In Situ Hybridization Results Research Articles

Abstract PO5-13-04: Breast Cancer Index (BCI) results from a HER2 low breast cancer community cohort of operable stage I-II hormone receptor positive (HR+) breast cancer

Abstract Background Hormone receptor positive (HR+) breast cancer (BC) traditionally included HER2-negative or HER2-positive classifications. The BCI test is a quantitative RNA profile assay applied to HR+ early-stage breast cancers from archival pretreatment surgical specimens. The BCI assay estimates the likelihood of distant recurrence (DR) during years 5-10 (prognostic result) and predicts extended endocrine therapy benefit (ETT)(i.e., BCI (H/I)-High, yes benefit) or no benefit (i.e., BCI (H/I)- Low) for patients who have completed 5 years of adjuvant endocrine therapy (AET). Based on the Destiny Breast 04 trial, an antibody drug conjugate (ADC) anti-HER2 therapy was recently approved for treating metastatic HR+HER2 low BCs, which are currently defined as tumors with either HER2 immunohistochemical (IHC) staining of 1+ or IHC 2+ without HER2 gene amplification. Primary archival specimens were eligible for enrollment on the Destiny Breast 04 trial (35%). Quality control of IHC result categories is needed due to new clinical implications in the metastatic setting. To date, BCI results for tumors classified as HER2 low has not been reported. Objectives Determine the average BCI distant DR risk (prognostic result) and the proportion of tumors with BCI (H/I)-High, yes benefit versus BCI (H/I)-Low, no benefit (predictive result) from a small community-based retrospective study of pretreatment samples from a cohort of HER2 low, HR+ breast tumors. Methods Around completion of 5 years of AET, BCI testing was applied to 110 surgically excised archived stage I and 2 HR+ untreated tumors that were collected between 2017 and 2022. Specimens had paired HER2 local pathology IHC and in-situ hybridization (ISH) results. Results 32 tumors were identified as HER2-low including 26 specimens with HER2 IHC 1+ results and six HER2 IHC 2+ tumors with non-amplified HER2 ISH results. Given this was a small exploratory analysis, statistical significance was deliberately not evaluated. Prognostic Results: The average estimated DR risk during years 5-10 was 8% for tumors classified as HER2-low (Range 1.3-17.5%). For 26/32 patients with HER2 IHC1+, the average DR risk was 7.4% (Range 1.3-17.5%). For 6/32 patients with HER2 IHC 2+ and non-amplified HER2 ISH results, the average DR risk was 4.6% (Range 1.6-8.6%). Predictive Results: Of the 32 HER2-low tumors, 15/32 (47%) were BCI (H/I)-High (yes benefit) compared to 17/32 (53%) which were BCI (H/I)-Low (no benefit). Conclusions The prevalence of HER2 low tumors appears to be as high as 67% in reports to date. In this small series, the prevalence of HR+ HER2 low tumors was 29% (32/110). The average DR risk in these HR+ HER2 low cases was roughly 8% which represents a DR risk that is very similar to the average DR risk of 7.4% for HR+ HER2 negative tumors reported from one of the large BCI validation studies (TransATAC). In this series of HER2 low BCs, the proportions of BCI (H/I)-High (yes benefit) and BCI (H/I)-Low (no benefit) tumors were 47% and 53%, compared with 46% and 54%, respectively, in three large BCI validation studies (Trans-aTTom, B42, IDEAL) for HER2 negative tumors. Although this is a small retrospective series, these results suggest that HER2 low tumor are, by BCI testing, very similar to HER2 negative tumors in DR risk (prognostic results). Also, it appears that BCI (H/I)-High (yes benefit) and BCI (H/I)-Low (no benefit) proportions (predictive results) for HER2 low tumors appear similar to reported results for HER2 negative tumors. Follow-up data from larger series are needed to determine if BCI testing is prognostic and predictive for HER-2 low BCs. Citation Format: Milana Dolezal, Mark Pegram. Breast Cancer Index (BCI) results from a HER2 low breast cancer community cohort of operable stage I-II hormone receptor positive (HR+) breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-04.

Abstract 1037: Clinical application of next-generation digital PCR capable of real-time analysis to HER2 measurement as a novel diagnostic in breast cancer: A multicenter retrospective study

Abstract HER2 testing is still challenging due to the subjective natures of immunohistochemistry (IHC) and in situ hybridization (ISH), standard methods for determining HER2 status. Here, we developed a clinically reliable HER2 testing method enabling ultra-fast detection of HER2 gene amplification with high accuracy by using the digital real-time PCR (drPCR) system. To test the clinical validity and standardize procedures of drPCR-based HER2 status evaluation, three independent breast cancer cohorts from different institutions were enrolled, which assigned as a training (SCHU hospital, n = 103) and two validation sets (SNU hospital, n = 170; CNUH hospital, n = 45), and the drPCR assay was compared with current standard HER2 testing methods. In the training cohort, the HER2/CEP17 ratio values from FISH and drPCR tests were highly correlated (r2 = 0.81; P < 0.001), and the drPCR results displayed 98.1% concordance to HER2 status defined by IHC and/or FISH with 92.6% sensitivity and 100% specificity. Eight samples further verified by targeted NGS showed 100% concordance of dPCR to NGS. Consistently, two validation cohorts also showed high concordance of drPCR to IHC and/or ISH results (accuracy = 97.1% and 97.8% in SNU and CNUH cohorts, respectively). The optimal cutoff for HER2 positivity in the drPCR assay was set as a HER2/CEP17 ratio ≥ 1.9 with AUC of 0.963 based on the results from training cohort, and the same cut-off for drPCR was applicable to two independent validation cohorts, supporting the clinical validity of our drPCR-based HER2 assessment. In some discordant cases, low tumor purity (≤ 25%) was observed and microdissection partly improved the drPCR results. The discordance between drPCR and ISH results was also found in marginal HER2+ cases with HER2/CEP17 ratio 2-3, but these cases showed inter-observer variability when re-evaluating the ISH/IHC data due to intratumoral HER2 heterogeneity. Of note, in HER2 IHC3+ cases with negative drPCR results, re-evaluation of IHC using an artificial intelligence (AI)-based HER2 scoring system revised the HER2 IHC 3+ score to 2+, and ISH assessment also confirmed that these cases are indeed HER2-negative, proving the high accuracy of HER2 CN drPCR assay. In conclusion, given the advantages of drPCR-based HER2 assessment with high accuracy, sensitivity, and simplicity, the drPCR assay could be a complementary or alternative method to IHC and ISH to greatly improve current HER2 testing. Citation Format: Hayeon Kim, Soo Young Park, Sohyeon Yang, Min Ji Song, Da Sol Kim, Jin hyuk Chang, YoonSik Kim, Hee-Joo Choi, Nam Hun Heo, Hee-Young Won, Ji-Hye Park, Minsik Song, Seung-Shick Shin, Do Young Lee, Si-Hyong Jang, Jeong-Yeon Lee, Han Suk Ryu. Clinical application of next-generation digital PCR capable of real-time analysis to HER2 measurement as a novel diagnostic in breast cancer: A multicenter retrospective study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1037.

Abstract A005: Ultra-rapid and precise measurement of HER2 copy number alteration by next-generation digital PCR capable of real-time analysis in patients with breast cancer: A multicenter retrospective study

Abstract The HER2 diagnostics is necessary for selection of patients harboring HER2 gene amplification or protein overexpression who will benefit from anti-HER2 therapies in breast cancer. However, HER2 testing is still challenging due to the subjective natures of immunohistochemistry (IHC) and in situ hybridization (ISH), standard methods for determining HER2 status. Thus, a new method is needed to accurately quantify HER2 levels. Here, we developed a clinically reliable HER2 testing method enabling ultra-fast detection of HER2 gene amplification with high accuracy by using the digital real-time PCR (drPCR) system, a potential new diagnostic platform with improved performance by integrating both real-time and digital PCR technologies. For drPCR-based HER2 copy number (CN) measurement, primer-probe sets specific to HER2 gene and a genomic region adjacent to chromosome 17 centromere (CEP17) were designed, and the optimal drPCR condition was determined in clinical breast tumor specimens. To test the clinical validity and standardize procedures of drPCR-based HER2 status evaluation, three independent breast cancer cohorts from different institutions were enrolled, which assigned as a training (SCHU hospital, n = 103) and two validation sets (SNU hospital, n = 170; CNUH hospital, n = 45), and the drPCR assay was compared with current standard HER2 testing methods. In the training cohort, the HER2/CEP17 ratio values from FISH and drPCR tests were highly correlated (r2 = 0.81; P < 0.001), and the drPCR results displayed 98.1% concordance to HER2 status defined by IHC and/or FISH with 92.6% sensitivity and 100% specificity. Eight samples further verified by targeted NGS showed 100% concordance of dPCR to NGS. Consistently, two validation cohorts also showed high concordance of drPCR to IHC and/or ISH results (accuracy = 97.1% and 97.8% in SNU and CNUH cohorts, respectively). The optimal cutoff for HER2 positivity in the drPCR assay was set as a HER2/CEP17 ratio ≥ 1.9 with AUC of 0.963 based on the results from training cohort, and the same cut-off for drPCR was applicable to two independent validation cohorts, supporting the clinical validity of our drPCR-based HER2 assessment. In some discordant cases, low tumor purity (≤ 25%) was observed and microdissection partly improved the drPCR results. The discordance between drPCR and ISH results was also found in marginal HER2+ cases with HER2/CEP17 ratio 2-3, but these cases showed inter-observer variability when re-evaluating the ISH/IHC data due to intratumoral HER2 heterogeneity. Of note, in HER2 IHC3+ cases with negative drPCR results, re-evaluation of IHC using an artificial intelligence (AI)-based HER2 scoring system revised the HER2 IHC 3+ score to 2+, and ISH assessment also confirmed that these cases are indeed HER2-negative, proving the high accuracy of HER2 CN drPCR assay. In conclusion, given the advantages of drPCR-based HER2 assessment with high accuracy, sensitivity, and simplicity, the drPCR assay could be a complementary or alternative method to IHC and ISH to greatly improve current HER2 testing. Citation Format: Jin hyuk Chang, YoonSik Kim, Hee-Joo Choi, Soo Young Park, Ji-Hye Park, Hee-Young Won, Min Ji Song, Da Sol Kim, Hayeon Kim, Sohyeon Yang, Nam Hun Heo, Minsik Song, Seung-Shick Shin, Do Young Lee, Han Suk Ryu, Si-Hyong Jang, Jeong-Yeon Lee. Ultra-rapid and precise measurement of HER2 copy number alteration by next-generation digital PCR capable of real-time analysis in patients with breast cancer: A multicenter retrospective study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A005.

Detection of Human Papillomavirus in Squamous Papilloma of the Esophagus.

Introduction: The etiology of esophageal squamous papilloma (ESP) is largely unknown. Previous studies have shown a variable association with human papillomavirus (HPV) with conflicting data. The aim of this study was to further investigate the possible association of HPV in our ESP series using RNA in-situ hybridization (ISH) and compare study groups from the United States of America and China. Methods: Demographic and clinical data of patients with ESP were retrieved from the University of California Los Angeles (UCLA) (1/2016-3/2019) and Peking Union Medical College Hospital (PUMCH) (9/2014-3/2019) pathology databases. Hematoxylin and eosin slides were reexamined. Confirmed cases were examined by high- and low-risk HPV RNA ISH. Results: For the UCLA cohort, 13 429 upper endoscopies were performed and 78 biopsies from 72 patients were identified as ESP (F:M = 45:27, 66.7% > 45 years). Seventy-four (94.9%) biopsies were designated as polyps or nodules and 46.6% were located in the mid-esophagus. Other abnormal findings included gastroesophageal reflux disease (48.6%), hiatal hernia (38.9%), and esophagitis (36.1%). For the PUMCH cohort, 63 754 upper endoscopies were performed and 73 biopsies from 71 patients were identified as ESP (F:M = 48:23, 71.8% > 45 years). Sixty-four (87.7%) biopsies were designated as polyps or nodules and 57.5% were located in the mid-esophagus. Other abnormal findings included esophagitis (19.7%), and hiatal hernia (8.5%). No features of conventional cytologic dysplasia or viral cytopathic change were found. None of the cases was associated with squamous cell carcinoma, and none showed positive HPV RNA ISH results. Conclusions: No association was found between ESP and active HPV infection in our 2 cohorts. Other etiopathogenetic mechanisms, such as aging, might contribute to the development of these innocent lesions.

Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues.

Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)-labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens. Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282-350). Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples. This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

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.

LncRNA WAC-AS1 expression in human tumors correlates with immune infiltration and affects prognosis

BackgroundWAC-antisense RNA1 (WAC-AS1) is a newly identified long non-coding RNA (lncRNA) implicated in the prognosis and development of a few types of tumors. However, the correlations of WAC-AS1 with immune infiltration and patient prognosis in pan-cancer remain unclear. In the present study, we aimed to investigate the prognostic value and immunological functions of WAC-AS1 across 33 different types of cancers.MethodsTo investigate the potential oncogenic roles of WAC-AS1, bioinformatics analyses were performed using the Cancer Genome Atlas (TCGA) and Genotype Tissue-Expression (GTEx) datasets. The correlations of WAC-AS1 with prognosis, clinical phenotype, tumor mutational burden (TMB), microsatellite instability (MSI), tumor regulation-related genes, tumor microenvironment, immune cell infiltration, and drug resistance to commonly used chemotherapy drugs in different types of tumors were explored. Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed to explore the biological functions of WAC-AS1 in tumors. In situ hybridization (ISH) was performed in tissue microarray (TMA) to confirm the expression of WAC-AS1 in multiple tumor tissues.ResultsWAC-AS1 showed aberrant expression in most cancers when compared to the normal tissues. It also has prognostic value in multiple types of cancers. Elevated WAC-AS1 expression was associated with poor prognosis and overall survival in adrenocortical carcinoma (ACC), breast invasive carcinoma (BRCA), and liver hepatocellular carcinoma (LIHC). A significant negative correlation between WAC-AS1 expression and overall survival was observed in brain lower-grade glioma (LGG), pancreatic adenocarcinoma (PAAD), and skin cutaneous melanoma (SKCM). The expression of WAC-AS1 also showed a correlation with clinical stage in six types of tumors, and with tumor mutational burden and microsatellite instability in several different types of cancers. The immune scores of those cancers were found to be significant. Additionally, the effectiveness of fluorouracil and four other anticancer drugs was significantly different based on the expression of WAC-AS1 in these cancers. Moreover, the ISH results showed in six types of tumors, the expression of WAC-AS1 was consistent with the Pan-cancer analysis using TCGA and GTEx database.ConclusionsThese results indicate an intensive involvement of WAC-AS1 in the regulation of immune responses, immune cell infiltration, and malignant properties in various types of cancers, suggesting that WAC-AS1 may serve as a prognostic marker across diverse types of cancers.

Abstract P1-04-01: High HER2/CEP17 ratio is associated with better treatment outcomes in advanced HER2-positive breast cancer treated with pertuzumab, trastuzumab, and docetaxel regardless of HER2 2+ or 3+ results

Abstract Background: Dual HER2 blockade with trastuzumab and pertuzumab combined with docetaxel (DHP) is the standard first-line treatment option for patients with HER2-positive metastatic breast cancer. However, HER2 positivity alone often fails to predict treatment outcome of HER2 targeted therapy. The magnitude of HER2 immunohistochemical staining (IHC) and HER2/CEP17 ratio from in situ hybridization (ISH) is a well-known predictive biomarker for HER2 targeted therapy. In the neo-adjuvant setting, patients with HER2 IHC 3+ have higher pathologic complete response (pCR) rate compared to HER2 IHC< 3+ with HER2 ISH positive. In addition, higher HER2/CEP17 ratio is associated with higher pCR rate in HER2-positive breast cancer patients treated with dual HER2 blockade regimens. However, there is no data whether there is an association between HER2/CEP17 ratio and treatment outcome of DHP regimen in patients with advanced of metastatic breast cancer especially among those with HER2 IHC 3+. Methods: We performed a retrospective cohort study with patients with locally advanced or metastatic HER2-positive breast cancer who were treated with first-line palliative DHP regimen between August 2008 and January 2021 at Seoul National University Hospital. In the clinical setting, HER2 IHC 3+ is defined as HER2 positive and no further ISH testing is required. Additional ISH was performed in patients with HER2 IHC 3+ without archival HER2 ISH results. The association between HER2/CEP17 ratio and treatment outcome was assessed. Results: In total, 165 patients were included with a median follow-up duration of 28.0 months. Among the 165 patients, 35 patients had archival ISH result and additional ISH was performed in 53 patients. The correlation between HER2/CEP17 ratio and treatment outcome was assessed in 88 patients. Cox proportional hazard analysis revealed that HER2/CEP17 ratio is correlated with PFS (HR 0.23, 95% CI 0.11-0.49, p < 0.001). When dichotomized by the median HER2/CEP17 ratio of 4.17, patients with higher HER2/CEP17 ratio had significantly longer PFS (37.5 vs. 17.4 months, p = 0.003) and numerically higher ORR (54.5% vs. 34.1%, p = 0.085). Multivariate analysis revealed that HER2/CEP17 ratio is an independent prognostic factor for PFS (HR 0.72, p = 0.001). Of 88 patients with ISH results, 25 had HER2 IHC 1+ or 2+ and 63 had HER2 IHC 3+. HER2/CEP17 ratio was associated with PFS in both HER2 IHC 1+/2+ patients (HR 0.12, 95% CI 0.02-0.88, p = 0.037) and HER2 IHC 3+ patients (HR 0.18, 95% CI 0.07-0.49, p = 0.001). Patients with higher HER2/CEP17 ratio had longer PFS in both HER2 IHC 1+/2+ patients (28.6 vs. 12.9 months, p = 0.003) and HER2 IHC 3+ patients (Not reached vs. 18.3 months, p = 0.005) when dichotomized by the median HER2/CEP17 of 2.95 and 4.75, respectively. Conclusion: This is the first study to report that higher HER2/CEP17 ratio is associated with longer PFS in HER2-positive advanced breast cancer patients treated with palliative first-line DHP. The strength of this study is that we identified prognostic role of ISH even in patients with HER2 IHC 3+. It would be helpful to perform ISH not only in patients whose HER2 IHC is ambiguous, but also in patients with HER2 IHC 3+ to make better prediction of treatment outcome. Table 1. Patient characteristics. Citation Format: Jeongmin Seo, Jiwon Koh, Dae-Won Lee, Han Suk Ryu, Kyung-Hun Lee, Tae-Yong Kim, Seock-Ah Im. High HER2/CEP17 ratio is associated with better treatment outcomes in advanced HER2-positive breast cancer treated with pertuzumab, trastuzumab, and docetaxel regardless of HER2 2+ or 3+ results [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-04-01.

Comparison of breast cancer HER-2 receptor testing with immunohistochemistry and in situ hybridization.

Human epidermal growth factor receptor-2 (HER2) status can be tested with immunohistochemistry (IHC) and in situ hybridization (ISH). The 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2 testing guidelines suggest initial HER2 testing using IHC and further testing IHC equivocal cases with ISH. However, many institutions perform both IHC and ISH on the same specimen. This study aims to analyze the concordance between HER2 IHC and ISH in order to evaluate the benefit of repeating HER2 testing on the same breast cancer specimens. Patients diagnosed with invasive breast cancer through BreastScreen NSW Sydney West program between January 2018 and December 2020 were identified and their HER2 IHC and HER2 ISH results on core needle biopsy (CNB) and surgical excisions (SE) were retrospectively collected. Specimens with both IHC and ISH results were then analyzed for agreement and concordance using unweighted kappa values. Equivocal IHC (2+) cases were excluded from concordance analysis. Overall, there were 240 invasive breast cancer specimens (CNB and SE) with both IHC and ISH recorded. Concordance between HER2 IHC and ISH was 100% (95% CI: 96.2-100%; κ=1.00 (P<0.001)). Of the IHC equivocal cases (n=146), 94.5% were ISH negative. There was perfect positive concordance and agreement between non-equivocal IHC and ISH results. This reinforces that IHC alone can be utilized reliably for testing HER2 status of non-equivocal cases consistent with the 2018 ASCO/CAP guidelines.

Abstract C015: Characterization of HER2-low breast tumors among a cohort of Colombian women

Abstract Introduction: HER2-low expression tumors, either with weak or incomplete membrane expression (immunohistochemistry (IHC) 1+ or 2+ with negative in situ hybridization (ISH) result), are considered and treated as HER2-negative tumors. However, recent clinical trials have shown promising results in the application of antibody-drug conjugates (ADCs) for patients with HER2-low breast tumors, highlighting the need to characterize the clinical phenotype of this group. This is the first study in Colombia aiming to explore the clinical-pathological features and survival of women affected with HER2-low breast cancer, compared with HER2-negative (0+) and HER2-positive groups (2+ with ISH+ or 3+). Methods: We included 516 breast cancer patients from different health institutions in Colombia, diagnosed at clinical stages that ranged between I-III. Protein expression of HER2, Ki67, and hormone receptors (estrogen (ER) and progesterone (PR) receptor) was evaluated by IHC in a paraffin block with tumoral tissue by a single pathologist. We defined HER2 expression as positive (3+ or 2+ with positive ISH test), low (1+/2+ with negative ISH result), and negative (0+). We applied a Chi-square or Fisher’s exact test to evaluate clinical-pathological features for HER2-low cases, compared with HER2-negative (low vs. negative) and HER2-positive tumors (low vs. positive). Differences in overall survival (OS) and risk of mortality were assessed using the Kaplan-Meier and log-rang test, and a Cox proportional hazard model. Results: 325 cases (63%) were classified as HER2-negative, 97 (18.8%) as HER2-low, and 94 (18.2%) as HER2-positive. We encountered more clinical-pathological significant differences between HER2-low and HER2-positive tumors than with HER2-negative cases. A higher proportion of patients diagnosed after the age of 50 (79.4% vs. 62.4%, p=0.018), with better-differentiated tumors (Bloom Richardson II: 58.8 vs. 41.2%, p=0.025), and a lower proliferation index (Ki67 &amp;lt;20%: 50.5 % vs. 21.2%, p&amp;lt;0.001) was observed for HER2-low tumors, compared to the HER2-positive group; these characteristics were not significantly different between HER2-low and HER2-negative tumors. The proportion of ER-positivity was significantly higher in the HER2-low group, compared to both HER2-positive (89.7% vs. 62.4%, p&amp;lt;0.001) and negative groups (89.7% vs. 79.7%, p=0.039). Moreover, although longer OS rates were found in HER2-low patients, compared to the negative group (5.5 vs. 5.0 years, p=0.0032), the multivariate Cox model adjusted by ER status showed no statistically significant differences between these groups (HR=0.62, 95% CI, 0.34 – 1.13, p=0.122). Conclusions: HER2-low tumors represent an important proportion of all breast cancer cases diagnosed in Colombian women. Our results are consistent with other reports that show differences in ER expression between HER2-low tumors and both HER2-positive and negative subtypes, although it seems that the prognosis of HER2-low patients is not different than of the other groups. Citation Format: Laura Rey Vargas, Lina Maria Bejarano, Juan Carlos Mejia Henao, Diego Felipe Ballen Lozano, Maria Carolina Sanabria Salas, Silvia Juliana Serrano Gomez. Characterization of HER2-low breast tumors among a cohort of Colombian women [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C015.

Hamster polyomavirus-associated T-cell lymphomas in Syrian hamsters (Mesocricetus auratus).

Hamster polyomavirus (HaPyV) infection has been associated with lymphomas in Syrian hamsters. In the present study, 14 cases of lymphoma in pet Syrian hamsters were pathologically examined and the involvement of HaPyV was investigated. Among 14 cases, 11 were abdominal and 3 were cutaneous lymphomas. The average ages of hamsters with abdominal lymphoma and cutaneous lymphoma were 7 months (range: 4-12 months) and 14 months (range: 6-23 months), respectively. Histologically, abdominal lymphomas were characterized by the diffuse growth of tumor cells with intermediate or large nuclei, low mitotic rates, the presence of tingible body macrophages, and the T-cell immunophenotype. Furthermore, 4/11 abdominal lymphomas were immunopositive for T-cell intracellular antigen-1, suggesting cytotoxic T-cell lymphomas. Cutaneous lymphomas were diagnosed as nonepitheliotropic T-cell lymphoma. Polymerase chain reaction (PCR) detected HaPyV DNA in 12/14 samples, and a sequence analysis of PCR amplicons confirmed >99% nucleotide identity to the published HaPyV sequences. In situ hybridization (ISH) for HaPyV DNA resulted in diffuse nuclear signals within tumor cells in 10/14 cases. Consistent with previous findings, all HaPyV-associated lymphomas were observed in the abdominal cavity of young hamsters. Polymerase chain reaction and ISH were useful for identifying the involvement of HaPyV in lymphomas, and ISH results indicated the presence of episomal HaPyV in neoplastic lymphocytes. The present study suggests that HaPyV infection is highly involved in abdominal lymphomas in young pet Syrian hamsters in Japan and provides diagnostic information on HaPyV-associated lymphoma.

Circular RNA hsa_circ_0007367 promotes the progression of pancreatic ductal adenocarcinoma by sponging miR-6820-3p and upregulating YAP1 expression

Circular RNAs (circRNAs) play critical regulatory roles in cancer biological processes. Nevertheless, the contributions and underlying mechanisms of circRNAs to pancreatic ductal adenocarcinoma (PDAC) remain largely unexplored. Dysregulated circRNAs between cancerous tissues and matched adjacent normal tissues were identified by circRNA microarray in PDAC. The biological effect of hsa_circ_007367 both in vitro and in vivo was demonstrated by gain- and loss-of-function experiments. Further, dual-luciferase reporter and RNA pull-down assays were performed to confirm the interaction among hsa_circ_007367, miR-6820-3p, and Yes-associated protein 1 (YAP1). The expression of hsa_circ_007367 and YAP1 were detected by in situ hybridization (ISH) and immunohistochemistry (IHC) using tissue microarray (TMA) in 128 PDAC samples. We first identified that a novel circRNA, hsa_circ_0007367, was markedly upregulated in PDAC tissues and cells. Functionally, in vivo and in vitro data indicated that hsa_circ_0007367 promotes the proliferation and metastasis of PDAC. Mechanistically, we confirmed that hsa_circ_0007367 could facilitate the expression of YAP1, a well-known oncogene, by sponging miR-6820-3p, which function as a tumor suppresser in PDAC cells. The results of ISH and IHC demonstrated that hsa_circ_0007367 and YAP1 were upregulated in PDAC tissues. Furthermore, clinical data showed that higher hsa_circ_0007367 expression was correlated with advanced histological grade and lymph node metastasis in PDAC patients. In conclusion, our findings reveal that hsa_circ_0007367 acts as an oncogene via modulating miR-6820-3p/YAP1 axis to promote the progression of PDAC, and suggest that hsa_circ_0007367 may serve as a potential therapeutic target for treatment of PDAC.

Comparison of DNA sequencing, immunohistochemistry, and in-situ hybridization techniques to determine HER2 status in metastatic breast cancer patients.

e13045 Background: Limited studies exist comparing methods for HER2 status determination using real-world data (RWD). Results of metastatic breast cancer (MBC) patients with Next Generation Sequencing (NGS) within the ConcertAI Genome360 dataset with DNA sequencing were compared to the standard immunohistochemistry (IHC) or in-situ hybridization (ISH) or both IHC and ISH for HER2 status. Methods: Patients diagnosed with MBC, with HER2 status determined with NGS DNA sequencing, IHC, or any of the ISH tests, by various laboratories within 2 months of each other, from August 19, 2011 to March 9, 2021, were analyzed. Patients were classified as positive, negative, or equivocal based on each of the testing methods. Comparisons were made between DNA sequencing and IHC results, DNA and ISH results, and DNA and IHC and subsequent ISH results. Results: The study sample included 252 patients (99% female, mean age [SD] of 62 [11.93]) with 382 test combinations as follows: 220 (58%) with DNA sequencing and IHC results, 127 (33%) with DNA sequencing and ISH results, and 35 (9%) with DNA sequencing and IHC and ISH results. Of the 220 combinations with DNA sequencing and IHC results, 15 were uniformly considered HER2+, 160 HER2-, and 1 equivocal, while 44 differed in the results, resulting in 80% match rate. Of the 127 combinations with DNA sequencing and ISH results, 14 were HER2+ and 96 HER2-, while 17 differed, resulting in 87% match rate. Of the 35 combinations with DNA sequencing and IHC and ISH, 3 were HER2+ and 26 were HER2-, while 6 differed, resulting in 83% match rate. Match rate was significantly higher for patients with negative results, than positive results. Specimen type, site, and type of expression had no impact on match rates. Conclusions: RWD indicate relatively high level of match rate among DNA sequencing, IHC, and ISH results in MBC patients. However, 13-20% of the combinations did not match. For these patients, mechanisms to reach consensus need to be explored to determine optimal treatments.

P53 and p16ink4a As Predictive and Prognostic Biomarkers for Nodal metastasis and Survival in A Contemporary Cohort of Penile Squamous Cell Carcinoma.

Human papilloma virus (HPV) infection is implicated in a proportion of invasive squamous cell carcinoma of the penis (PC). A subset of PC involves dysregulation of the p53 pathway. HPV in situ hybridization (ISH) and p16ink4a positivity are surrogate markers for HPV infection, and p53 immunohistochemistry (IHC) denotes abnormality in the p53 pathway. There remains an ambiguity with regard to the contribution of both the pathways in the prognosis of PC. We sought to analyze the clinicopathologic characteristics of a cohort of Indian PC patients with respect to p16 ink4a and p53 expression. A cohort of 123 PC patients was studied for p16ink4aand p53IHC and HPVISH. The results of these biomarkers were correlated with various clinicopathologic parameters. p16ink4a and HPV ISH were positive in 47% and 53% of the tumors, respectively. The proportion of warty, basaloid, or mixed warty-basaloid tumor subtypes showed significant p16ink4apositivity (P< .0001) compared to other subtypes. Twenty-eight patients were dual negative (p53- /p16ink4a-), 32 were dual positive (p53+/p16ink4a+), 38 were p53+/p16ink4a-, and 25 were p53-/p16ink4a +. In patients where p16ink4a was negative, a p53-positive phenotype had a higher propensity for lymph node metastases (OR, 5.42; 95% CI, 1.75-16.80; P=.003). Similarly, p53 positivity dictates nodal involvement in the p16ink4a-positive subset of tumors (OR, 5.00; 95% CI, 1.23-20.17; P=.024). On multivariate analyses, pathologic subtypes (warty, warty-basaloid, and basaloid) (P < .0001), p16ink4aexpression (P < .0001), and absence of nodal metastasis (P < .0001) were significant predictors of improved overall (OS) and cancer specific survival (CSS). In Kaplan-Meier analysis, the OS was significantly longer in patients with p16ink4a+tumors (P < .0001), as was the CSS (P < .0001). Patients with dual positive tumors had a significantly higher OS (P < .001) and CSS (P=.012), in the entire cohort. In the node positive patients, dual positivity was associated with significantly higher OS (P < .0001); however, the median CSS for p53+/p16ink4a+tumors were not significantly different compared to p53- /p16ink4a- tumors (P=.064), although there was a trend towards improved CSS. There is a strong concordance between p16ink4aIHC and HPV ISH results. p16ink4a status is an independent predictor of survival (OS and CSS) in our cohort of PCs. p53 is a predictor of nodal metastasis irrespective of p16 status. Dual positive tumors have a significantly better outcome in comparison to dual negative tumors.

HER2 testing in metastatic breast cancer – is reflex in situ hybridization necessary in cases that are equivocal by immunohistochemistry?

Abstract Introduction/Objective Current guidelines recommend HER2 testing on all primary invasive breast cancers and at least one metastatic lesion. Typical HER2 testing involves immunohistochemistry (IHC), with reflex in situ hybridization (ISH) in the event of equivocal (2+) IHC. ISH testing is time consuming and resource intensive, and there may be situations where it is unnecessary. The incidence of discordance between HER2 negative primary tumors and HER2 IHC2+ metastases that are ISH positive is unknown. We hypothesize that the majority of such cases are non- amplified. Methods/Case Report A retrospective review of IHC2+ metastatic lesions further assessed with ISH at our center from 2013-2021 was undertaken. A total of 105 cases were identified after exclusion of cases missing HER2 results, with primaries of unconfirmed origin, and cases of synchronous primary and metastatic disease. IHC and ISH results were recorded, with a detailed slide review of discordant cases. Results (if a Case Study enter NA) 91/105 metastases had HER2 negative primaries (87%). A metastasis was significantly more likely to be HER2 negative when the primary was HER2 negative (93%) versus positive (43%) (p &amp;lt; 0.0001). 54/91 primaries were IHC2+/ISH negative, and 50/54 (93%) corresponding metastases had identical results. Of the 37 HER2 negative primaries that were IHC0/1+, 35 (95%) corresponding metastatic tumors were ISH negative. Six metastatic lesions in cases with HER2 negative primaries were discordant and slides were reviewed. Characteristics of metastatic tumors suggesting ISH testing was warranted to assess for discordance included IHC heterogeneity, morphological discordance, and increased staining of moderate intensity. These factors were present in all six discordant metastases. Conclusion Our results suggest that selective rather than reflex ISH testing on HER2 IHC2+ breast cancer metastases in the context of HER2 negative primary disease may be appropriate when there is careful review of the IHC. Validation of our findings await further studies with larger sample sizes.

4MO p53 and p16ink4a as predictive and prognostic biomarkers for nodal metastasis and survival in a contemporary cohort of penile squamous cell carcinoma

Human papilloma virus (HPV) infection is implicated in a proportion of invasive squamous cell carcinoma of the penis (PC). A subset of PC involves dysregulation of the p53 pathway. HPV in situ hybridization (ISH) and p16 ink4a positivity are surrogate markers for HPV infection, and p53 immunohistochemistry (IHC) denotes abnormality in the p53 pathway. Clinicopathologic characteristics of a cohort of Indian PC patients with respect to p16 and p53 expression were analyzed. 123 PC patients were studied for p16 and p53 IHC and HPV ISH and the results were correlated with various clinicopathologic parameters. p16 and HPV ISH were positive in 47% and 53% of the tumors, respectively. The proportion of warty, basaloid, or mixed warty-basaloid tumor subtypes showed significant p16 positivity (p < 0.0001) compared to other subtypes. In patients where p16 was negative, a p53-positive phenotype had a higher propensity for lymph node metastases (OR, 5.4; 95% CI, 1.7 to16.8; p=0.003). Similarly, p53 positivity dictates nodal involvement in the p16-positive subset of tumors (OR, 5; 95% CI, 1.2 to 20.1; p=0.024). On multivariate analyses, pathologic subtypes (warty, warty-basaloid, and basaloid) (p<0.0001), p16 expression (p<0.0001), and absence of nodal metastasis (p<0.0001) were significant predictors of improved overall (OS) and cancer specific survival (CSS). The OS was significantly longer in patients with p16+ tumors (p<0.0001), as was the CSS (p<0.0001). Patients with dual positive tumors had a significantly higher OS (p<0.001) and CSS (p=0.01), in the entire cohort. In the node positive patients, dual positivity was associated with significantly higher OS (p<0.0001); however, the median CSS for p53+/p16+tumors was not significantly different compared to p53- /p16- tumors (p=0.064), although there was a trend towards improved CSS. There is a strong concordance between p16 IHC and HPV ISH results. p16 status is an independent predictor of survival (OS and CSS) in our cohort of PCs. p53 is a predictor of nodal metastasis irrespective of p16 status. Dual positive tumors have a significantly better outcome in comparison to dual negative tumors.

HER2-Low Breast Cancers.

Recent clinical trials have demonstrated significant clinical benefits from novel therapeutic compounds in breast cancer patient with human epidermal growth factor receptor 2 (HER2) immunohistochemical (IHC) score of 1+ or 2+ and negative in situ hybridization (ISH) result. A new concept of "HER2-low" breast cancer has been proposed and applied in the recent and ongoing clinical trials. In this article, we review the literature on the topic of HER2-low breast cancer. A literature search in PubMed was performed using key words related to HER2-low breast cancer. Major relevant studies that were presented in international breast cancer conferences were also included. HER2-low breast cancer is currently defined as breast cancer with HER2 IHC score of 1+ or 2+ and negative ISH result. It likely represents a group of tumors with significant biological heterogeneity. Reports of clinical activity using the next generation of HER2-targeting antibody-drug conjugates in HER2-low breast cancers suggest that some strategies of targeting HER2 might be effective in this patient population while raising considerable concerns over limitations in our current testing methodologies and our ability to accurately identify such patients. The promising efficacy of novel HER2-targeted therapy in advanced HER2-low breast cancers has raised the possibility for changing the clinical interpretation of HER2 status in breast cancer to include a HER2-low category; however, the definition of HER2-low breast cancer, the corresponding reliable and accurate quantitative HER2 testing methodology, and the biology of HER2-low breast cancer remain poorly defined.

ALK Gene Rearrangements in Lung Adenocarcinomas: Concordance of Immunohistochemistry, Fluorescence In Situ Hybridization, RNA In Situ Hybridization, and RNA Next-Generation Sequencing Testing

IntroductionThe 2018 updated molecular testing guidelines for patients with advanced lung cancer incorporated ALK immunohistochemistry (IHC) analysis as an equivalent to fluorescence in situ hybridization (FISH) method recommended in 2013. Nevertheless, no specific recommendation for alternative methods was proposed owing to insufficient data. The aim of this study was to compare the results of ALK IHC, FISH, RNA next-generation sequencing (NGS), and RNA in situ hybridization (ISH) with available clinical data. MethodsA search for lung carcinomas with ALK testing by greater than or equal to one modality (i.e., ALK IHC, FISH, NGS) was performed; a subset underwent RNA ISH. When available, clinical data were recorded. ResultsThe results were concordant among all performed testing modalities in 86 of 90 cases (95.6%). Of the four discordant cases, two were ALK positive by FISH but negative by IHC, RNA NGS, and RNA ISH. The remaining two cases failed RNA NGS testing, one was IHC negative, FISH positive, RNA ISH negative and the second was IHC positive, FISH positive, RNA ISH equivocal. RNA NGS identified one rare and one novel ALK fusion. Sufficient therapy data were available in 10 cases treated with tyrosine kinase inhibitors; three had disease progression, including one with discordant results (FISH positive, RNA NGS negative, IHC negative, RNA ISH negative) and two with concordant ALK positivity among all modalities. ConclusionsOur results reveal high concordance among IHC, RNA NGS, and RNA ISH. In cases of discordance with available RNA NGS, FISH result was positive whereas IHC and ISH results were negative. On the basis of our data, multimodality testing is recommended to identify discrepant results and patients (un)likely to respond to tyrosine kinase inhibitors.

Tilapia lake virus immunoglobulin G (TiLV IgG) antibody: Immunohistochemistry application reveals cellular tropism of TiLV infection

Tilapia lake virus (TiLV) is a notable contagious agent that causes massive economic losses in the tilapia industry globally. Evaluations of the histological changes associated with TiLV infection are not only crucial for diagnosis, but also to gain an understanding of the disease. We therefore synthesized a rabbit polyclonal immunoglobulin G antibody against TiLV and developed an immunohistochemical (IHC) procedure to detect TiLV localization in the tissues of infected fish for comparison with in situ hybridization (ISH) testing. A total of four different sample cohorts derived from TiLV-infected fish was used to validate the IHC procedure. The TiLV IHC application was successfully developed and facilitated nuclear and cytoplasmic immunolabelling in the intestines, gills, brain, liver, pancreas, spleen, and kidneys that corresponded with the ISH results. Apart from the ISH results, TiLV-IHC signals were clearly evident in the endothelial cells of various organs, the circulating leukocytes in the blood vessels, and the areas of tissue inflammation. Among the tested sample cohorts, the intestines, gills, and brain had IHC-positive signals, highlighting the possibility of these organs as common TiLV targets. Immunological staining pattern and distribution corresponded with the TiLV viral load but not the inoculation route. The TiLV IHC was also capable of detecting TiLV infection in the experimentally challenged ornamental cichlids, Mozambique tilapia, giant gourami, and naturally infected tilapia, indicating the dynamic range of IHC for TiLV detection. Overall, our study delivers the first IHC platform to detect TiLV infection and provides novel evidence of cellular tropism during TiLV infection. Our findings also reveal the TiLV distribution pattern of infected fish and propose the endotheliotropism and lymphotropism of this virus, which requires further elaboration. Importantly, this new IHC procedure could be applied to study the pathogenesis and interaction of TiLV in future research.

Abstract PS4-22: Prevalence of HER2-low and HER2-zero subgroups and correlation with response to neoadjuvant chemotherapy (NACT) in patients with HER2-negative breast cancer

Abstract INTRODUCTION Current definition of HER2-positive BC follows ASCO/CAP guidelines using immunohistochemistry (IHC) and/or in situ hybridization (ISH)-based techniques. However, HER2 expression can be variable in cells that lack ERBB2 amplification. For example, HER2-negative tumors can express some level of HER2 protein by IHC (i.e. 1+ or 2+ and a negative ISH result) and are identified as HER2-low. Others have no expression and are considered HER2-zero. Innovative therapies have shown promising activity in patients in HER2-low BC. The aim of this study is to evaluate the association of HER2-low and HER2-zero status with response to NACT in HER2-negative BC. METHODS Retrospective cohort of patients with HER2-negative BC treated with NACT in four institutions in Brazil. Protocols of diagnosis, treatment and follow-up were standardized and based on international guidelines. Tumors with HER2 IHC score 0 were classified as HER2-zero whereas tumors with HER2 score 1+ and those with HER2 score 2+ with FISH-negative were classified as HER2-low. Patients were treated with anthracycline- and taxane- based chemotherapy. The following clinicopathological data were evaluated, when available: age, ER, Ki67, tumor size, lymph node (LN) status and response to NACT according to pCR status and residual cancer burden (RCB) index. Primary objective was to evaluate the prevalences and compare pCR rates among HER2-zero and HER2-low cases. Secondary objectives were to perform the same comparison within the HR-positive (HR+) and HR-negative subgroups. Pearsons chi squared tests were performed and a p value of &amp;lt;0.05 was considered statistically significant. RESULTS 331 patients were included in this analysis. 63% were HR+and 37% were TNBC. 50% were HER2-zero and 50% HER-low (36% HER2 IHC 1+ and 14% HER2 IHC 2+/FISH-negative). Median age, initial tumor size, clinical LN status and Ki67 expression were similar among HER2-zero and HER2-low subgroups. In HR+ tumors, 42% (86/207) were HER2-zero and 58% (121/207) were HER2-low. In TNBC, 63% (78/124) were HER2-zero and 37% (46/124) were HER2-low (p&amp;lt;0.001, Pearsons chi squared test). The pCR rate was 26% (85/331) in the entire cohort. As expected, there was a higher rate of pCR in TNBC vs HR+ (50% vs 11%, p&amp;lt;0.001). We found a statistically significant difference in the pCR rates when comparing the HER2-zero versus HER2-low subgroups (31% vs 20%, p=0.03). However, this difference is mostly related to an imbalance between groups (HER2-zero subgroups had a higher proportion of TNBC). Among HR+ tumors, there was no difference in the pCR rates between HER2-zero and HER2-low subgroups (8% vs 13%, p=0.35). In TNBC, we identified an interesting but non-statistically significant difference in pCR in HER2-zero vs. HER2-low tumors (56% vs. 39%, p=0.09). In the TNBC cohort we identified a non-statistically significant difference in RCB 0-I in HER2-zero vs. HER2-low tumors (p=0.06). With a 30 month median follow-up, PFS and OS data are immature. CONCLUSION The distribution of HER2-zero and HER2-low cases is different in HR+ and TNBC. HER2-low is more frequent in HR+ and HER2-zero in TNBC. We identified a higher pCR rate in HER2-zero compared to HER2-low tumors, even though this difference is associated with an imbalance between the two groups. Still, we identified a trend to higher pCR rate in HER2-zero compared to HER2-low tumors even within the TNBC subgroup. Identification of HER2-low and HER2-zero tumors may have clinical implications that should be further explored. Citation Format: Tomas Reinert, Guilherme Parisotto Sartori, Alessandra AB Souza, Rodrigo Pellegrini, Mahira L Rosa, Nathalia Rossatto, Guilherme P Coelho, Isnard E Litvin, Felipe Zerwes, Eduardo Millen, Francisco P Cavalcante, Antonio L Frasson, Marcia S Graudenz, Carlos H Barrios. Prevalence of HER2-low and HER2-zero subgroups and correlation with response to neoadjuvant chemotherapy (NACT) in patients with HER2-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-22.