HER2 Copy Number Research Articles

HER2 copy number determination in breast cancer using the highly sensitive droplet digital PCR method

Human epidermal growth factor receptor 2 (HER)-positive breast cancer (BC) is characterized by an aggressive clinical course. In the case of HER2 overexpression/amplification, patients benefit from HER2-targeting therapies. Standardized diagnostic HER2 assessment includes immunohistochemistry (IHC) and/or in situ hybridization (ISH). The aim of this study was to compare this “gold standard” with the Droplet Digital™ polymerase chain reaction (ddPCR), a method that allows sensitive and precise detection of copy number variations (CNV) in FFPE (formalin-fixed, paraffin-embedded) DNA samples. Partitioning of the PCR reaction into 20,000 droplets enables a precise quantitative “CN” discrimination also in heterogeneous samples. FFPE breast cancer samples (n = 170) with routinely assessed HER2 status by IHC/ISH were retrospectively analyzed using the ddPCR CNV ERBB2 assay. Comparison of HER2 status assessment by the two methods revealed concordant results in 92.9% (158/170) of the cases. Discrepant cases were verified and interpreted. For ddPCR, a cut off value of 3 HER2 copies was set to distinguish between HER2-negative and HER2-positive BC. Results obtained with the ddPCR CNV ERBB2 assay were consistent and reproducible, and serial dilutions demonstrated a high stability and sensitivity of the method. The ddPCR CNV ERBB2 assay may be a specific and convenient tool to quantify HER2 copy numbers in BC samples. In our study, this method showed high reproducibility in accuracy of HER2 assessment compared to IHC/ISH analysis.

Clinicopathologic Features of 2018 American Society of Clinical Oncology/College of American Pathologists Fluorescence In Situ Hybridization Group 3 Breast Carcinoma (Human Epidermal Growth Factor Receptor 2 Chromosome 17 Centromere Ratio

The American Society of Clinical Oncology/College of American Pathologists 2018 update of the human epidermal growth factor receptor 2 (HER2) testing guideline includes a fluorescence in situ hybridization (FISH) group with a HER2 to chromosome 17 centromere (CEP17) ratio less than 2.0 and HER2 copy number 6.0 or greater (group 3), which requires integrated review of HER2 immunohistochemistry (IHC). To assess the clinicopathologic features of group 3 patients and determine features associated with HER2-positive status after workup. Cases submitted for HER2 FISH between January 2019 and June 2022 were identified, and relevant clinicopathologic information was obtained. One hundred forty-two HER2 FISH cases (1.6%) were group 3. In 52 cases (36.6%) IHC was negative (0/1+), in 3 (2.8%) IHC was positive (3+), and in 86 (60.6%) IHC was 2+. Annotated IHC 2+ slides were recounted by a second reviewer in targeted areas, where 16 of 86 (18.6%) had a HER2:CEP17 ratio less than 2.0 and a HER2 copy number of 4.0 or greater to less than 6.0 (HER2 negative). After combined IHC/FISH review, 74 of 142 (52.1%) were classified as HER2 positive. HER2 copy number/cell was higher in HER2-positive compared with HER2-negative cases after the workup. The extent and intensity of staining in IHC 2+ cases did not correlate with the level of gene amplification. Twenty percent of HER2-positive patients achieved pathologic complete response. About half of group 3 cases were classified as HER2 positive after additional workup. Pathologic complete response rates in HER2-positive cases were lower than expected for group 1 (HER2:CEP17 ratio ≥2.0; HER2 copy number ≥4.0) patients. IHC-targeted FISH recounts may be redundant and may potentially lead to classification of some patients as HER2 negative, resulting in withholding of targeted therapy.

Liquid biopsy based HER2 amplification status in gastric cancer patients indicates clinical response

Gastric carcinomas are among the most common cancers in Germany, with approximately 18,000 new cases per year.About 10 years ago, based on results of the Trastuzumab for gastric cancer (ToGA) trial, the addition of the monoclonal antibody trastuzumab to a platinum-fluoropyrimidine chemotherapy backbone became the standard-of-care 1st-line therapy for human epidermal growth factor receptor 2 (HER2)-positive gastric cancers. Only patients with primary HER2 gene amplification benefit from this therapy. Thus, accurate HER2 gene amplification detection is predictive and critical for therapy selection. As a gold standard the HER2 status is currently determined in tumor tissue specimens using immune histochemistry and fluorescent in situ hybridisation. However, HER2 amplification is detectable in only about 20 % of gastric carcinomas. The recent approval of an antibody-drug conjugate Trastuzumab deruxtecan (T-DXd) and the establishment of a new subgroup of HER2-low tumors due to the bystander effect associated with T-DXd increases the relevance of precise HER2 diagnostics.Aim of this analysis was to determine the HER2 amplification status from circulating DNA fragments in blood using a HER2 Copy Number Variation assay to establish a minimal invasive approach.For the present study, a digital droplet PCR-based method was validated relative to established tissue-based methods. Furthermore and most importantly, the changes of HER2 status during therapy were investigated in seven patients indicating that the changes of HER2 status and number of HER2 copies detected in blood can reflect on therapy efficiency and uncover treatment resistance.

Efficacy of neoadjuvant therapy on HER2-positive breast cancer: a clinicopathological analysis

Objective: To investigate the efficacy of neoadjuvant therapy (NAT) on HER2-positive breast cancer and to analyze their clinicopathological features. Methods: A total of 480 cases of HER2-positive breast cancer who received neoadjuvant therapy (NAT), diagnosed at the Department of Pathology of Fudan University Shanghai Cancer Center from 2015 to 2020, were retrospectively identified. Clinicopathological parameters such as age, tumor size, molecular subtype, type of targeted therapy, Ki-67 proliferation index, ER and HER2 immunohistochemical expression, and HER2 amplification status were analyzed to correlate with the efficacy of NAT. Results: Among 480 patients with HER2-positive breast cancer, 209 achieved pathology complete response (pCR) after NAT, with a pCR rate of 43.5%. Of all the cases,457 patients received chemotherapy plus trastuzumab and 23 patients received chemotherapy with trastuzumab and pertuzumab. A total of 198 cases (43.3%) achieved pCR in patients with chemotherapy plus trastuzumab, and 11 cases (47.8%) achieved pCR in patients with chemotherapy plus trastuzumab and pertuzumab. The pCR rate in the latter group was higher, but there was no statistical significance. The results showed that the pCR rate of IHC-HER2 3+patients (49%) was significantly higher than that of IHC-HER2 2+patients (26.1%, P<0.001). The higher the mean HER2 copy number in the FISH assay, the higher the pCR rate was achieved. The expression level of ER was inversely correlated with the efficacy of NAT, and the pCR rate in the ER-positive group (28.2%) was significantly lower than that in the ER-negative group (55.8%, P<0.001). The pCR rate (29.1%) of patients with luminal B type was lower than that of HER2 overexpression type (55.8%, P<0.001). In addition, higher Ki-67 proliferation index was associated with higher pCR rate (P<0.001). The pCR rate was the highest in the tumor ≤2 cm group (57.7%), while the pCR rate in the tumor >5 cm group was the lowest (31.1%). The difference between the groups was significant (P=0.005). Conclusions: HER2 copy numbers, HER2 immunohistochemical expression level, molecular subtype, ER expression level and Ki-67 proliferation index are significantly associated with pCR after NAT. In addition, fluorescence in situ hybridization results, HER2/CEP17 ratio and tumor size could also significantly affect the efficacy of NAT.

Correlation of In Situ HER2 RNA Expression With HER2 Immunohistochemistry and Fluorescence In Situ Hybridization Categories in Breast Cancer.

RNA sequencing study has demonstrated that human epidermal growth factor receptor 2 (HER2) RNA levels influence anti-HER2 therapeutic efficacy. However, in situ HER2 RNA expression (isHRE), which evaluates HER2 RNA expression in tissue, has remained unclear in breast cancers (BCs) of various HER2 immunohistochemistry (IHC)/in situ hybridization (ISH) categories. To correlate isHRE with all HER2 IHC/fluorescence ISH (FISH) categories in BC. Formalin-fixed, paraffin-embedded tissue sections from 259 BCs, covering all IHC/FISH categories, were analyzed for isHRE by RNAscope. We validated HER2 RNAscope scoring as a semiquantitative method to evaluate isHRE and demonstrated significantly higher RNAscope scores in IHC 3+ than in IHC 2+ cases, and in IHC 2+ than in IHC 0/1+ cases. Among the 5 IHC 2+/FISH groups, group 1 (G1) cases had the highest scores. The scores in G3 cases were higher than those in G2, but not significantly different from those in G4 and G5. G4 cases had significantly higher scores than those in G2. Higher HER2 copy numbers and HER2:CEP 17 (centromere 17) copy number ratios were significantly correlated with higher isHRE in G1 cases, but not in G2 to G5 cases. RNAscope scores were significantly lower in HER2-negative (IHC 0) than in HER2-low (IHC 2+/FISH- and IHC 1+) BCs but were not different between IHC 0 and 1+ BCs when analyzed separately. We demonstrate the HER2 RNA expression status among BCs of various HER2 IHC/FISH categories in tissue. Such information may be relevant for anti-HER2 treatment decisions considering the role of HER2 RNA expression in predicting anti-HER2 therapeutic efficacy.

HER2 Genetic Intratumor Heterogeneity Is Associated With Resistance to Trastuzumab and Trastuzumab Emtansine Therapy in Recurrent High-Grade Endometrial Cancer

Anti-HER2 targeted therapies have recently demonstrated clinical activity in the treatment of high-grade endometrial carcinomas (ECs), particularly serous carcinomas with HER2 amplification and/or overexpression. Intratumor heterogeneity of HER2 amplification or HER2 genetic intratumor heterogeneity (G-ITH) has been associated with resistance to anti-HER2 therapies in breast and gastroesophageal cancers; however, its clinical relevance in EC is unknown. To characterize HER2 G-ITH in EC, archival specimens from a clinically annotated cohort of 57 ECs treated with trastuzumab or trasutuzmab emtansine in the recurrent (n = 38) or adjuvant (n = 19) setting were subjected to central pathology review, HER2 assessment by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and next-generation sequencing. HER2 G-ITH, defined as HER2 amplification in 5% to 50% of tumor cells examined by FISH, was identified in 36% (19/53) of ECs and was associated with lower HER2 copy number and levels of protein expression. HER2 IHC revealed spatially distinct areas of strong expression juxtaposed with areas of low/absent expression in tumors with the “cluster” pattern of G-ITH, whereas the “mosaic” pattern was typically associated with a diffuse admixture of cells with variable levels of HER2 expression. HER2 G-ITH was frequently observed in cases with IHC/FISH or FISH/next-generation sequencing discrepancies and/or with an equivocal/negative FISH result (9/13, 69%). Although the objective response rate to anti-HER2 therapy in recurrent ECs was 52% (13/25) for tumors lacking HER2 G-ITH, none (0%, 0/10) of the patients with HER2 G-ITH achieved a complete or partial response (P = .005). HER2 G-ITH was significantly associated with worse progression-free survival (hazard ratio, 2.88; 95% CI, 1.33-6.27; P = .005) but not overall survival. HER2 IHC score, HER2/CEP17 ratio, HER2 copy number, histologic subtype, and other genetic alterations, including PIK3CA hotspot mutations, were not significantly associated with therapeutic response or survival outcomes. Treatment responses were not restricted to serous carcinomas, supporting consideration of anti-HER2 therapy in patients with HER2-positive high-grade ECs of non-serous histology. Our results demonstrate that HER2 G-ITH is an important determinant of response to trastuzumab and trastuzumab emtansine in EC, providing a rationale for the development of novel therapeutic strategies to target HER2-nonamplified resistant tumor subpopulations, such as HER2 antibody-drug conjugates with bystander effects.

Improved concordance of challenging human epidermal growth factor receptor 2 dual in-situ hybridisation cases with the use of a digital image analysis algorithm in breast cancer.

Accurate assessment of human epidermal growth factor receptor 2 (HER2) expression by HER2 immunohistochemistry and in-situ hybridisation (ISH) is critical for the management of patients with breast cancer. The revised 2018 ASCO/CAP guidelines define 5 groups based on HER2 expression and copy number. Manual pathologist quantification by light microscopy of equivocal and less common HER2 ISH groups (groups 2-4) can be challenging, and there are no data on interobserver variability in reporting of these cases. We sought to determine whether a digital algorithm could improve interobserver variability in the assessment of difficult HER2 ISH cases. HER2 ISH was evaluated in a cohort enriched for less common HER2 patterns using standard light microscopy versus analysis of whole slide images using the Roche uPath HER2 dual ISH image analysis algorithm. Standard microscopy demonstrated significant interobserver variability with a Fleiss's kappa value of 0.471 (fair-moderate agreement) improving to 0.666 (moderate-good) with the use of the algorithm. For HER2 group designation (groups 1-5), there waspoor-moderate reliability between pathologists bymicroscopy [intraclass correlation coefficient (ICC) = 0.526], improving to moderate-good agreement (ICC = 0.763) with the use of the algorithm. In subgroup analysis, the algorithm improved concordance particularly in groups 2, 4 and 5. Time to enumerate cases was also significantly reduced. This work demonstrates the potential of a digital image analysis algorithm to improve the concordance of pathologist HER2 amplification status reporting in less common HER2 groups. This has the potential to improve therapy selection and outcomes for patients with HER2-low and borderline HER2-amplified breast cancers.

HER2 in situ hybridization testing in breast cancer: Applying algorithm-assisted assessment to reduce interobserver variability in difficult cases.

e13561 Background: Accurate assessment of HER2 expression by HER2 immunohistochemistry and in-situ hybridization (ISH) is critical for the management of patients with breast cancer. The revised 2018 ASCO/CAP guidelines defined 5 subgroups based on HER2 expression and copy number. Few reports have investigated interobserver variability when applying the revised guidelines. Digital algorithms may have a role in assisting pathologists in assessing cases with equivocal and uncommon patterns of HER2 amplification. This study was designed to evaluate interobserver variability in the gold standard manual assessment of HER2 ISH, particularly in less common HER2 ISH groups. Subsequently, we evaluated whether a new digital image analysis algorithm (Roche uPath HER2 Dual ISH image analysis) could reduce interobserver variability and potentially improve patient selection for targeted anti-HER2 therapy. Methods: Of 2982 breast cancer cases, we derived an enriched cohort of 99 patient cases with HER2 IHC 2+ or 3+ and selective ISH results (groups 2-4, evidence of HER2 heterogeneity, borderline negative group 5 and borderline positive group 1). As per guidelines, a minimum of 20 tumour cells per slide were manually and independently counted by 4 anatomical pathologists. After a washout period and second round of de-identification, the slides were scanned (DP200; Roche) and re-scored using the digital algorithm. Results: Pathologist reporting of patient HER2 status (HER2 positive vs. HER2 negative) using manual microscopy demonstrated significant interobserver variability, with a Fleiss’s kappa value of 0.471 (95% CI: 0.378-0.564; P= 1.24E-21) indicating fair-moderate agreement. Interobserver variability in manual scoring of HER2 ISH at the level of HER2 group designation (groups 1-5) showed poor-moderate reliability between pathologists, with an intraclass correlation coefficient (ICC) of 0.526 (95% CI: 0.407-0.641; P= 9.28E-21). Repeat analysis of the cohort using algorithm-assisted quantification resulted in moderate-substantial concordance in HER2 patient status reporting, with a Fleiss’s kappa value of 0.666 (95% CI: 0.572-0.759; P= 1.70E-40). Algorithm-assisted quantification resulted in moderate-good agreement of HER2 group designation, with an ICC of 0.763 (95% CI: 0.683-0.832; P= 8.76E-48). In sub-group analysis, use of the algorithm improved concordance particularly in groups 2,4 and 5. Time to report cases was also significantly reduced with the use of the algorithm. Conclusions: Our study demonstrates the potential to improve the concordance of HER2 amplification status reporting in less common HER2 subgroups using a digital algorithm and potentially improve therapy selection and outcomes for patients with HER2-low and borderline HER2-amplified breast cancers.

Metastatic breast cancer (MBC) with ultra-high tumor mutational burden (UHTMB): A comprehensive genomic profiling (CGP) study.

1036 Background: Pts with MBC whose tumors feature high TMB (≥ 10 mutations/Mb) are eligible for on label immune checkpoint inhibitor (ICI) treatment. This study evaluated the genomic landscape of MBC with “Ultra high” TMB, defined at &gt; 20 mutations/Mb. Methods: 2049 MBC underwent hybrid capture-based CGP to evaluate all classes of genomic alterations (GA), TMB, microsatellite instability (MSI) and trinucleotide mutational signatures. HER2 IHC results were available in a subset of pts. PD-L1 expression on immunocytes was determined by IHC (Ventana SP142). Results: 45/2049 (2.2%) of MBC were UHTMB. 45 (100%) pts had metastatic disease. 38 (84%) had documented Stage IV disease and 7 documented axillary LN metastases at the time of sequencing. Local breast tumor was used for CGP in 19 (42.2%) MBC and metastatic site biopsy was used in 26 (57.8%). When compared with 2004 non-UHTMB pts with UHTMB were older (mean 64.6 yrs vs 58.2 yrs p &lt; .0001), more often had lobular histology (40.0% vs 14.5% p &lt; .0001) and ER+ disease (86.6% vs 70.0%). Of the 35 UHTMB cases with HER2 IHC data available, 11 (31.4%%) were HER2 IHC negative (0+), 21 (60.0%) were HER2-low status (9 1+ and 12 2+/ISH negative) and 3 (8.6%) were HER2 IHC positive (3+). 1/3 HER2 IHC2+ cases and 2/45 (4.4%) of all UHTMB cases were positive for HER2 copy number gain on CGP. UHTMB cases had more driver GA/tumor (mean 9.8 vs 5.7 p &lt; .0001) and were less often TNBC (13.3% vs 27.0% p = .041) compared to non-UHTMB high cancers. Mutation signature analysis revealed APOBEC was predominant in UHTMB samples (82.5%); MMR signature was also observed in 10% of cases. MSI high status was significantly more frequent in UHTMB high cases (11.6% vs 0.4% p &lt; .0001). GA more frequently identified in UHTMB cases included CDH1 (45.5% vs 14.3% p &lt; .0001), PIK3CA (81.8% vs 37.9% p &lt; .0001), CDKN2A (11.4% vs 3.2% p = .017), ARID1A (25.0% vs 5.0% p &lt; .0001) and NF1 (20.5% vs 5.9% p = .0014). PD-L1 ( CD274) gene amplification (2.3% vs 1.3%) or protein expression by the Ventana SP142 assay (57.14% vs 51.10%) were not significantly different among groups. Conclusions: UHTMB MBC is a rare, yet clinically important subset of clinically advanced breast cancer driven by APOBEC mutagenesis, with high incidence of ER+ lobular histology and frequent alterations in CDH1 and PIK3CA. In addition to potential benefit from ICI based treatment, UHTMB MBC present with a high frequency of HER2-low status which may impact therapy decisions for this rare disease. [Table: see text]

Tissue-agnostic efficacy of trastuzumab deruxtecan (T-DXd) in advanced solid tumors with HER2 amplification identified by plasma cell-free DNA (cfDNA) testing: Results from a phase 2 basket trial (HERALD/EPOC1806).

3014 Background: HER2 ( ERBB2) gene amplification (amp) is a potential therapeutic target beyond breast and gastric cancers. HER2 amp can be detected in plasma cfDNA which may be an alternative to tissue biopsy. HERALD/EPOC1806 was a multicenter, investigator-initiated phase 2 trial of T-DXd for patients with HER2-amplified advanced solid tumors identified by cfDNA testing. Methods: We enrolled adults with advanced solid tumors harboring HER2 amp detected by next-generation sequencing of cfDNA (Guardant360) in the GOZILA study (UMIN000029315). We excluded breast or gastric cancer patients with HER2 overexpression (IHC 3+ or IHC 2+/ISH+). Patients received T-DXd 5.4 mg/kg every 3 weeks until disease progression or unacceptable toxicity. The primary endpoint was investigator-assessed objective response rate (ORR). Results: From December 2019 to January 2022, 4,734 patients were screened by cfDNA in the GOZILA study. Among 252 with HER2 amp, 62 with 16 cancer types were enrolled in HERALD. Median baseline plasma HER2 copy number (CN) was 8.55 (range, 2.4–73.9). All patients but 3 (all with salivary gland cancer) received prior anti-cancer therapy (median, 3 lines; range, 0–8). At a median follow-up of 8.9 months (data cut-off: July 17, 2022), the confirmed ORR was 56.5% (95% CI, 43.3–69.0%), statistically higher than the threshold value of 5%. Responses were observed for 13 cancer types, including KRAS-mutant colorectal (1/3), PIK3CA-mutant endometrial (5/6), and tissue HER2-negative gastric cancers (1/2). Plasma HER2 CN above vs. below the baseline median value did not impact response (ORR: 58.1% vs. 54.8%); however, the clearance vs. persistence of HER2 amp in cfDNA on Cycle 2 Day 1 corresponded to higher response (ORR: 88.0% vs. 22.7%). ORR by independent review was 58.1% (95% CI, 44.8–70.5%), and the disease control rate was 90.3% (95% CI, 80.1–96.4%). Median progression-free survival was 7.0 (95% CI, 4.9–9.7) months, and the median duration of response was 8.8 months (95% CI, 5.8–11.2). Most adverse events were mild to moderate. Interstitial lung diseases occurred in 16 patients (25.8%, G1/G2/G3; 14/1/1). Conclusions: T-DXd achieved a high ORR, durable response with a manageable safety profile in patients with advanced solid tumors and HER2 amp detected in cfDNA. Clinical trial information: JapicCTI-194707 .[Table: see text]

Breast Cancers with a HER2/CEP17 Ratio of 2.0 or Greater and an Average HER2 Copy Number of Less Than 4.0 per Cell: Frequency, Characteristics and Survival Outcomes in those Receiving HER2-targeted Treatment

Purpose: 2018 ASCO CAP testing guidelines for HER2 recommended a change from ‘positive’ to ‘negative’ in cases of IHC 2+, FISH HER2/CEP17 ratio ≥2.0 and HER2 copy number <4 – known as ‘group 2’ cases [1,2]. In the UK, these cases are positive, but borderline amplified with uncertain benefit of anti-HER2 treatment [3]. Ahead of a forthcoming review of HER2 testing guidelines by the UK National Coordinating Committee for Breast Pathology, we aimed to identify real-world data on group 2 patients, including frequency, characteristics and outcomes in those treated with anti-HER2.

3MO HER2 expression and early response to patritumab deruxtecan (HER3-DXd) in early-stage hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC): A correlative analysis from SOLTI-TOT-HER3 trial

HER3-DXd and trastuzumab deruxtecan (T-DXd) are antibody-drug-conjugates (ADCs) directed against HER3 and HER2, respectively. Both drugs have shown efficacy in patients (pt) with HR+/HER2- BC. Baseline HER3 protein or mRNA levels do not predict efficacy from HER3-DXd. Here, we aimed to evaluate the association of early response to HER3-DXd with HER2 levels. TOT-HER3 (NCT04610528), a window-of-opportunity trial, evaluated a single dose of HER3-DXd in 77 pts with untreated HR+/HER2- early BC in Part A (6.4 mg/kg) and 17 in Part B (5.6 mg/kg). Primary objective was CelTIL score [a surrogate of response that combines tumor cellularity and tumor-infiltrating lymphocytes] variation between baseline and day 21 tumor samples. Here, CelTIL response was defined as an absolute increase of 20 at day 21. HER2 expression was determined by immunohistochemistry (IHC). RNA and DNA were purified from baseline FFPE tumor samples and analyzed using the nCounter and the VHIO-300 NGS platforms, respectively. Logistic regression models and area under the ROC Curve (AUC) estimated the performance of HER2 IHC, mRNA or copy-number (CN) levels with CelTIL response. In TOT-HER3 Part A, HER2 IHC status was 32% HER2 0, 38% HER2 1+ and 30% HER2 2+. Distribution of high CelTIL responders was 40% in HER2 0, 45% in HER2 1+ and 13% in HER2 2+ (p=0.034). High ERBB2 mRNA and RNA-based HER2 amplicon signatures were significantly associated with low CelTIL response (ERBB2: Odds Ratio [OR]=0.34, p<0.001, AUC=0.71; and HER2 amplicon: OR=0.20, p=0.005, AUC=0.74). The association of ERBB2 mRNA with CelTIL response was independent of HER2 IHC expression (p=0.002), PAM50 subtype (p=0.001) or proliferation (p=0.003). High HER2 CN signal was found associated with low CelTIL response (p=0.005). The association of HER2 IHC and ERBB2 mRNA with CelTIL response will be independently validated in TOT-HER3 Part B. Low HER2 IHC, mRNA or CN levels in early-stage HR+/HER2- BC are associated with high response to HER3-DXd. HER3-DXd might be highly active in HR+ BC with very low HER2 levels, thereby offering a new therapeutic paradigm to this subset of patients.

Frequency of HER2 protein overexpression and HER2 gene amplification in endometrial clear cell carcinoma

HER2 (ERBB2) overexpression and/or HER2 gene amplification has been well established in several tumors types and when present HER2 directed therapy may be to be efficacious. While recent findings suggests that HER2 overexpression and HER2 amplification are a relatively common in serous endometrial carcinoma, similar data regarding clear cell endometrial carcinoma (CCC) is difficult to interpret due to issues such as diagnostic criteria, sample type and HER2 interpretation criteria. Our goals were to study HER2 expression and HER2 copy number status in hysterectomy specimens from a large series of patients with pure CCC to determine the frequency of HER2 overexpression and HER2 amplification and evaluate applicability of current HER2 interpretation criteria. Pure CCC specimens derived from hysterectomy specimens from 26 patients were identified. All diagnoses were confirmed by two gynecologic pathologists. Immunohistochemistry for HER2 protein and fluorescence in situ hybridization (FISH) studies for HER2 were performed on whole-slide sections from all cases. Results were interpreted according to the 2018 ASO/CAP HER2 guidelines for breast cancer and International Society of Gynecologic Pathologists (ISGyP) HER2 guidelines for serous endometrial carcinoma. Additional testing was performed when indicated by the guidelines. HER2 expression by immunohistochemistry was 3+ in 4% and 0% of cases, and 2+ in 46% and 52% of cases, by 2018 ASCO/CAP and ISGyP criteria, respectively, while the remaining cases were negative. HER2 testing by FISH showed a positive result in 27% of tumors with 2018 ASCO/CAP guidelines, while 23% were positive with the ISGyP criteria. Our findings indicate that HER2 overexpression and HER2 amplification occur in a subset of CCC. Therefore, additional study into the potential benefit of HER2 targeted therapy in patients with CCC is warranted.

Abstract 6162: High resolution detection of HER2 copy number variation in admixed breast cancer cell lines and HER2+ FFPE donor tissue using a digital PCR multigene reference panel

Abstract The measurement of HER2 (ERBB2) gene amplification guides both the prognosis and treatment of breast cancer, as amplification correlates with worse outcomes and the recommendation for anti-HER2 treatment. Digital PCR (dPCR) can serve as a useful tool for the direct quantification of copy number variants (CNV) in this gene, without the need for calibration standards as required for qPCR, or labor-intensive histological techniques such as FISH. Furthermore, increasing the number of available partitions in a digital PCR reaction can yield substantial gains in precision, thus offering the potential for ultra-high resolution discrimination of copy number changes. In this study, we evaluated the utility of a multiplex dPCR assay for the high-resolution detection of HER2 copy number variation in clinically relevant samples. The precision achievable by digital PCR correlates with the number of partitions, and the resolution of small CNV differences greatly relies on high precision. We utilized a dPCR setup with ~100,000 physical partitions per reaction, which allowed for a theoretical precision (relative confidence) of 1%, assuming optimal input and template. Five single-plex primer and probe sets, published by the National Institute of Standards and Technology, were assembled and optimized into a multiplex configuration. The final 5-plex consisted of four reference genes and HER2 as the target of interest. The accuracy and precision of the optimized five-plex assay was tested in nine breast cancer cell lines with established HER2 amplification ratios, ranging from 1.3 to 70 fold. The assay demonstrated the precise and accurate measurement of the HER2 amplification ratio across all nine cell lines over a wide dynamic range of sample input. Along with increasing the reaction partition number, the use of multiple reference genes also improved the precision of the HER2 amplification ratio measurement and avoided inaccurate copy number calls due to outlier references. The resolution of the assay was evaluated using admixed samples simulating low tumor fraction. A minimum of a 1.03 fold difference (3% difference from wild type) was detected with statistical significance. Assay concordance was evaluated against IHC-characterized HER2+ FFPE donor tissue with concordant FISH/CISH results. The assay demonstrated robust performance in clinically relevant sample types, supporting further investigation of dPCR as a complementary or even alternative diagnostic approach to current methods. Citation Format: Tyler Landrith, Samantha Smith, Jennifer Chan, Mari Christensen, Yu Chuan Tai, Megan Gonzales, William Yee, Nicolas Newton, Wouter Pattje, Patrick Bogard, Wei Yang. High resolution detection of HER2 copy number variation in admixed breast cancer cell lines and HER2+ FFPE donor tissue using a digital PCR multigene reference panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6162.

Genomic landscape and efficacy of HER2-targeted therapy in patients with HER2-mutant non-small cell lung cancer.

HER2-targeted therapy provides survival benefits to HER2-mutant non-small cell lung cancer (NSCLC). A better understanding of the clinical and genomic characterization of treatment-naïve HER2-positive NSCLC, as well as the efficacy of and resistance to HER2-targeted therapy in HER2-altered NSCLC, could promote further improvement of HER2 targeted therapy. HER2-altered NSCLC patients was retrospectively included and their genomic profiles were performed by next-generation sequencing. The clinical outcomes included overall response rate, disease control rate and progression-free survival. Among 176 treatment-naïve patients with HER2 alterations, 64.8% harbored HER2 mutations with/without HER2 amplification, and 35.2% carried HER2 amplification only. Molecular characterization was correlated with tumor stage that late-stage NSCLC with HER2 oncogenic mutations showed a higher prevalence of TP53 mutations and a higher tumor mutation burden. However, this correlation was not found in patients with HER2 amplification only. Twenty-one patients with HER2 alterations treated with pyrotinib or afatinib were retrospectively enrolled. Pyrotinib yielded a longer median progression-free survival than afatinib (5.9 [95% CI, 3.8-13.0] vs. 4.0 months [95% CI, 1.9-6.3], P = 0.06) in these patients. Analysis of the genomic profiles before and after anti-HER2 targeted therapies identified de novo HER2 copy number gain and G518W mutation, as well as mutations involving DNA damage repair signaling, SWI-SNF complex, and epigenetic regulations as potential resistance mechanisms. HER2-mutant NSCLC had different molecular features from HER2-amplified NSCLC, and its genomic profile was dependent of tumor stage. Pyrotinib had superior therapeutic effects than afatinib in HER2-altered NSCLC, although larger cohorts are warranted to validate it. HER2-dependent and -independent resistance mechanisms to afatinib and pyrotinib were unveiled.

Incidence, Clinicopathologic Features, HER2 Fluorescence In Situ Hybridization Profile, and Oncotype DX Results of Human Epidermal Growth Factor Receptor 2-Low Breast Cancers: Experience From a Single Academic Center

Human epidermal growth factor receptor 2 (HER2)-low breast cancer, defined by an immunohistochemical (IHC) score of 1+ or 2+ with negative in situ hybridization, is emerging as a predictive marker for the use of the antibody-drug conjugate. To understand how this category differs from HER2-zero cases, we investigated clinicopathological characteristics and HER2 fluorescence in situ hybridization results in a large cohort of 1309 continuous HER2-negative invasive breast carcinomas from 2018 to 2021 evaluated by the Food and Drug Administration-approved HER2 IHC test. Additionally, we compared Oncotype DX recurrence scores and HER2 mRNA expression between HER-low and HER2-zero cases in a separate cohort of 438 estrogen receptor-positive (ER+) early-stage breast carcinoma cases from 2014 to 2016. Based on the cohort from 2018 to 2021, the incidence of HER2-low breast cancers was approximately 54%. HER2-low cases had less frequent grade 3 morphology, less frequent triple-negative results, ER and progesterone receptor negativity, and a higher mean HER2 copy number and HER2/CEP17 ratio than HER2-zero cases (P < .0001). Among ER+ cases, HER2-low cases showed significantly less frequent Nottingham grade 3 tumors. In the cohort from 2014 to 2016, HER2-low cases showed significantly higher ER+ percentages, fewer progesterone receptor-negative cases, lower Oncotype DX recurrence scores, and higher HER2 mRNA expression scores than HER2-zero cases. In summary, this is the first study, to our knowledge, using a large cohort of continuous cases evaluated by the Food and Drug Administration-approved HER2 IHC companion diagnostic test for HER2-low expression and HER2 fluorescence in situ hybridization profile in a real-world setting. Although HER2-low cases showed a higher HER2 copy number, ratio, and mRNA level than HER2-zero cases statistically, such small differences are unlikely to be biologically or clinically meaningful. However, our study suggests that HER2-low/ER+ early-stage breast carcinoma may represent a less aggressive group of breast carcinoma, given its association with a lower Nottingham grade and Oncotype DX recurrence score.

Effect of Surface Decalcification With Hydrochloric Acid on the Determination of Estrogen Receptor, Progesterone Receptor, Ki67, and Human Epidermal Growth Factor Receptor 2 Expressions in Invasive Breast Carcinoma Based on Immunohistochemistry and Fluorescence In Situ Hybridization.

Bone is the most common site of metastatic breast cancer (MBC). EDTA is often used to decalcify bony tissue samples to ensure the accurate assessment of antigenicity in MBC. It takes ~24 to 48 hours to decalcify small bone tissues such as bone marrow, which is considered unacceptable given the priority that is often placed on the rapid processing of bone marrow trephine cores. Thus, an effective decalcification method that preserves genetic material is needed. We performed immunohistochemical studies on surface decalcification (SD) in breast tumors and evaluated the effect of SD on receptor status and human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization was performed on a subset of these tumors to establish a protocol for handling bone specimens for MBC. Forty-four cases of invasive breast tumors were studied. We compared the immunohistochemical expressions of estrogen receptor (ER), progesterone receptor (PR), Ki67, and HER2 between control tissue (nondecalcified) and parallel tissue subjected to SD with hydrochloric acid. We also evaluated the effect of SD on the fluorescence in situ hybridization expression of HER2. Categorical decreases in ER and PR expression were identified in 9/31 (29.0%) cases without SD and 10/26 (38.5%) cases with SD. HER2 expression changed from equivocal to negative in 4/12 (33.4%) cases. Among the HER2-positive cases, all remained positive after SD. The most significant declines in immunoreactivity occurred with Ki67, with an average decrease from 22% to 13%. The average HER2 copy numbers were 5.37 and 4.76 in the control and SD groups, respectively, and the average HER2/CEP17 ratios were 2.35 and 2.08, respectively. Overall, SD is an alternative decalcification method in bony metastases to assess ER, PR, and HER2 in MBC.

HER2 Amplification Level Predicts Pathological Complete Response in the Neoadjuvant Setting of HER2-Overexpressing Breast Cancer: A Meta-Analysis and Systematic Review.

Anti-HER2 therapies have dramatically improved the prognosis of human epidermal growth factor receptor 2 (HER2)-overexpressing cancers. However, the correlation between the HER2 copy number and the response rate to anti-HER2 remains unclear. Here, following the PRISMA method, we performed a meta-analysis in the neoadjuvant setting in breast cancer to study the association between the HER2 amplification level and the pathological complete response (pCR) to anti-HER2 therapies. Nine articles (four clinical trials, five observational studies) were retrieved after full-text screening, involving 11,238 women with locally advanced breast cancer in the neoadjuvant setting. The median HER2/CEP17 ratio cut-off value was 5.0 ± 5.0 (min-max = 1.0-14.0). For the overall population, the median pCR rate was 48% using the random effect model. The studies were categorized in quartiles as follows: ≤2 (Class 1); 2.1 to 5.0 (Class 2); 5.1 to 7.0 (Class 3); and >7.0 (Class 4). After grouping, the pCR rates were 33%, 49%, 57%, and 79%, respectively. When we excluded the study by Greenwell et al., which accounted for 90% of the patients, using the same quartiles, we still observed an increasing rate of pCR as the HER2/CEP17 ratio increased. This is the first meta-analysis demonstrating the relationship between the HER2 amplification level and the percentage of pCR in the neoadjuvant setting among women with HER2-overexpressing breast cancer, with potential therapeutic applications.

Prospective observational study of monitoring gene alterations in plasma cell-free DNA using droplet digital PCR system during anti-EGFR antibody treatment in patients with RAS wild-type advanced colorectal cancer.

172 Background: Anti-EGFR antibody is recommended as one of the standard treatments in patients with RAS wild-type advanced colorectal cancer (CRC). Few reports have evaluated low-frequency subclones of alterations in EGFR signaling pathway genes in plasma cell-free DNA (cfDNA) during anti-EGFR treatment in prospective cohort of advanced CRC patients. Methods: Key inclusion criteria of this study was as follows: 20 years old and over, performance status 0 to 2, metastatic or recurrent CRC (adenocarcinoma), RAS wild-type CRC diagnosed by PCR-rSSO using tumor tissue, no prior use of anti-EGFR antibody. Plasma samples were prospectively collected at pre-treatment, at 4, 10 weeks, every 10 weeks thereafter, and at the end of the anti-EGFR treatment. With QX200 Droplet Digital PCR platform, we analyzed hotspot mutations of KRAS, NRAS, BRAF, and PIK3CA and copy number (CN) variant of HER2 and MET using plasma samples of pre- and post-treatment as well as during treatment (limited to patients with any gene alterations at pre- and post-treatment). Results: Total 50 patients were registered in this study, but 3 patients were excluded because inclusion criteria were not met. Finally, 47 patients were included in full analysis set. In 15 patients (31.9%), genetic alterations including KRAS (G12G13: 10.1%, A59Q61: 2.2%), NRAS (G12G13: 2.2%, Q61: 2.1%), BRAF (V600: 6.4%) , PIK3CA (E545: 4.3%, H1047: 4.3%) , and HER2 (amplification: 6.5%) were detected in pre-treatment samples. Analysis of pre-treatment samples showed that response rates of patients with any gene alterations (N = 13) and that without any gene alterations (N = 29) were 30.8% and 58.6%, respectively (p = 0.095). When the cut-off of variant allele frequency (VAF) was set as 0.5% in patients with any gene mutations, the response rates for cases with VAF ≥0.5% (N = 6) and those with VAF &lt; 0.5% (N = 7) were 0% and 57.1%, respectively (p = 0.049). Among 41 patients with detectable target lesions by CT scan, there was significant difference in percentage of best tumor shrinkage between patients with gene alterations and those without gene alterations (32.1% vs 10.5%, p = 0.037), when cut-off VAF of any gene mutations was set as 0.5% and cut-off CN of HER2 or MET was set as 4 at pre-treatment. Due to dynamics of each gene alteration in cfDNA during treatment, mutations of KRAS A59Q61, NRAS Q61, and MET amplification were characterized as acquired alterations which were newly detected after the administration of anti-EGFR antibodies. Conclusions: Our study demonstrated the clinical relevance of minor mutant subclones in EGFR signaling pathway genes in plasma for predicting response to anti-EGFR treatment. Although sample size of this study was small, preferable threshold for distinguishing responders from non-responders may be 0.5% as VAF of these gene mutations. Clinical trial information: 000034923 .

A clinicopathological study and survival analysis of 99 breast cancers with HER2/CEP17 ratio ≥ 2.0 and an average HER2 copy number < 4.0 per cell in China

BackgroundBreast cancer patients of American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) Group 2 were all HER2-negative according to the 2018 guideline, not HER2-positive as defined in the 2013 guideline.MethodsWe aims to elucidate the unique clinicopathological features of ASCO/CAP Group 2 patients by comparing with classic HER2-nonamplified cancers, and reveal the efficacy of the former to anti-HER2 therapy. The clinicopathological features, treatment and prognosis information of 99 patients between 2014 and 2018 were collected. HER2 status was re-defined using the updated recommendations.ResultsOf the 99 ASCO/CAP Group 2 tumors, 25.5% (25/99) tumors were immunohistochemical (IHC) 0/1+ and 74.7% (74/99) tumors were IHC 2+. According to the updated 2018 guideline, all of them were HER2 negative. When compared to ASCO/CAP Group 5, patients of ASCO/CAP Group 2 displayed higher ratio of histological grade 3 (P = .03), high Ki67 proliferation index (P = .03) and pN3 (more than 9 lymph nodes metastasis, P = .02), and lower estrogen receptor (ER) positivity (P = .04). There was no statistical difference in the survival of patients received anti-HER2 therapy and patients not received anti-HER2 therapy.ConclusionsPatients of ASCO/CAP Group 2 did not received apparent benefit from anti-HER2 treatment. Although according to the updated guidelines and latest reports, HER2 is negative, but when compared with classic HER2-nonamplified cancers, patients of this group seemed to be more aggressive. We suggest that this group still be regarded as an independent category, in order to accumulate more cases in the future to expand the scope of research.