T. These biomarkers may allow to better identify pts with higher benefit from HER2 inhibition. Among 780 pts enrolled in JACOB and 580 with available baseline tumor DNA, 327 samples were sequenced by Oncomine Focus DNA assay. In a post-hoc analysis, HER2 CNV, HER2 expression by IHC and AMNESIA were correlated with overall response rate (ORR), progression-free survival (PFS) and OS by uni-/multivariable models. Median HER2 CNV was 4.7 (IQR 2.2-16.9). By pooling treatment arms, HER2-high vs low status using the median as the cut-off value was associated with longer PFS (mPFS: 10.5 vs 6.4 mos; hazard ratio (HR)=0.48, 95%CI: 0.38-0.62; p<.001) and OS (mOS: 20.3 vs 13.0 mos; HR=0.54, 0.42-0.72; p<.001). Combining HER2 CNV and IHC improved the discriminative ability, with better outcomes restricted to HER2-high/HER2 3+ subgroup. AMNESIA positivity was found in 51 (16%), with unadjusted HR=1.35 (0.98-1.86) for PFS; 1.43 (1.00-2.03) for OS. HER2-high subgroup was enriched for AMNESIA-neg and HER2 3+ status. In multivariable models with main variables including treatment arm, only HER2 CNV status remained statistically significant for PFS (p<.001) and OS (p=.004). Higher ORR was significantly associated with IHC 3+ [61% vs 34% in 2+; odds ratio(OR)=3.11 (1.89-5.17)] and HER2-high [59% vs 43% in HER2-low; OR=1.84 (1.16-2.94), with highest OR in the top CNV quartile. The selected biomarkers were not associated with activity and efficacy of P added to T-CT. HER2-high CNV assessed by NGS is associated with significantly better ORR, PFS, OS in a pts subgroup from the JACOB trial, independent from treatment arm and especially if combined with HER2 3+.

The frequency and clinical significance of centromere enumeration probe 17 alterations in human epidermal growth factor receptor 2 immunohistochemistry-equivocal invasive breast cancer.

Background and aimsChromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry‐equivocal score.Methods and resultsA large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in‐situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2‐amplified tumours with monosomy 17 (CEP17 copy number < 1.5 per nucleus), normal 17 (CEP17 1.5–< 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2‐non‐amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2‐amplified and HER2‐non‐amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2‐amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37–102.00; P = 0.02], and histological grade 3 in HER2 non‐amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61–19.00; P = 0.007).ConclusionThe impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry‐equivocal score who would benefit from NAT.

Abstract 5418: Comprehensive genomic profiling of HER2low breast carcinomas unveils unique genomic features and actionable genetic alterations

Abstract Introduction &amp; Purpose: “HER2low breast cancer” is an operational term identifying breast carcinomas (BCs) displaying a HER2 score 1+ or score 2+ in immunohistochemistry (IHC) and lacking HER2 amplification. This paradigm shift in HER2 assessment in BC stems from substantial antitumor activity of potent antibody-drug conjugates (ADCs) targeting the HER2 receptor on the tumor cell membrane. Whether these tumors constitute a distinct pathological entity is a matter of debate. Experimental procedures: To dissect the biology of HER2low BCs we performed a high throughput mutational analysis coupled with gene expression profiling on 98 HER2low BCs [34 cases score 1+ (1), 15 cases score 2+ HER2 not amplified (2N), 49 cases score 2+ with HER2 copy numbers in the equivocal range (2E)], of which 89 were estrogen receptor (ER)-positive, and 9 ER-negative. DNA and RNA were extracted and subjected to targeted DNA sequencing (Illumina TSO500 panel) and targeted gene expression analysis (NanoString BC360 panel). Mutation rates were compared with in silico data from 1317 samples of the MSK Breast Cancer cohort (PMID: 30205045). Results: The most frequently mutated genes were PIK3CA (33/98, 33%), GATA3 (18/98, 18%), TP53 (17/98, 17%), HER2 (8/98, 8%, private to 2E cases). Tumor mutational burden mean values were significantly higher in score 1+ compared to score 2+ cases (p= 0.04), with a single score 1+ tumor harboring microsatellite instability (MSI). Mutational signatures 1, 5 and 13 (APOBEC) were the most prevalent, with signature 30 being private to HER2 score 1+ cases. Comparative statistics revealed mutational drift of HER2low BCs from both HER2 negative (19 differentially mutated genes) and HER2 positive BCs (11 differentially mutated genes). Potentially actionable genetic alterations (OncoKB classes) accounted for 43 SNVs (predominantly HER2 and PIK3CA mutations) and 17 CNVs (e.g. MET amplification and PTEN homozygous deletions), including a MSI tumor, distributed over 47 independent patients.Subgroup stratifications based on gene expression were observed when considering genes involved in angiogenesis, stromal marker and cytokines signalling. HER2 mRNA levels were significantly higher in 2E versus 2N and in 2E versus 1. Differential gene expression Kegg pathway analysis identified RTK activation restricted to 2E cases. Conclusions: HER2low BCs harbour a different mutational landscape from ER- and grade- matched HER2 positive and HER2 negative BCs. Differences across IHC score classes within HER2low carcinomas were demonstrated, with the 2E category showing distinct features (private HER2 mutations, HER2 mRNA levels significantly higher compared to the other groups, RTK pathway activation). Of note, besides the option of ADCs we demonstrate that up to 48% of cases may be targeted with different approaches exploiting actionable alterations. Citation Format: Enrico Berrino, Laura Annaratone, Ivana Sarotto, Maria Cecilia Zecchillo, Laura Casorzo, Riccardo Ponzone, Filippo Montemurro, Anna Sapino, Caterina Marchio. Comprehensive genomic profiling of HER2low breast carcinomas unveils unique genomic features and actionable genetic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5418.

Characteristics of HER2 Gene Amplification by Fluorescence In Situ Hybridization in Endometrial Serous Carcinoma.

Targeted anti-human epidermal growth factor receptor 2 (HER2) therapy has recently become the standard for advanced-stage and recurrent HER2-positive endometrial serous carcinoma (ESC) in the United States, and an endometrial carcinoma-specific HER2 testing algorithm has been proposed. However, comprehensive studies on the specific features of HER2 gene amplification in these tumors are lacking. To evaluate the characteristics of HER2 amplification in ESC in the context of breast and gastric HER2 fluorescence in situ hybridization (FISH) guidelines. Ninety-four ESCs with available HER2 immunohistochemistry (IHC) and FISH were included. HER2 IHC was scored according to the proposed endometrial carcinoma-specific algorithm and FISH was evaluated by using the 2018 ESC clinical trial criteria, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2016 gastric criteria, and ASCO/CAP 2013 and 2018 breast criteria. Most tumors (90.4%; 85 of 94) had a 2+ HER2 IHC score. Polysomy of chromosome 17 was present in 16% (15 of 94) and monosomy 17 was seen in 2% (2 of 94) of tumors. HER2 FISH interpretation per the clinical trial criteria (HER2/CEP17 ratio ≥ 2.0) showed 99% concordance with the current gastric and breast HER2 FISH interpretations. Our results support the clinical trial criteria for HER2 FISH in ESC with a modification to include HER2 IHC 2+ and HER2/CEP17 ratio less than 2.0 and average HER2 copy number of 6.0 or greater in the HER2-positive category. Future prospective clinical investigations are necessary to assess the correlation between specific HER2 FISH result categories and therapeutic response.

Dynamic circulating tumor DNA (ctDNA) in monitoring trastuzumab deruxtecan (TDXd) activity for patients (pts) with advanced breast cancer: Preliminary results of a feasibility study.

1046 Background: Trastuzumab Deruxtecan (TDXd), a novel antibody-drug conjugate (ADC) that combines trastuzumab with a topoisomerase I inhibitor, has recently demonstrated high efficacy in HER2-overexpressing breast cancer after trastuzumab failure. Resistance to TDM1 have recently suggested to be dynamically associated with distinct circulating ctDNA species ( Allegretti et al, Mol Cancer 2021). A prospective study aiming to evaluate the feasibility of Liquid Biobsy (LB) in monitoring ctDNA in pts receiving TDXd in the national Expanded Access Program was conducted. Methods: In this prospective study, LB for ctDNA analysis (evaluating ‘ per pt’ ctDNA species and Variant Allelic Frequencies, VAF) was performed using 52-gene targeted NGS panel, in patients who progressed after two or more prior anti-HER2-based regimens and candidates to TDXd (3-weekly 5.4 mg/kg). This preliminary analysis reports data referring to Time-0 (T0) and T6 (cycle 6) assays. Results: From April 2021, LBs were collected for 14 pts and to date 8 are evaluable for LB. Median pts’ age was 59 yrs (range 38-72) and median number of previous anti-HER2 lines was 6 (2-11); 4 pts had Pertuzumab/Trastuzumab plus taxane as first-line and all pts received TDM-1. Median cycles of TDXd administered was 7.5 (1-10). At T0, 5/8 pts had at least one detectable ctDNA specie, and the remaining 3 developed at least one ctDNA at T6. ctDNA species and VAFs ranged from 1 to 5, and A0.1% to 68.94%, respectively. Decreases and increases were observed simultaneously in the same pt. The former varied from marginal to drastic, whereas the latter were invariably below 0.5% in VAFs. ctDNA monitoring was possible in 8/8 pts and at T6 ctDNA progression was detectable in 5/8 pts. Two pts displayed multiple HER2 copy number variations. Conclusions: The early results of this study suggest that considerable ctDNA burden, marked clonal complexity, and variable clonal response to TDXd can be found in pretreated HER2 positive patients, who progressed after antiHER2 therapies. Although the very small sample, this complex tumor evolution is surprising in light of the bystander payload effect of TDXd.

Copy number gains of chromosome 17 identified by dual in situ hybridization in non-small cell lung cancer tissue correlate with overexpression of c-Myc.

Backgroundc-Myc regulates multiple genes involved in cell proliferation in various cancer types including non-small cell lung cancer (NSCLC). Copy number gains of cytoband 17q25.3, along with chromosome 17, have been reported in NSCLC patients, emphasizing the clinical significance as a potential molecular target for therapy. The upregulation of c-Myc has been found to accelerate tumor development associated with duplication of the syntenic human cytoband 17q25.3. This study aimed to explore and compare the correlations of chromosome 17 copy number and c-Myc expression in NSCLC with the paired-normal respiratory epithelium and to examine their role as potential molecular targets for NSCLC therapy.MethodsA total of 66 NSCLC tissue samples with paired-normal respiratory epithelium were examined. The copy number of chromosome 17 was determined by human epidermal growth factor receptor 2 (HER2)/centromeric enumeration probe of chromosome 17 (CEP17) dual in situ hybridization (DISH).ResultsCopy number gains of chromosome 17 were identified in 8 of 60 (13.3%) available NSCLC specimens. No copy number gains of chromosome 17 were demonstrated in the paired-normal respiratory epithelium. The mean HER2 (1.2±0.3) and CEP17 (1.4±0.3) copy numbers of the normal respiratory epithelium were significantly lower than those of the NSCLC tissue [1.8±1.0 vs. 2.0±0.8, respectively (P<0.001)]. Twelve of 66 (18.2%) NSCLC patients had overexpression of c-Myc. Five (41.7%) of the patients whose tumors positive for c-Myc had HER2 gene amplification [1] or copy number gain of chromosome 17 [4]. HER2 gene amplification or copy number gain of chromosome 17 and high expression of c-Myc were associated with decreased overall survival.ConclusionsBoth biomarkers deserve further investigation to identify NSCLC patients with poorer survival outcomes requiring better therapeutic approaches.

RAI1 alternate probe identifies additional gastroesophageal adenocarcinoma cases as amplified following equivocal HER2 fluorescence in situ hybridization testing: experience from a national reference laboratory

The College of American Pathologists/American Society of Clinical Oncology recommends HER2 testing prior to initiation of targeted therapy for patients with advanced Gastroesophageal adenocarcinoma (GEA), using immunohistochemistry (IHC) followed by fluorescence in situ hybridization (FISH) in cases with an equivocal (score 2 + ) result on IHC. The FISH results are considered indeterminate if the HER2/CEP17 ratio is <2.0 with an average CEP17 copy number of ≥3.0 and a HER2 copy number ≥4.0 and ≤6.0 after counting additional tumor cells. Indeterminate results may be resolved by using an alternative chromosome 17 probe such as RAI1. The purpose of this study is to review our experience with RAI1 alternate probe in HER2 FISH testing of GEA in a large reference laboratory setting. Esophageal, gastroesophageal, and gastric adenocarcinomas received for HER2 FISH testing in our lab between 9/2018 and 1/2020 were included. HER2/CEP17 and HER2/ RAI1 ratios, and the average HER2, CEP17, RAI1 signals per cell were recorded. 328 GEA had HER2 testing performed in our lab during the study period. 101 (30.8%) were amplified, 169 (51.5%) were non-amplified and 58 (17.7%) were indeterminate. Following RAI1 testing, 42 (72.4%) of 58 indeterminate cases were reclassified as non-amplified and 16 (27.6%) were reclassified as amplified, increasing the total amplified cases to 117 (35.7%). The correlation between the average CEP17 and RAI1 copy number for all cases was weak (R2 = 0.095). In summary, using the alternate probe RAI1 reclassifies 27.6% of original HER2 FISH indeterminate gastroesophageal carcinomas as amplified, which makes them eligible for targeted therapies.

Six-year absolute invasive disease-free survival benefit of adding adjuvant pertuzumab to trastuzumab and chemotherapy for patients with early HER2-positive breast cancer: A Subpopulation Treatment Effect Pattern Plot (STEPP) analysis of the APHINITY (BIG 4-11) trial

AimThe APHINITY trial showed that adding adjuvant pertuzumab (P) to trastuzumab and chemotherapy, compared with adding placebo (Pla), significantly improved invasive disease-free survival (IDFS) for patients with HER2+ early breast cancer both overall and for the node-positive (N+) cohort. We explored whether adding P could benefit some N− subpopulations and whether to consider de-escalation for some N+ subpopulations. MethodsSubpopulation Treatment Effect Pattern Plot (STEPP) is an exploratory, graphical method that plots estimates of treatment effect for overlapping patient subpopulations defined by a covariate of interest. We used STEPP to estimate Kaplan–Meier differences in 6-year IDFS percentages (P minus Pla: Δ ± standard error [SE]), both overall and by nodal status, for overlapping subpopulations defined by (1) a clinical composite risk score, (2) tumour infiltrating lymphocytes (TILs) percentage, and (3) human epidermal growth factor receptor 2 (HER2) FISH copy number. Because of multiplicity, a Δ of at least three SE is required to warrant attention. ResultsThe average absolute gains in 6-year IDFS percentages were 2.8 ± 0.9 overall; 4.5 ± 1.2 for N+ and 0.1 ± 1.1 for N−. Largest gains were for patients with intermediate clinical composite risk (5.3 ± 1.9 overall; 6.9 ± 2.3 N+; 4.0 ± 3.0 N−), highest TILs percentage (6.3 ± 1.7 overall; 7.4 ± 2.4 N+; 3.2 ± 1.7 N−), and intermediate HER2 copy number (2.8 ± 1.9 overall; 7.4 ± 2.5 N+; −1.3 ± 1.9 N−), but clear evidence indicating a pattern of differential subpopulation treatment effects was lacking. ConclusionsSTEPP plots for N− did not identify subpopulations clearly benefiting from adding P, and those for N+ did not identify subpopulations warranting de-escalation. TILs percentage appeared to be more predictive of P treatment effect than clinical composite risk score. Trial registrationclinicaltrials.gov Identifier NCT01358877.

Association of HER-2/CEP17 Ratio and HER-2 Copy Number With pCR Rate in HER-2-Positive Breast Cancer After Dual-Target Neoadjuvant Therapy With Trastuzumab and Pertuzumab.

ObjectiveTo explore the correlation between HER-2 status and pathological complete response (pCR) in HER-2-positive breast cancer after dual anti-HER-2 neoadjuvant therapy with trastuzumab and pertuzumab.MethodsA total of 57 HER-2-positive breast cancer patients admitted to the Second Affiliated Hospital of Anhui Medical University and Xijing Hospital Affiliated to Air Force Military Medical University, between January 1, 2019 and September 30, 2020, were enrolled in this multicenter retrospective study. HER-2 status, including HER-2/CEP17 ratio and HER-2/cell number ratio, was detected by FISH. The correlation between HER-2 status/clinicopathological data and pCR was analyzed. The ROC curve was drawn to determine the cutoff value.ResultsIHC assessment revealed 40 (70.18%) patients with IHC 3+ and 17 (29.82%) with IHC 2+. 41/57 (71.93%) patients achieved pCR. FISH revealed that the ratio of HER-2/chromosome 17 centromere (HER-2/CEP17) (p<0.001) and the ratio of HER-2/cell number (p<0.001) was significantly correlated with the pCR rate. ROC analysis showed that patients with an HER-2/CEP17 ratio ≥4.495 or HER-2/cell number ≥11.650 have a high pCR rate after dual anti-HER-2 neoadjuvant therapy, suggesting its predictive significance.ConclusionThe response to dual-targeted neoadjuvant therapy with trastuzumab and pertuzumab was adequate in hormone receptor-negative, HER-2-positive breast cancer patients. HER-2/CEP17 ratio and HER-2/cell number ratio were crucial for predicting efficacy.

Abstract P3-09-05: Genomic and clinical characterization of breast tumors with unusual HER2 FISH pattern (ratio &amp;lt; 2, HER2 copy number ≥ 6): Are they mostly HER2 “positive?”

Abstract Background: Breast tumors with HER2/CEP17 fluorescent in situ hybridization (FISH) ratio &amp;lt; 2 and HER2 copy number ≥ 6, defined as Group 3 FISH pattern by the 2018 ASCO/CAP HER2 testing guidelines, are clinically rare. Their biologic and molecular characteristics are under-characterized. They require only a concomitant HER2 immunohistochemistry score of at least 2+ to merit HER2 “positive” status by the ASCO/CAP guidelines. We seek to characterize the genomic and tumor microenvironment landscape of breast tumors with this unique HER2 FISH pattern. Our second aim is to assess the clinicopathologic features with emphasis on HER2-targeted therapy response.Method: Breast cancers with Group 3 FISH pattern were evaluated by the following methods: 1) High-resolution genome-wide copy number alterations by molecular inversion probe (MIP) array; 2) molecular profiling of tumor immune microenvironment, tumor signaling pathways, and PAM50-based intrinsic subtypes by Nanostring nCounter Breast Cancer 360 Panel; 3) tumor infiltrating lymphocytes (TIL) histologic quantitation, and 4) clinical chart review. Classically amplified HER2 breast tumors (Group 1 FISH pattern; ratio ≥ 2 and HER2 copy number ≥ 2) were used as comparison. Results: Thirty-five (1.3%) cases were identified from 2731 consecutive clinical cases that underwent HER2 FISH testing from 2014 to 2019. Of those, thirteen consecutive cases (spanning 2014 - 2017) with sufficient genomic material were analyzed using MIP array. Group 3 tumors had a more complex karyotype and greater chromosomal instability, compared to classically amplified HER2 breast tumors. None of the Group 3 tumors showed HER2 locus amplification at 17q12. Instead, most showed gain of the 17q arm. Six of the Group 3 tumors were profiled by Nanostring nCounter. Compared to HER2 classically amplified tumors, Group 3 tumors were more immune cold, enriched in ER signaling and TGF-beta signaling pathways. In contrast, HER2 classically amplified tumors were enriched in immune infiltration, cytokine and chemokine signaling, PI3K and MAPK signaling, epithelial-mesenchymal transition signaling, and proliferation (P &amp;lt; 0.5 for all). PAM50 analysis showed that classically amplified tumors were more enriched for HER2-subtype (2/4; 50%), while the majority of the Group 3 tumors were enriched for Luminal B-subtype (5/6; 83%). TIL percentage was statistically higher in HER2 classically amplified tumors compare to Group 3 tumors (avg 53% vs 3%; P = 0.02). Clinicopathologic correlation revealed a high rate of ER positivity and high tumor grade in Group 3 tumors. Group 3 FISH pattern can occur as de novo or in the context of FISH status change following therapy. In the 17 evaluable patients for HER2-targeted treatment efficacy, none of the eight patients who received HER2-targeted neoadjuvant therapy achieved complete pathologic response. Nine of ten patients who received TDM-1 in the metastatic setting progressed with minimal treatment response. Significantly, most of these patients (16/17; 94%) were considered overall HER2 positive by the latest ASCO/CAP guideline. Conclusion: Breast tumors with Group 3 HER2 FISH pattern are molecularly and clinically dissimilar from classically amplified HER2 positive breast tumors. HER2-targeted therapy did not appear efficacious in either the neoadjuvant or metastatic/recurrent settings. The lack of apparent efficacy of HER2-targeted therapy, in the context of their HER2 positive status by the current HER2 guideline assessment, warrants further investigation of this HER2 FISH subtype. Citation Format: Christina H Wei, Lixin Yang, Daphne Stewart, Victoria Bedell, Daniel Schmolze, Sophia Apple, Joyce L. Murata-Collins, Raju Pillai, Joanne E. Mortimer. Genomic and clinical characterization of breast tumors with unusual HER2 FISH pattern (ratio &amp;lt; 2, HER2 copy number ≥ 6): Are they mostly HER2 “positive?” [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-05.

Abstract P1-02-03: Tumor-infiltrating lymphocytes but not HER2 copy number or ratio show prognostic value in trastuzumab-treated HER2-positive breast cancer

Abstract Background: Around 15% of all breast cancers are HER2+, a subtype that prior to HER2-targeted therapy was associated with a poor prognosis. With advances in treatment the survival has dramatically improved, and previous studies suggest that the level of HER2 copies are predictive of the effect of anti-HER2 therapies. However, some patients progress despite HER2-targeted therapy and routine HER2 testing is hampered by inaccuracy to discriminate patients with therapy-resistant disease. Furthermore, tumor-infiltrating lymphocytes (TILs) in the tumor stroma shows prognostic value not only in triple negative but also in HER2+ breast cancer. However, the clinical utility in the adjuvant setting for HER2+ disease needs further validation. The aim of this study was to investigate the prognostic significance of HER2/CEP17 ratio, HER2 copy numbers and TILs among early breast cancer patients treated with HER2-targeted therapy. Materials and methods: A retrospective study cohort comprising a total of 584 patients with HER2+ early breast cancer, all treated with the monoclonal antibody trastuzumab targeting HER2 during 2006-2014 in the Stockholm Region, Sweden, was identified and HER2 re-testing (silver in situ hybridization and immunohistochemistry (IHC)) was performed on 474 whole tissue sections prior to neoadjuvant (18.1%) or adjuvant therapy. Stromal TILs were manually assessed on all hematoxylin-eosin stained slides. Registry-based information on patient- and tumor characteristics including treatment were completed from medical records with a median follow-up of 8.0 years. Results: Among the 474 analyzed tumors, 11.4% were HER2 IHC score 2+ and 86.7% were 3+. In addition, 61.6% were ER+ and the median TIL score was 15%. The median HER2 copy number was 9.8 signals/cell and only 12.9% had &amp;lt;6.0 average HER2 signals/cell and 2.5% had a HER2/CEP17 ratio &amp;lt;2.0. Survival analysis showed no significant association between HER2 IHC score (recurrence: p=0.58; breast cancer specific death: p=0.69), HER2 signals (p=0.77; p=0.73), HER2/CEP17 ratio (p=0.73; p=0.85), ER status (p=0.55; p=0.11), PR status (p=0.97; p=0.46), Ki67 (p=0.14; p=0.77) or tumor grade (p=0.71; p=0.16) with risk of recurrence or breast cancer specific death. The hazard ratio (HR) for recurrence did not significantly differ by HER2 signals/cell (HR 1.12, CI 0.53-2.37, p=0.77) or ER status (HR 1.18, CI 0.68-2.05, p=0.55). Tumors with low TILs were independently associated with recurrence (cutoff &amp;lt;40%: HR 4.5, CI 1.4-14.9, p=0.012) and breast cancer specific survival (cutoff &amp;lt;30%: HR 3.5, CI 1.2-10.1, p=0.02). Among the 56 recurrences (11.8%), 37.5% had ER- primary tumors and 82.4% of the 34 HER2-analyzed recurrent tumors remained HER2+. Conclusions: This study demonstrates that routine clinicopathological data, including HER2 status, is insufficient to predict breast cancer recurrence among patients treated with HER2-targeted therapy. Neither HER2 levels, HER2/CEP17 ratio or ER status correlated to risk of recurrence. Importantly, TILs showed robust prognostic potential in trastuzumab-treated patients. Our results emphasize the need for detailed molecular analyses to understand the biology behind resistance to HER2-targeted therapy and to further tailor therapy for HER2+ breast cancer patients. Citation Format: Stephanie Robertson, Caroline Rönnlund, Irma Fredriksson, Theodoros Foukakis, Johan Hartman. Tumor-infiltrating lymphocytes but not HER2 copy number or ratio show prognostic value in trastuzumab-treated HER2-positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-03.

Abstract P2-13-11: Response to anti-HER2 neoadjuvant chemotherapy in invasive breast cancers with different HER2 FISH-positive patterns

Abstract Backgrounds: Since human epidermal growth factor receptor 2 (HER2)-positive breast cancers may have different HER2/CEP17 ratios and HER2 copy numbers, outcomes of HER2-positive breast cancer patients treated with anti-HER2 neoadjuvant chemotherapy (NACT) may be different. The aim of this study is to explore the relationship between different groups of HER2 fluorescence in situ hybridization (FISH) positive patterns and response to anti-HER2 NACT. Methods: 513 HER2-positive invasive breast cancers who received anti-HER2 NACT in Fudan University Shanghai Cancer Center, during January 2015 to September 2020, were collected. According to FISH results, 513 patients were divided into three groups. Group A: HER2/CEP17 &amp;lt; 2.0 and HER2 average copy number ≥6.0; Group B: HER2/CEP17≥2.0 and HER2 average copy number ≥4.0 and &amp;lt; 6.0; Group C: HER2/CEP17≥2.0 and HER2 average copy number ≥6.0. Clinicopathological characteristics and pathological complete response(pCR) rates of three groups were analyzed. Results: All 513 patients were treated with anti-HER2 NACT. The anti-HER2 treatment included trastuzumab in 463 (90.3%) patients, trastuzumab plus pertuzumab in 21 (4.1%) patients, trastuzumab plus lapatinib in 3 (0.6%) patients, and trastuzumab plus pyrotinib in 1 (0.2%) patient. 25 (4.9%) cases were unblinded in clinical trials, who were treated either with trastuzumab plus pertuzumab or with trastuzumab plus pyrotinib. Among 513 patients, 237 cases (46.2%)were luminal B (hormone receptor positive and HER2 positive) and 276 cases (53.8%) were hormone receptor negative and HER2 overexpressed (HER2 overexpression type). According to IHC results, cases with HER2 1+,2+ and 3+ were 8 (1.6%), 123 (24.0%) and 382(74.5%), respectively. Among them, 0.0%, 25.2%, and 48.7% achieved pCR (p&amp;lt;0.001). The pCR rate of HER2 overexpression type was higher than that of luminal B type (54.0% vs 28.7%, P&amp;lt;0.001). Lymph nodes with metastasis after NACT in luminal B type was higher than that of HER2 overexpression type (43.0% vs 21.4%, P&amp;lt;0.001). According to HER2-FISH results, 11 cases (2.1%) were group A, 28 cases (5.5%) were group B and 474 cases (92.4%) were group C. Compared with the pCR rate of group A (36.4%) and group C (44.5%), the pCR rate in group B (7.1%) was significantly lower (p&amp;lt;0.001). Conclusions: Among HER2-positive breast cancers, HER2 protein expression level was positively correlated with pCR rate. Luminal B(HER2+)patients benefited less from anti-HER2 NACT than HER2 overexpression patients. Although all were invasive breast cancers with positive HER2-FISH results, patients with HER2/CEP17≥2.0 and HER2 copy number ≥4.0 and &amp;lt;6.0 seemed to respond less favorably to anti-HER2 NACT compared with other groups. The biological characteristics of this group of patients are worthy of further study. Citation Format: Hong Lv, Qian-Ming Bai, Yin Liu, Zhong-Hua Wang, Ruo-Hong Shui, Hong-Fen Lu, Xiao-Li Xu, Bao-Hua Yu, Xiao-Yu Tu, Rui Bi, Yu-Fan Cheng, Xiao-Yan Zhou, Zhi-Min Shao, Wen-Tao Yang. Response to anti-HER2 neoadjuvant chemotherapy in invasive breast cancers with different HER2 FISH-positive patterns [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-11.

Abstract PD4-03: Characterization of HER2 amplification in the cerebrospinal fluid of patients with Leptomeningeal Disease in stage IV patients with breast cancer

Abstract Introduction. Breast cancers that metastasize to the central nervous system (BrM) and progress to leptomeningeal disease (LMD) are commonly HER2 amplified. Triple negative breast cancer (TNBC) also may also acquire HER2 amplification and/or copy number changes (HER2+) in BrM and LMD due to genetic heterogeneity. LMD has a dismal prognosis if untreated, but advances in anti-HER2 therapy have been highly effective for improving outcomes in patients with HER2+ LMD. Current methods for evaluating HER2 status in LMD are extremely limited. Cerebrospinal fluid (CSF) cytology is of limited sensitivity with poor cellular yield and preservation due to technical artifacts of preparation that severely limit evaluation of HER2 by FISH or IHC. We evaluated the ability of CNSide™ to selectively enrich CSF tumor cells (CSF-TCs) and assess HER2 gene amplification or copy number gain in breast cancer patients with LMD. Methods. CSF specimens from 77 unique breast cancer patients with suspected LMD were collected using CEE-Sure™ CSF collection tubes and analyzed using the CNSide assay, both produced by Biocept, Inc (San Diego CA). CSF tumor cells (CSF-TCs) stabilized at ambient temperature in CEE-Sure for shipping. In the CLIA laboratory CSF-TCs are labeled using a proprietary mouse anti-human antibody cocktail with secondary biotinylated anti-mouse Ig. All CSF cells are then captured in a streptavidin-coated micro-fluidic channel and fluorescently labeled with DAPI, streptavidin, keratin and CD45 to discriminate nucleated CSF-TCs that are SA+ and cytokeratin+, but CD45 negative. CSF-TCs are digitally imaged, localized and counted with specific X-Y coordinates in the channel that allow further characterization of the HER2 status of each cell using FISH probes for centromere 17 and HER2. Results. Of 77 unique patient CSF specimens, 74.0% (57/77) demonstrated the presence of metastatic tumor cells (defined as cells which were DAPI positive, streptavidin positive, and CD45 negative). HER2 cells ranged from fewer than 10 cells to tens of thousands of cells recovered from a single collection tube of CSF. Of the 57 specimens in which malignant cells were identified, 56.1% (32/57) demonstrated HER2 amplification by FISH (defined as HER2:CEP17 ≥ 2.0 or ≥ 6 HER2 signals) and 43.9% (25/57) did not demonstrate HER2 amplification by FISH. Ten (10) patients also had polysomy of chromosome 17 as well as other gene alterations. Conclusion: HER2 amplification is frequently found in breast cancer patients with LMD and can be reliably detected in tumor cells isolated from CSF. Evaluation of HER2 status in CSF-TCs allows treating physicians the opportunity to rapidly confirm the presence of this biomarker and select a more effective targeted therapy against HER2 in patients who present with life-threatening complications of breast cancer. Additional studies are needed to fully establish the clinical utility of CNSide when using various anti-HER2 therapies or therapy combinations to treat breast cancer patients with LMD. Citation Format: Michael Dugan, Deanna Fisher, Nathan Sweed, Barbara Blouw, Julie Mayer. Characterization of HER2 amplification in the cerebrospinal fluid of patients with Leptomeningeal Disease in stage IV patients with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD4-03.

Abstract P2-13-06: Clinical implication of HER2 status change after neoadjuvant chemotherapy with Trastuzumab and Pertuzumab (HP) in patients with HER2-positive breast cancer

Abstract Background: Patients with early HER2-positive breast cancer (BC) and residual disease after HER2-targeted neoadjuvant chemotherapy (NAC) are at high risk of recurrence. It is estimated that 10-30% of HER2-positive breast cancers change HER2 status after trastuzumab alone, but the effects of adding pertuzumab on this phenomenon and clinical outcomes remain unclear. We previously reported a high rate (~50%) of HER2 status change after HP in a small subset of patients. Herein, we present an updated analysis incorporating pathological review of additional cases.Methods: We identified patients with HER2-positive BC who received NAC with pertuzumab and trastuzumab (NAC-HP) followed by surgery at our institution between September 1, 2013 to November 1, 2019. Patients with HER2 status performed either at MSKCC or outside institutions were included. Change in HER2 status on residual disease from baseline was evaluated. We defined HER2 positivity as immunohistochemistry (IHC) IHC3+ or IHC0-2+ FISH amplified (ratio ≥ 2 or ratio &amp;lt; 2 and HER2 copy number ≥ 6 signals/cell). HER2-low was defined as IHC 1+ or 2+, FISH non-amplified. Disease free survival (DFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. Differences between patients with concordant and discordance HER2 status were assessed using the log-rank test.Results: Of 525 patients receiving NAC with HP, 229 (44%) patients had residual disease post NAC-HP. Among these 229 patients, 141 had both pre and post NAC-HP HER2 status available and were included in this analysis. HER2 status on biopsy specimens was determined at MSKCC in 35/141 (25%) and at external institution in 106/141 (75%). The majority of patients (84%) received dose-dense AC-THP; the remainder received TCHP or other HP-based regimens. Most (96%) of patients continued HP after surgery, and 2 patients received T-DM1. Of the 141 patients, 84/141(60%) were found to be HER2 concordant, while 57 (40%) were found to be HER2 discordant. In 13/57 (23%) patients, HER2 expression was lost (IHC 0), while in 44/57 (77%) patients, HER2-low profile was detected (IHC 1+ in 31, and IHC 2+, FISH non-amplified in 13). Further details are reported in the table. Patients with HER2 discordance after NAC-HP had similar survival outcome compared with patients who remained HER2 concordant (5-years DFS: 92.3% versus 88.7%, p=0.49 and 5-yr OS 93.6% versus 88.4%, p=0.70).Conclusions: In a single center cohort, discordant HER2 status after NAC-HP appeared frequently without statistically significant impact on survival outcome, although this finding may be due to the small size and hence low statistical power. Of these, HER2-low profile is the most frequent post treatment HER2 status change. This raises the possibility that patients with change in HER2 status may have heterogenous expression of HER2 at baseline, and HER2-loss or low sub-clones survive as residual disease due to the selection pressure of HP. Alternatively, anti-HER2 therapy may suppress HER2 expression in surviving cells. These findings could inform studies of tailored approaches in the post-neoadjuvant setting based on the biological profile of residual disease. Pre NAC-HP HER2 statusNPost NAC-HP HER2 statusNDiscordantN=57IHC 3+: 19IHC0: 4IHC1+: 9IHC2+ FISH not ampl: 6IHC 0-2+ FISH ampl: 38IHC0: 9IHC1+: 22IHC2+ FISH not ampl: 7Concordant N= 84IHC 3+: 59IHC 3+: 47IHC 0- 2+ FISH ampl: 12IHC 0-2+ FISH ampl: 25IHC 3+: 4IHC 0- 2+ FISH ampl: 21 Citation Format: Emanuela Ferraro, Anton Safonov, Hanna Y Wen, Edi Brogi, Mithat Gonan, Andrea V. Barrio, Pedram Razavi, Sarat Chandarlapaty, Shanu Modi, Andrew D. Seidman, Larry Norton, Mark E. Robson, Chau T. Dang. Clinical implication of HER2 status change after neoadjuvant chemotherapy with Trastuzumab and Pertuzumab (HP) in patients with HER2-positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-06.