HER2-positive Carcinoma Research Articles

HER2-positive grade 1 invasive carcinomas of the breast.

The American Society of Clinical Oncology and College of American Pathologists HER2-guidelines recommend repeat testing for most grade 1 mammary carcinomas that are HER2-positive in the core biopsy. This study aimed to assess the value of repeat HER2-testing and the histological features of HER2-positive grade 1 carcinomas. A case-series of HER2-results of grade 1 carcinomas was conducted of patients with no pre-operative systemic treatment over a 5-year period. HER2-positive carcinomas had histological review. Twelve HER2-positive carcinomas were initially reported as grade 1. On review, two were reclassified as grade 2. The remaining 10 carcinomas represented 2% of the 508 grade 1 carcinomas. Eight HER2-positive grade 1 carcinomas from other years were also studied. HER2-positive carcinomas more often had marked nuclear pleomorphism (50 versus 6%) and were more often oestrogen receptor-negative (17 versus 0.8%) and progesterone receptor-negative (28 versus 8%) compared with HER2-negative grade 1 carcinomas. Six carcinomas that were HER2 3+ in the core biopsy were also 3+ on repeat assessment. Five of seven carcinomas that were 2+ amplified in the core biopsy were also HER2-positive in the excision. HER2-positive grade 1 carcinomas are uncommon, and more often have marked nuclear pleomorphism and lack oestrogen receptor and progesterone receptor expression compared with HER2-negative grade 1 carcinomas. A HER2-poitive result in the core biopsy was confirmed in 11 of 13 tumours that had repeat testing.

HER2-Low Luminal Breast Carcinoma Is Not a Homogenous Clinicopathological and Molecular Entity.

With the development of some new antibody-drug conjugates, the HER2 classification of breast carcinomas now includes the HER2-low (H2L) category: IHC 1+, 2+ non-amplified by ISH, and double-equivocal carcinomas, mostly luminal, expressing hormone receptors (HR+). We analyzed mutational status and transcriptomic activities of three HER2 effector pathways: PI3K-AKT, MAPK, and JAK-STAT, in association with clinicopathologic features, in 62 H2L carcinomas compared to 43 HER2-positive and 20 HER2-negative carcinomas, all HR+. H2L carcinomas had significantly lower histoprognostic grades and mitotic and Ki67 proliferation indexes than HER2-positive carcinomas. Their PIK3CA mutation rates were close to those of HER2-negative and significantly higher than in HER2-positive carcinomas, contrary to TP53 mutations. At the transcriptomic level, we identified three distinct groups which did not reflect the new HER2 classification. H2L and HER2-negative carcinomas shared most of clinicopathological and molecular characteristics, except HER2 membrane expression (mRNA levels). The presence of a mutation in a signaling pathway had a strong pathway activation effect. PIK3CA mutations were more prevalent in H2L carcinomas, leading to a strong activation of the PI3K-AKT signaling pathway even in the absence of HER2 overexpression/amplification. PIK3CA mutations may explain the failure of conventional anti-HER2 treatments, suggesting that new antibody-drug conjugates may be more effective.

Abstract PO4-15-11: HER2 STATUS AND TUMOR HETEROGENEITY IN INVASIVE BREAST CARCINOMAS: CLINICAL PATHOLOGICAL IMPACT

Abstract Human epidermal growth factor receptor 2 (HER2) is a major player in breast carcinogenesis with a range of 15-20% of cases harboring gene amplification and overexpression turning the tumor eligible to over eight anti-HER2 drugs in the past 15 years. Recently, novel anti-HER2 antibody-drug conjugates (ADCs) treatment was recently approved to metastatic breast cancer bearing some HER2 expression albeit not due to gene amplification. Immunohistochemically classified, by ASCO-CAP Guidelines (2018), as 1+ cases were enrolled in clinical trials and benefited by the addition of ADCs to chemotherapy. Despite being classified as HER2 negative, most tumors exhibit detectable levels of HER2 protein, reinforcing the importance of HER2 expression evaluation. HER-2 intratumoral heterogeneity (ITH) is reported in up to 40% of breast cancers and is defined as the coexistence of subpopulations of tumor cells with different HER2 gene or protein expression. In this scenario, it becomes very important to evaluate the spectrum of HER-2 expression in correlation with pathological factors as ITH is associated with poor prognosis and anti-HER2 resistance. We designed a retrospective linear study to reanalyze all HER2 immunohistochemical core biopsy slides of patients with primary invasive breast carcinomas (IBC) diagnosed and treated at São Paulo Federal University Hospital between 2019 and 2023. Clinical variables (age, laterality, tumor size by ultrasound imaging, BI-RADS) were collected from clinical files. HER2 slides were reviewed by three observers and reported regarding status by ASCO-CAP 2018 guidelines, HER2 heterogeneity and patterns. Statistical analyses were performed with Chi-Square test of independence, Fisher's exact test and Mann–Whitney U test using IBM® SPSS® Statistics (26.0) software. 353 cases were included; the mean age of the patients at the time of diagnosis was 58 years old. 191 cases (54,1%) were left sided and 162 cases (45,9%) were right sided. Regarding BI-RADS report, the major category was 4 (140 cases), followed by 5 (119 cases). Out of the 164 patients who underwent clinical follow-up at our institute, 14 deaths were reported. Concerning pathological features, IBC of no special type was the prevalent histological subtype with 304 cases (86,1%), followed by invasive lobular carcinoma, with 26 cases (7,4%). 296 cases (83,9%) were HER2 negative and 42 (11,9%) were HER2 positive; 15 cases were HER2 2+ (4,2%). Amongst negative cases, 235 were HER2 0+ and 61 were HER2 1+. Considering ITH, 287 cases (81,3%) were HER2 homogenous with 0+ score as the most prevalent, presenting with 232 cases (80%). 66 cases (18,7%) had ITH, with 1+ as the most prevalent primary score, with 34 cases (51,5%), followed by 3+, with 17 cases (25,8%). Not only HER2 positive carcinomas, but also the presence of heterogeneity in HER2 expression was associated with death outcome (p=0,003 and p=0,001, respectively). Also, HER2 positive IBC had larger tumor size compared to HER2 negative carcinomas (mean size of 25,6 mm x 31,3 mm, respectively; p=0,012); HER2 heterogeneity showed no statistical significance when associated with tumor size (p=0,165). We conclude that ITH is prevalent in HER2 expression and should be addressed in pathology reports since it may play an additional role in tumor progression and drug response, especially in the ADCs scenario. Citation Format: Karla Calaça Kabbach Prigenzi, Lisandra Gonzalez Porta Nova, Adilson Monteiro dos Santos Filho, Ana Luiza da Cruz, Lucas de Figueiredo Barbosa, Angela Flavia Waitzberg. HER2 STATUS AND TUMOR HETEROGENEITY IN INVASIVE BREAST CARCINOMAS: CLINICAL PATHOLOGICAL IMPACT [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 PO4-15-11.

Abstract 2524: Molecular and clinicopathologic landscape of HER2 positive breast cancer: A single institution study

Abstract Background: Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase that is encoded by the ERBB2 gene, located on chromosome 17. Expression levels of HER2 are a key pathogenic and treatment-associated factor in breast carcinomas, with HER2 expression reported as per the ASCO/CAP scoring guidelines. Outside the intense focus on the HER2 overexpression few studies currently exist that look at the comprehensive molecular profile of these aggressive tumors. Design: Our institutional pathology database was searched for patients with breast carcinoma diagnosed between 2009 and 2013 and with HER2 scored as 3+ based on immunohistochemistry, or HER2 2+ with confirmatory positive FISH results. Comprehensive next-generation sequencing (NGS) was performed using the Illumina TruSight Oncology 523 gene panel, and clinicopathologic data were gathered from the institutional electronic health record system. A CoMutation plot was made using the CoMut python library. Results: The mean age of the patients at time of diagnosis was 56.1±12.7 years. The tumors were overwhelmingly high-grade, with 26/30 being grade 3 and the remaining 4/30 being grade 2 and a mean size of 2.6 cm. All were histologically invasive ductal carcinoma, and 26/30 were scored as HER2 3+ on IHC while 4/30 were HER2 2+ with positive FISH. ER was positive in 30% of cases (9/30), PR was positive in 23.3% (7/30), p53 was positive by IHC in 53.3% (16/30), and the mean Ki-67 proliferative index was 47.1%. 20 of the 30 (66%) cases meeting the inclusion criteria had pathogenic mutations detected by NGS testing. Several distinct molecular subgroups were seen on the CoMutation plot (figure 1). The mean tumor mutational burden (TMB) was 14.1 mutations per megabase. A significant subset of the tumors 70% (14/20) showed TP53 mutations, and 30% (6/20) show PIK3CA mutations. Of interest, only 1/14 tumors showed a co-occurring homologous recombination repair gene mutation and one tumor (1/20) showed mutations in 2 homologous recombination pathway genes (FANCA and BRCA1) as well as numerous additional mutations (tumor mutational burden of 65.3 mutations per megabase). Conclusion: Overall the mean TMB was 14.1 mutations/megabase in this HER2-positive breast cancer study set. Over 2/3 of our HER2-positive breast cancer cases also had TP53 mutations. In addition, 1/3 had PIK3CA mutations, suggesting that a subset of HER2-positive carcinomas also harbor other potentially actionable mutations which may increase the effectiveness of our existing interventions. Additional studies are needed to further subclassify genetically distinct subsets of HER2-positive breast cancers and to further understand clinical, pathologic and treatment-related differences in this aggressive breast cancer subset. Citation Format: Gokce Deniz Ardor, Zachary M. Coty-Fattal, Luis Z. Blanco, Kalliopi Siziopikou. Molecular and clinicopathologic landscape of HER2 positive breast cancer: A single institution 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 2524.

PRAME expression and its prognostic significance in invasive breast carcinoma

ObjectivesPRAME (PReferentially expressed Antigen in MElanoma) is a carcinoma testis antigen expressed in numerous tumour types. The aim of this study was to assess PRAME expression in different surrogate subtypes of breast carcinoma and its correlation with other prognostic factors. Material and methodsA total of 220 cases of invasive breast carcinoma were selected and categorized according to ER, PgR, HER2 status, and Ki67 proliferation index in luminal A like, luminal B HER2+ like, luminal B HER2- negative like, HER2 positive like and triple-negative or basal-like. All cases were examined for PRAME expression by immunohistochemistry (IHC). ResultsA PRAME-high profile was detected in 53 (24,1 %) of all examined breast carcinoma samples. A significantly higher expression of PRAME was detected in HER2-positive carcinomas (50 %) and TN breast carcinomas (40,54 %) compared to ER-positive (luminal-like) subtype of breast carcinomas (3,38 % luminal A and 15,38 % luminal B). Percentage of PRAME positive tumour cells showed positive correlation with tumor size, Ki67 proliferation index, HER2 status, nuclear grade, TILs and presence of metastasis, and negative correlation with ER status and disease-free survival (DFS). ConclusionOur study showed that HER2 positive and TN breast carcinomas more commonly express PRAME than ER positive carcinomas and that PRAME expression shows positive correlation with certain prognostic factors, however PRAME wasn't revealed as an independent prognostic factor in our study. The importance of PRAME expression in breast carcinoma lies in its potential use as an immunotherapeutic target, particularly in patients with limited therapeutic options.

Abstract ED3-1: Updates in the pathology of PABC

Abstract Updates in the Pathology of Pregnancy Associated Breast Cancer (PABC) Pregnancy associated breast cancer is defined as a breast cancer diagnosed during gestation, lactation and within 1 to 5 years postpartum. While the development of malignancy during pregnancy is rare, the incidence is increasing; breast cancer is one of the most common cancers diagnosed during pregnancy and the postpartum period occurring in up to 1 in 3000 deliveries. Of interest, breast cancer is the leading cause of cancer death in US women age 15-29. Pregnancy has a dual effect on breast cancer development: on one hand cancer protective and on the other cancer promoting. While a number of hypotheses have been proposed over the years to explain these effects, the most likely hypothesis for the development of PABC is the involution hypothesis. This hypothesis proposes that the involution pathways activated during pregnancy and the immediate postpartum period are remodeling programs similar to wound healing and inflammation that may be associated with tumor development and progression. Although PABCs can be any subtype of breast carcinomas, they are usually invasive ductal carcinomas of high tumor grade and large tumor size with higher stage at presentation and higher rates of lymph node involvement. Most PABCs are hormone receptor negative tumors with high Ki-67 proliferation rates; most frequently, they are either triple negative or HER2-positive carcinomas. A number of studies have shown that PABCs have different genomic signatures than the non-PABC tumors with PABCs having an increased expression of immune response mediators. Better understanding of the molecular pathways of tumor initiation and progression and prompt diagnosis and state-of-art treatment protocols in PABC is expected to lead to better outcomes for these young breast cancer patients. Citation Format: Kalliopi Siziopikou. Updates in the pathology of PABC [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 ED3-1.

Comparing treatment and outcomes in advanced esophageal, gastroesophageal junction, and gastric adenocarcinomas: a population-based study.

Treatment of advanced or metastatic esophageal adenocarcinoma (EAC) follows the guidelines for gastroesophageal junction adenocarcinoma (GEJC) and gastric adenocarcinoma (GAC), but patients with EAC are often excluded from clinical studies of GEJC/GAC. Here we describe treatment and survival of patients with advanced EAC, GEJC, and GAC to provide population-based evidence on distinctions and similarities between these populations. Retrospective cohort study of patients with unresectable advanced (cT4b) or metastatic (cM1) EAC, GEJC, or GAC (2015-2020) were selected from the Netherlands Cancer Registry. Overall survival (OS) was assessed using Kaplan-Meier methods, log-rank tests, and multivariable Cox regression. In all, 7391 patients were included (EAC: n = 3346, GEJC: n = 1246, and GAC: n = 2798). Patients with EAC were more often males and more often had ⩾2 metastatic locations. First-line systemic therapy was received by 42%, 47%, and 36% of patients with EAC, GEJC, and GAC, respectively. Median OS was 5.0, 5.1, and 4.0 months for all patients with EAC, GEJC, and GAC, respectively (p < 0.001). Median OS from start of first-line therapy of patients with human epidermal growth factor receptor 2 (HER2)-negative adenocarcinomas was 7.6, 7.8, and 7.5 months (p = 0.12) and of patients with HER2-positive carcinoma receiving first-line trastuzumab-containing therapy was 11.0, 13.3, and 9.5 months (p = 0.37) in EAC, GEJC, and GAC, respectively. After multivariable adjustment, no difference in OS for patients with EAC, GEJC, and GAC was observed. Despite differences in clinical characteristics and treatment strategies, survival between patients with advanced EAC, GEJC, and GAC was similar. We advocate that EAC patients should not be excluded from clinical trials for patients with molecularly similar GEJC/GAC.

Association between the Expression of KISS1, KISS1R and MMP-9 in Invasive Breast Carcinomas

Metastases are the leading cause of fatal outcome in patients with breast cancer, therefore different proteins related to metastasis are widely studied for the potential to control the spread of cancer cells. It has been shown in various types of cancers that the anti-metastatic role of the KISS1/KISS1R system has been performed by suppressing metalloproteinase 9 (MMP-9) expression, but there is evidence that the relationship between KISS1/KISS1R signalling and MMP-9 in breast cancer seems to be different. The aim of the current study was to assess the association between protein expression of KISS1, KISS1R and MMP-9 using software-based measurement of staining intensity and the possible link to tumour size, grade and receptor status in invasive breast carcinomas. Standard histopathological parameters were determined by an experienced pathologist and immunohistochemical staining for KISS1, KISS1R and MMP-9 was performed on tissue samples from 54 patients. Evaluation of staining intensity was performed using ImageJ software on RGB images by two operators. Results showed significant strong positive correlation between KISS1, KISS1R and MMP-9 and strong expression of studied proteins in tumour associated stromal cells. No association was found between the expression of the studied proteins and size, receptor status or grade. Higher expression of KISS1R, however, was observed in HER2-negative compared to HER2-positive carcinomas, which might indicate an alternative pathway for stimulating pro- proliferation of tumour cells when HER2 expression is low.

Disitamab vedotin: a novel antibody-drug conjugates for cancer therapy

Human epidermal growth factor receptor 2 (HER2) regulates cell mitosis, proliferation, and apoptosis. Trastuzumab is a HER2-targeted monoclonal antibody (mAB), which can prolong the overall survival rate of patients with HER2 overexpression in later periods of gastric cancer and breast cancer. Although anti-HER2 monoclonal antibody has a curative effect, adjuvant chemotherapy is still necessary to upgrade the curative effect maximumly. Antibody-drug conjugate (ADC) is a kind of therapeutic drug that contains antigen-specific antibody and cytotoxic payload, which can improve the survival time of tumor patients. To date, there are several HER2-ADC products on the market, for which two anti-HER2 ADC (trastuzumab emtansine and trastuzumab deruxtecan) have been authorized by the FDA for distinct types of HER2-positive carcinoma in the breast. Disitamab vedotin (RC48) is a newly developed ADC drug targeting HER2 that is comprised of hertuzumab coupling monomethyl auristatin E (MMAE) via a cleavable linker. This paper aims to offer a general insight and summary of the mechanism of action and the currently completed and ongoing clinical studies of RC-48 in HER-2 positive solid tumors.

Poor histologic tumor response after adjuvant therapy in basal-like HER2-positive breast carcinoma

AimsHER2-positive breast carcinomas are all treated with first-line anti-HER2 therapy. However, immunohistochemical and molecular profiling demonstrates significant heterogeneity among HER2-positive carcinomas. Basal-like HER2-positive breast carcinomas are poorly differentiated from pure HER2-positive breast carcinomas. Materials and methodsSeventy-five patients with HER2-positive, ER- and PR-negative breast carcinomas who received anti-HER2 based neoadjuvant therapy were retrospectively analyzed. Thirty-seven cases were classified as basal-like HER2-positive breast carcinoma with any positivity for CK5/6, and thirty-eight cases were classified as pure HER2-positive breast carcinoma with completely negativity for CK5/6. The clinicopathological features and tumor responses after neoadjuvant therapy and outcomes were analyzed. ResultsCompared to non-basal HER2-positive breast carcinoma, basal-like HER2-positive breast carcinoma showed distinctive histologic features including poor differentiation and syncytial tumor cells with pushing, invasive borders and a significantly higher proportion of apocrine metaplasia. They also demonstrated significantly higher histologic grade; 18/37 (48.6%) of basal-like carcinomas were grade 3, whereas only 5/38 (13.2%) of non-basal carcinomas were grade 3 (p = 0.001), Furthermore, basal-like HER2-positive breast carcinomas were more likely to be positive or completely negative for p53 (p = 0.009), and demonstrated a higher percentage of TP53 mutation (p = 0.17). These tumors were less responsive to anti-HER2 based neoadjuvant therapy, with Miller-Payne grades 1–3 higher than pure HER2-positive breast carcinoma (25/37 [67.6%] vs 16/38 [42.1%]), and the percentage of grade 4–5 was lower (12/37 [32.4%] vs 22/38 [57.9%]; p = 0.027). ConclusionsBasal-like HER2-positive breast carcinoma has distinctive clinicopathological features and less histologic tumor response after neoadjuvant therapy. There is urgent need to recognize basal-like HER2-positive breast carcinoma to be treated precisely.

The age-specific differences in histopathological tumor characteristics and TNM classification of breast carcinomas in Quality assured mamma diagnostic (QuaMaDi) program in the state of Schleswig\u2013Holstein in Germany

BackgroundWe explored the hypothesis that high-quality standards in diagnostic mammography can lead to an early diagnosis of breast cancers and identifies at risk populations outside screening programs. The histopathological features and distribution of the TNM classification were examined in relation to patient age in a large group of women with breast cancers participating in the Quality Assured Mamma Diagnostic (QuaMaDi) program of the state of Schleswig–Holstein.Patients and methodsSurgical pathological reports were studied for clinicopathological characteristics, receptor status, molecular subtype and tumor stage. The analysis was conducted by dividing the study population into three age groups: women under 50 years (pre-screening), 50–69 years (peri-screening) and over 70 years (post-screening).Results7.111 biopsies and 2.887 resection specimens were included. Breast cancer was diagnosed in 4.241 (59.7%) cases, one fourth of them in women < 50 years. Elderly women (> 70 years) had more well-differentiated, estrogen receptor (ER)-positive and HER2-negative carcinomas, whereas younger women (< 50 years) tended to have more poorly differentiated, ER negative, and HER2-positive carcinomas. 47% of breast carcinoma were luminal B tumors and were most common regardless of age. 70.4% of resected specimen had pT1 stage. Nodal negative were 71.2%.ConclusionIn QuaMaDi breast cancer was diagnosed at an early and potentially curable stage of the disease due to high-quality standards in diagnostic mammography. In addition, regardless of age, an increased number of prognostically unfavorable molecular subtypes were detected. Thus, QuaMaDi helps to identify at risk populations. QuaMaDi significantly improves diagnostic mammography and complements mammography screening programs.

Abstract LB170: HER2-targeted interferon-beta-1a mutein, a potent immunocytokine for the treatment of HER2-positive cancers

Abstract Background: Interferon beta (IFN-β), a promising potent cytokine, has been attracting attention for treatment of cancer. The pleiotropic antitumor effects of IFN-β have been studied, with specific reference to their direct role on cancer cells and indirect action through the immune effector cells. However, its systemic toxicities and poor biophysical properties prevent IFN-β from being widely used for cancer therapy. Since the potential of cytokine therapies are impaired due to dose-limiting systemic toxicities, novel treatment methods should be developed to safely deliver effective drug quantities at the tumor sites. Like antibody-drug conjugates (ADCs), immunocytokine can be attempted to induce cytokine's organ-targeting and alleviate its systemic side effects. Here, we designed recombinant IFN-β mutein immunocytokines that comprise a HER2-targeting antibody and IFN-β mutein, and evaluate the antitumor properties against HER2-positive cancers.Method: A panel of human gastric cancer cell lines was treated with trastuzumab-IFN-β mutein to evaluate direct antitumor effect. In addition, to test the immune cell-mediated antitumor effect, cancer cells were co-cultured with effector cells (e.g. PBMC) in the absence or drug. The antitumor efficacy of trastuzumab-IFN-β mutein in vivo was tested in HER2-positive cancer xenograft models using nude mice or humanized mice. Result: Trastuzumab-IFN-β mutein directly inhibited the growth of HER2-positive gastric cancer cell lines and was more effective than trastuzumab or IFN-β mutein alone. Trastuzumab-IFN-β mutein also displayed enhanced immune cell-mediated cytotoxicity. Collectively, trastuzumab-IFN-β mutein may have indirect immune cell-mediated antitumor effects and direct cell growth inhibitory effects. Moreover, trastuzumab-IFN-β mutein significantly suppress tumor growth in HER2-positive cancer xenograft models. Tumor-infiltration of lymphocytes was enhanced by trastuzumab-IFN-β mutein, implying that the tumor-targeting IFN-β may have an antitumor effect through increased immune response. Conclusion: We have characterized and evaluated the antitumor properties of trastuzumab-IFN-β mutein in HER2-expressing cancers. Since IFN-β activates antitumor immune responses, it is expected to be administered in combination with immunotherapeutic drugs. Therefore, the study suggests that trastuzumab-IFN-β mutein is a promising candidate for the treatment of HER2-positive carcinoma. Citation Format: Chan Gyu Lee, Tae Eun Kim, Sungyoul Hong, Jongwan Chu, Ju Eun Kang, Hae Min Jeong, Young Kee Shin. HER2-targeted interferon-beta-1a mutein, a potent immunocytokine for the treatment of HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB170.

Clinicopathologic features of unexpectedly HER2 positive breast carcinomas: An institutional experience

Human epidermal growth factor receptor 2 (HER2) overexpression occurs in 15–20 % of all breast carcinomas. These tumors are usually high-grade which often correlates with reduced overall survival and increased rates of recurrence. In a retrospective review, we identified 19 cases of unexpectedly HER2 positive (by immunohistochemistry and/or fluorescence in-situ hybridization) invasive breast carcinomas on core needle biopsies from a registry at Northwestern Memorial Hospital. These cases included low-grade tumors, invasive lobular carcinomas, classic type, and invasive carcinomas with special subtype features. Twelve of the tumors were histologic grade 1 and 7 were histologic grade 2. One of the grade 1 tumors had tubular features (8 %), 1 had cribriform features (8 %), 2 had mucinous features (17 %), 2 were invasive lobular carcinomas, classic type (17 %), and the rest were invasive carcinoma, no special type (50 %). The histologic grade 2 tumors included 5 invasive lobular carcinomas, classic type (71 %) and 2 invasive ductal carcinomas with mucinous features (29 %). By immunohistochemistry, 13 (65 %) were HER2 score 3+, 7 were score 2+ (35 %), and reflex fluorescence in-situ hybridization (FISH) testing showed amplification in 6 cases, with 1 equivocal case amplified on excision. Despite the HER2 positive status in the selected cases, no unique morphologic features that would indicate aggressive behavior were identified. In clinical follow up, two patients were found to have recurrences, five had lymph node metastasis, and one had distant metastasis. None of the patients with recurrent disease were treated with trastuzumab, despite their positive HER2 results. These findings support that our population of HER2 positive carcinomas showed a similar rate of lymph node metastases and recurrence as poorly-differentiated tumors, supporting HER2 positivity as a poor prognostic indicator, irrespective of morphologic features. We recommend continuing to test all breast cancers, regardless of grade or special subtype features, to provide the most comprehensive treatment and prognostic information for both clinicians and patients.

Abstract P4-10-30: PD-L1 expression in tumor infiltrating lymphocytes (TILs) inversely correlates with androgen receptor (AR) expression in HER2-positive breast cancer

Abstract Background: Human epidermal growth factor receptor 2 (HER2)-positive breast cancer accounts for 20-30% of breast carcinomas and is associated with a poor prognosis. While anti-HER2 targeted therapies have improved outcomes within this group, a subset of these tumors eventually develop resistance urgently requiring novel therapeutic options. In addition to classic HER2+ subtype, estrogen receptor (ER)-positive and HER2-positive tumors (luminal B) are another subtype of HER2 expressing breast carcinomas which could also benefit from additional therapies. Targeting the androgen receptor (AR) pathway and the PD-L1 axis may represent potential new therapeutic strategies in breast cancer. Our group previously reported that AR expression is seen in ¾ of HER2+ carcinomas and is more common in luminal B compared to classic HER2+ tumors. In this report, we evaluated expression of PD-L1 in tumor infiltrating lymphocytes (TILs) in both classic HER2 + and Luminal B subtypes of breast carcinomas and correlated the expression of PD-L1 with AR expression in these tumors. Methods: The study population consisted of 114 patients with HER2-positive invasive breast carcinoma diagnosed from 2009-2014 at Northwestern Memorial Hospital. Electronic medical records were reviewed for patient demographics (mean age 54, range 23-80). Pathologic tumor characteristics (histologic type, size, and grade) and tumor marker profile (ER and HER2) were evaluated. Tissue microarrays were constructed (3 cores from each case to account for tumor heterogeneity) for immunohistochemical evaluation of AR and PD-L1. AR was graded as positive when at least 1% of tumor cells showed nuclear immunoreactivity. For PD-L1, the extent of staining (0%=0, 1-10%=1, 11-50%=2, and 51-100%=3) and staining intensity (0, 1+, 2+, 3+) were assessed in TILs. Then a composite score (CS) was calculated by multiplying extent and intensity results (range 0-9; 0-3=negative, 4-9=positive). Results: Of the 114 studied breast carcinoma cases, 54 were classic HER2+ tumors (47%) and 60 were luminal B (53%) tumors. Approximately 69% of classic HER2+ and 87% of luminal B cases were positive for AR. Of interest, in classic HER2+ cases, the expression of PD-L1 in TILs was inversely correlated with the expression of AR, with 82% (14/17) of AR- tumors being positive for PD-L1 in TILs compared to only 46% (17/37) of AR+ tumors with PD-L1 expression in TILs (p&amp;lt;0.01). No association of expression of PD-L1 in TILs with AR expression was observed in luminal B cases (62% in AR+ versus 50% in AR- tumors; p=0.70). Moreover, in classic HER2+/AR- group, grade 3 tumors were seen almost exclusively in the PD-L1 expressing group (13 versus 3), in contrast to the equal number of grade 3 tumors in classic HER2+/AR+ group regardless of PD-L1 expression in TILs (14 versus 14). No association of the PD-L1 expression with age, race, and tumor histologic types was observed in classic HER2+/AR+ or classic HER2+/AR- cases. Conclusions: Our results demonstrated that the expression of PD-L1 in TILs was inversely correlated with the expression of AR in classic HER2 positive tumors but not in luminal B tumors. We also found that in classic HER2+/AR- group, grade 3 tumors were seen almost exclusively in the PD-L1 expression group. Our findings add to the body of knowledge of the molecular mechanisms that drive the different molecular subtypes of breast carcinomas and raise the interesting possibility that new treatment strategies such as targeting the PD-L1 axis may show more benefit against the AR negative subtype of the classic HER2 positive carcinomas. Citation Format: Tiansheng Shen, Jennifer L. Pincus, Kalliopi P. Siziopikou. PD-L1 expression in tumor infiltrating lymphocytes (TILs) inversely correlates with androgen receptor (AR) expression in HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-30.

A novel detection methodology for HER2 protein quantitation in formalin-fixed, paraffin embedded clinical samples using fluorescent nanoparticles: an analytical and clinical validation study

BackgroundClinical assays for the assessment of the human epidermal growth factor receptor-2 (HER2) status in breast cancer include immunohistochemistry (IHC) and in situ hybridization (ISH), both of which have limitations. Recent studies have suggested that a more quantitative approach to the measurement of HER2 protein expression may improve specificity in selecting patients for HER-2 targeted therapy. In the current study, we have used HER2 expression in breast cancer cell lines and clinical samples as a model to explore the potential utility of a novel immunodetection technique, using streptavidin coated Phosphor Integrated Dot fluorescent nanoparticles (PID), which can be quantitatively measured using computer analysis.MethodsThe expression of HER2 protein in cell lines was evaluated with antibody-binding capacity using fluorescence-activated cell sorting (FACS) for comparison with PID measurements to test for correlations with existing quantitative protein analysis methodologies. Various other analytic validation tests were also performed, including accuracy, precision, sensitivity, robustness and reproducibility. A methods comparison study investigated correlations between PID versus IHC and ISH in clinical samples. Lastly, we measured HER2 protein expression using PID in the pretreatment biopsies from 34 HER2-positive carcinomas that had undergone neoadjuvant trastuzumab-based chemotherapy.ResultsIn the analytic validation, PID HER2 measurements showed a strong linear correlation with FACS analysis in breast cell lines, and demonstrated significant correlations with all aspects of precision, sensitivity, robustness and reproducibility. PID also showed strong correlations with conventional HER2 testing methodologies (IHC and ISH). In the neoadjuvant study, patients with a pathologic complete response (pCR) had a significantly higher PID score compared with patients who did not achieve a pCR (p = 0.011), and was significantly correlated to residual cancer burden (RCB) class (p = 0.026, R2 = 0.9975).ConclusionsAnalytic testing of PID showed that it may be a viable testing methodology that could offer advantages over other experimental or conventional biomarker diagnostic methodologies. Our data also suggests that PID quantitation of HER2 protein may offer an improvement over conventional HER2 testing in the selection of patients who will be the most likely to benefit from HER2-targeted therapy. Further studies with a larger cohort are warranted.

Standardized determination of tumor-infiltrating lymphocytes in breast cancer : A prognostic marker for histological diagnosis

Tumor-infiltrating lymphocytes (TILs) have been identified as prognostic parameter in breast cancer. The aim of this review article is to provide an overview on the clinical and analytical validation of TILs in breast cancer. Summary of international guidelines of the TIL working group as well as clinical and translational studies. Breast carcinomas with ahigh TIL level have an improved response to neoadjuvant chemotherapy. Triple-negative and HER2-positive carcinomas with increased TIL levels have improved survival. TILs are anew prognostic biomarker for routine histopathological diagnosis.

Phase I study of TrasGEX, a glyco-optimised anti-HER2 monoclonal antibody, in patients with HER2-positive solid tumours

PurposeTrasGEX is a second-generation monoclonal antibody of trastuzumab, glyco-optimised to enhance antibody-dependent cellular cytotoxicity while fully retaining trastuzumab’s antigen-binding properties to human epidermal growth factor receptor 2 (HER2). A phase I dose-escalation study was conducted to establish the optimal TrasGEX dose and regimen for phase II studies and to define the safety, pharmacokinetics (PK) and preliminary antitumour activity of TrasGEX.Patients and methodsA total of 37 patients with advanced HER2-positive carcinomas and progressive disease received TrasGEX intravenously every 3 weeks until disease progression in doses of 12–720 mg in a three-plus-three dose escalation design, including an expansion cohort at the highest dose.ResultsNo dose limiting toxicity was observed, and no maximum tolerated dose was reached. Drug-related adverse events were mainly infusion-related reactions occurring during the first infusion in 51% of patients; all but two were mild-to-moderate. Compared with trastuzumab, the PK parameters were dose dependent, with a mean terminal half-life (t1/2) of 263±99 hours for the 720 mg dose. Clinical benefit in 15 out of 30 (50%) evaluable patients included one ongoing complete response, two partial remissions lasting 16 and 77 weeks and disease stabilisation (SD) in 12 patients lasting a median (range) of 17 (7–26) weeks; three of them had SD of 24, 25 and 26 weeks, respectively.ConclusionTrasGEX was safe, well-tolerated and showed antitumour activity in 50% of evaluable patients, all with progressive disease at study entry. Infusions at an interval of 2–3 weeks should achieve clinically relevant trough levels for future studies (NCT01409343).

Selection of DNA aptamers for extra cellular domain of human epidermal growth factor receptor 2 to detect HER2 positive carcinomas

Human epidermal growth factor receptor 2 (Her2, an orphan receptor of ErbB family) is considered as an important biomarker as it plays a key role in the development and progression of aggressive types of breast, ovarian, stomach and gastric cancer. In the present study, we developed novel DNA aptamers against the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive carcinomas. We cloned and expressed Her2-ECD protein in E. coli system. After purification, the protein was used as a bait for screening of specific DNA aptamer candidate from a pool of 1014-15 random oligonucleotides through in vitro Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was estimated by FACS and immunofluorescence assay. The specificity of the candidate was also verified with the tissue samples of breast cancer patients by immunohistochemistry process. Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24) showed the highest binding affinity (K d=6.33±0.86nM) to Her2-ECD protein. The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at 490nm wavelength) in the dynamic range of 100-700nM ECD_Apt1 with a detection limit of 12.5±2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic staining in Her2-positive breast cancer cell lines (SKBR3) compared to Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue sections, it showed specific and selective localization in the cytoplasmic niche of malignant duct cancer cells without any cross-reactivity to fibroblasts, inflammatory cells and adipocytes. Binding assays, cytochemical and histochemical studies support ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1 could be an effective low-cost alternative to conventional anti-Her2 antibody in solid phase immunoassays for cancer diagnosis and related applications.

Cross-reactivity of EGFR L858R mutation-specific antibody in HER2 positive gastric adenocarcinomas

Recently, the study of the EGFR mutations in lung cancer has become mandatory due to their usefulness in target therapy. Lately, the use of specific antibodies to study the most prevalent of these mutations has been introduced. The newly published discovery of the existence of a cross-reaction between the HER2 protein and the EGFR L858R-specific antibody in breast cancer led us to corroborate this cross-reactivity in other tumors; specifically, we have chosen gastric carcinomas where HER2 is routinely evaluated as a molecular therapy driver. Our series consists of 15 primary gastric carcinomas, 7 HER2 cases positive for overexpression/amplification and 8 negative cases for both techniques. EGFR L858R mutation was studied with immunohistochemistry and complemented with real time PCR when positive. Immunohistochemistry assay with EGFR L858R was positive in 5 of the HER2 positive carcinomas (71%), none of which was confirmed by PCR, and negative in all HER2 negative carcinomas. The EGFR L858R antibody gives false positive results in most gastric carcinomas with HER2 overexpression/amplification, confirming the results described previously in breast cancer. It is also important to bear in mind that HER2 has also been described in other carcinomas, including lung cancer. En los últimos años, el estudio de las mutaciones de EGFR en cáncer de pulmón se ha convertido en esencial por su utilidad como diana terapéutica. Recientemente se ha introducido el uso de anticuerpos para la detección de las mutaciones más prevalentes. El descubrimiento de la existencia de una reacción cruzada entre la proteína HER2 y el anticuerpo específico EGFFR L858R, recientemente publicado en cáncer de mama, nos condujo a explorar la existencia de dicha reacción cruzada en otros tumores. Seleccionamos en primer lugar el cáncer gástrico, en el que se evalúa HER2 de forma rutinaria para la identificación de candidatos a tratamiento con terapia dirigida. La serie incluyó 15 carcinomas gástricos primarios, 7 casos positivos para sobreexpresión/amplificación de HER2 y 8 casos negativos para ambas técnicas. La presencia de la mutación EGFR L858R se estudió por inmunohistoquímica, y en caso de resultado positivo se complementó con el estudio molecular por PCR a tiempo real para su confirmación. El estudio inmunohistoquímico de EGFR L858R resultó positivo en 5 de los carcinomas HER2 positivos (71%), sin que ninguno se confirmara por PCR, y fue negativo para todos los carcinomas HER2 negativos. El anticuerpo EGFR L858R presenta resultado falso positivo en la mayoría de carcinomas con sobreexpresión/amplificación de HER2, confirmando los resultados descritos previamente en cáncer de mama. Es importante tener presente que la presencia de HER2 ha sido descrita en otros carcinomas, incluido el cáncer de pulmón.

Gene status in HER2 equivocal breast carcinomas: impact of distinct recommendations and contribution of a polymerase chain reaction-based method.

The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.