High HER2 Research Articles

Abstract TF2-3: Molecular heterogeneity in HER2+ breast cancer - can outcomes be predicted?

Abstract HER2+ breast cancer is clinically and biologically heterogenous. In the last 10 years, translational research in this disease has deciphered many biological features, which are associated with clinical outcomes both in early and metastatic disease. Among them, RNA-based gene expression has identified 4 main molecular subtypes (HER2-enriched, Basal-like, Luminal A and Luminal B), and HER2-enriched tumors within HER2+ breast cancer show high HER2 activation and response to anti-HER2 therapy. At the same time, ERBB2 mRNA levels by itself have also been linked to a higher response to anti-HER2 therapy, including T-DM1. Whether these 2 biological features will predict response to novel anti-HER2 therapies such as DS8201a is currently unknown. One important aspect is that levels of the target (i.e., HER2/ERBB2) and the activation of the downstream signaling pathway (i.e., HER2-enriched) are related but different at the same time. In other words, higher levels of ERBB2 mRNA are associated with a HER2-enriched phenotype; however, a substantial proportion of HER2+ tumors with low levels of ERBB2 are also HER2-enriched. Thus, they should not be considered the same, and this could have clinical implications. Similarly, hormone receptor negative HER2+ breast cancer has higher levels of ERBB2 and higher proportion of HER2-enriched disease than hormone receptor-positive. The high sensitivity of hormone receptor-negative and HER2-enriched disease to anti-HER2 therapy has been demonstrated in the neoadjuvant setting as well as studies looking at on-treatment biology after short-course of anti-HER2 treatment. Indeed, in advanced HER2+ breast cancer, the major gains in overall survival with new anti-HER2-based therapies are seen in hormone receptor-negative disease. Finally, tumor microenvironment plays a critical role. Indeed, tumor-infiltrating lymphocytes and immune gene expression signatures are associated with treatment response and better survival outcome. Whether this immune-related variables predict response to antiPD1/PDL1 is currently unknown, although translational studies do suggest that there might be a link. To conclude, the biological heterogeneity that exists within HER2+ disease is now well-known, and these variables, together with clinical-pathological features, should allow the design of new biomarkers to help answer critical clinical questions. Citation Format: A Prat. Molecular heterogeneity in HER2+ breast cancer - can outcomes be predicted? [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr TF2-3.

Immune microenvironment characteristics and their implications for immune checkpoint inhibitor efficacy in HER2-overexpressing gastric cancer.

HER2-positive gastric cancer is a distinct tumor subtype, accounting for ~10% of gastric cancer cases. It is characterized by HER2 overexpression and responds well to HER2-targeting therapies. Recently, the addition of immune checkpoint inhibitors to HER2-targeting therapies produced satisfactory outcomes in these patients. In the present study, we used gene expression profiles and patient surgical sections to analyze the tumor immune microenvironment characteristics of gastric tumors with high HER2 expression. Several differentially enriched pathways were identified between the HER2 high-expression group and the low-expression group, such as pathways related to cytokine-cytokine receptor interactions, calcium signaling, and cell adhesion molecules. Tumors with high HER2 expression comprised fewer stromal cells and fewer immune cells, and had higher tumor purity. They also presented with lower expression of PD-1, PD-L1, CTLA-4, TIGIT, and LAG-3. In conclusion, our study provides a comprehensive blueprint of the immune microenvironment of HER2-positive gastric tumors. This analysis highlights the importance of considering the tumor microenvironment when assessing response to immune checkpoint inhibitors.

Correlation analysis of lymphocyte-monocyte ratio with pathological complete response and clinical prognosis of neoadjuvant chemotherapy in patients with breast cancer

PurposeInflammation plays an important role in tumor proliferation, metastasis, and chemotherapy resistance. Peripheral blood lymphocyte-monocyte ratio (LMR) has been reported to be closely associated with the prognosis of many tumors, such as certain hematologic malignancies and gastric cancer. However, the association in breast cancer is still not clear. This study investigated the relationship between LMR with pathological complete response and clinical prognosis of neoadjuvant chemotherapy in patients with breast cancer, to provide convenient and accurate predictive indicators for pathological complete response (pCR) and prognosis. MethodsThe clinicopathological data of 192 female breast cancer patients who received neoadjuvant chemotherapy and surgery in Harbin Medical University Tumor Hospital from January 2013 to August 2017 were retrospectively analyzed. Blood lymphocytes and monocytes were obtained by peripheral venous punctures. ResultsCompared with the low LMR group, pCR was more easily obtained in the high LMR group (P=0.020); Subgroup analysis showed that patients with the high LMR and HER-2(+) group were more likely to obtain pCR (P=0.011).Univariate andmultivariate results showed that the overall survival (OS) and disease free survival (DFS) of the high LMR group were longer than that of the low LMR group. ConclusionLMR and HER-2 status are correlated with pCR of neoadjuvant chemotherapy in breast cancer patients and are independent predictors of pCR after neoadjuvant chemotherapy in breast cancer patients. Meanwhile, both LMR and T stage of tumor are independent prognostic factors of breast cancer patients, with good predictive value.

Dual Inhibition of Myc Transcription and PI3K Activity Effectively Targets Colorectal Cancer Stem Cells

Simple SummaryCompelling evidence has shown that cancer stem cells (CSCs) are responsible for high resistance to conventional anti-cancer therapies. Here, we demonstrate that the tumor microenvironment protects CR-CSCs from EGFR/HER2, BRAF and PI3K targeting, promoting CD44v6 and Myc expression. Alternatively, as a substitution for HER2 and BRAF, the Myc transcription inhibitor can overcome the protective effects of microenvironmental cytokines, impairing the survival of CR-CSCs. These data highlight the targeting of Myc and PI3K activity as a novel therapeutic strategy against advanced colorectal cancer.Despite advances in the curative approach, the survival rate of advanced colorectal cancer (CRC) patients is still poor, which is likely due to the emergence of cancer cell clones resistant to the available therapeutic options. We have already shown that CD44v6-positive CRC stem cells (CR-CSCs) are refractory toward standard anti-tumor therapeutic agents due to the activation of the PI3K pathway together with high HER2 expression levels. Tumor microenvironmental cytokines confer resistance to CR-CSCs against HER2/PI3K targeting by enhancing activation of the MAPK pathway. Here, we show that the CSC compartment, spared by BRAF inhibitor-based targeted therapy, is associated with increased expression levels of CD44v6 and Myc and retains boosted clonogenic activity along with residual tumorigenic potential. Inhibition of Myc transcription, downstream of the MAPK cascade components, and PI3K pathway activity was able to overcome the protective effects of microenvironmental cytokines, affecting the survival and the clonogenic activity of CR-CSCs, regardless of their mutational background. Likewise, the double targeting induced stabilization of mouse tumor avatars. Altogether, these data outline the rationale for dual kinase targeting of CR-CSCs to prevent their adaptive response, which would lead to disease progression.

The influence of degree of labelling upon cellular internalisation of antibody-cell penetrating peptide conjugates.

Antibody-based agents are increasingly used as therapeutics and imaging agents, yet are generally restricted to cell surface targets due to inefficient cellular internalisation and endosomal entrapment. Enhanced cell membrane translocation of antibodies can be achieved by the covalent attachment of cell-penetrating peptides, including the HIV-1-derived transactivator of transcription (TAT) peptide. This study evaluated the cellular internalisation properties of five anti-HER2 Herceptin-TAT conjugates with degrees of TAT labelling (DOLTAT) ranging from one to five. Herceptin-TAT conjugates were synthesised via a strain-promoted alkyne-azide cycloaddition reaction, characterised by UV-vis spectroscopy, MALDI-TOF, and gel electrophoresis, then radiolabelled with zirconium-89 to permit measurement of cellular internalisation by gamma counting. [89Zr]Zr-DFO-Her-TAT(0-5) conjugates were isolated in high radiochemical purity (>99%) and exhibited high stability in murine and human serum over 7 days at 37 °C. Significant increases in cellular internalisation were observed for [89Zr]Zr-DFO-Her-TAT conjugates with DOLTAT values of 2 and above in SKBR3 (high HER2) cells over 48 h, in contrast to low-level non-specific uptake in MDA-MB-468 (low HER2) cells that did not increase over time. Notably, [89Zr]Zr-DFO-Her-TAT conjugates with DOLTAT of 3, 4, and 5 reached uptake values in SKBR3 cells of 5, 6, and 8% of the applied dose at 48 h respectively, representing 9, 10, 14-fold increases relative to the TAT-free control conjugate, [89Zr]Zr-DFO-Her-TAT(0).

A study of Her-2/Neu oncogene expression in benign and malignant endometrial lesions and its association with clinicopathologic variables

Introduction: Endometrial carcinoma is commonly diagnosed gynaecological malignancy in women and the incidence of endometrial cancer and associated mortality rates have increased in recent years. Endometrial hyperplasia is recognized precursor lesion of endometrial cancer and its timely detection offers an opportunity to prevent the development of endometrial cancer. Her-2/neu oncogene amplification has been implicated in the development and progression of many tumours and its overexpression may be involved in tumorigenesis of endometrial carcinoma as well. The present study was aimed to evaluate Immunohistochemical (IHC) expression of Her-2/neu oncogene in Benign and Malignant Endometrial Lesions and its correlation with clinicopathological features in order to define its potential prognostic significance. Material and Methods : A total of 62 cases including 20 cases of endometrial hyperplasia and 42 cases of Endometrial Carcinoma were enrolled in the study. Clinical and pathologic information was obtained and recorded. Her-2/neu expression was evaluated by immunohistochemistry (IHC) on paraffin embedded tissue sections with Her-2/neu antibody. Overexpression was defined as complete, membrane, moderate to strong intensity staining in greater than 10% of cells. Results: Positive HER-2/neu expression is seen in 45% cases of Endometrial Hyperplasia and in 54.76% cases of endometrial carcinoma. The Her-2/neu overexpression was observed in patients with advanced stage (p Conclusion: In the present study Abnormal high Her-2/neu expression was observed in Endometrial carcinoma, correlating with High FIGO stage, high grade, lymph node metastasis, myometrial invasion and cervical invasion, indicating Her-2/neu expression analysis could be an important prognostic factor of Endometrial carcinoma. Keywords: Endometrial carcinoma, Endometrial Hyperplasia, HER­2/neu, prognostic factor

Abstract P167: Site-specific Dolasynthen ADCs demonstrate consistent exposure across a wide range of drug-to-antibody ratios

Abstract Key defining attributes of an antibody-drug conjugate (ADC) include the choice of targeting antibody, linker, and the drug-to-antibody ratio (DAR). The choice of DAR, within the constraints of acceptable physicochemical properties for the given platform, is a function of balancing delivery of sufficient payload to targeted cells with the ability to achieve sustained in vivo exposures. Previous reports have described lower DAR mc-VC-MMAE conjugates, DAR = 1-2, that demonstrated higher in vivo exposure and lower clearance when compared to higher DAR (e.g. 4-8) counterparts. In theory, high DAR conjugates may be especially desirable when targeting low antigen expressing tumors or when lower potency payloads are used, as each binding and internalization event results in greater payload delivery. Here we report a systematic exploration of DAR across a much wider range than has been previously reported, by combining THIOMAB® protein engineering technology with the Dolasynthen platform. Homogeneous, site-specific ADCs spanning a discrete range of DARs – 2, 4, 6, 12, and 18 – were made by conjugation of Trastuzumab IgG1 THIOMAB constructs with 1, 2, or 3 engineered cysteines to monomeric or trimeric Dolasynthen. The cytotoxicity of the resulting well-defined ADCs was assessed in vitro in cell lines with high or low expression of HER2 antigen. Pharmacokinetic data for all test articles in mice were generated in tumor bearing mice. In high HER2 expressing cell lines, in vitro cytotoxicity by payload was comparable across DARs. In a lower HER2 expressing system, the higher DAR ADCs performed better. In vivo, our data demonstrated comparable pharmacokinetics for the Dolasynthen conjugates across all DARs. These results illustrate the utility of a DAR ranging platform, such as Dolasynthen when evaluating ADCs as it enables the interrogation of a range of antibody and payload dosing regimens. Citation Format: Kalli C. Catcott, Susan Clardy, Jack Sadowsky, Rebecca K. Rowntree, Naniye Malli Centibas, Ling Xu, Andy Polson, Kenneth Avocetien, Tyler Carter, Mark Nazzaro, Dokyong "DK" Kim, Thomas H. Pillow, Neelie Zacharias, Cong Wu, Jeffrey Zurita, Elizabeth Ditty, Stephen Bradley, Alex Uttard, Bingfan Du, William S. Sawyer, Doug Leipold, Gail Lewis Phillips, LiuLiang Qin, Kelly Slocum, Geoffrey Del Rosario, Ginny Li, Shang-Fan Yu, David Lee, Radha Iyengar, Marc Damelin, Dorin Toader, Timothy B. Lowinger. Site-specific Dolasynthen ADCs demonstrate consistent exposure across a wide range of drug-to-antibody ratios [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P167.

Hsa-miR-21-3p associates with breast cancer patient survival and targets genes in tumor suppressive pathways.

Breast cancer is the cancer most often diagnosed in women. MicroRNAs (MIRs) are short RNA molecules that bind mRNA resulting in their downregulation. MIR21 has been shown to be an oncomiR in most cancer types, including breast cancer. Most of the effects of miR-21 have been attributed to hsa-miR-21-5p that is transcribed from the leading strand of MIR21, but hsa-miR-21-3p (miR-21-3p), transcribed from the lagging strand, is much less studied. The aim of the study is to analyze whether expression of miR-21-3p is prognostic for breast cancer. MiR-21-3p association with survival, clinical and pathological characteristics was analyzed in a large breast cancer cohort and validated in three separate cohorts, including TCGA and METABRIC. Analytical tools were also used to infer miR-21-3p function and to identify potential target genes and functional pathways. The results showed that in the exploration cohort, high miR-21-3p levels associated with shorter survival and lymph node positivity. In the three validation cohorts, high miR-21-3p levels associated with pathological characteristics that predict worse prognosis. Specifically, in the largest validation cohort, METABRIC (n = 1174), high miR-21-3p levels associated with large tumors, a high grade, lymph node and HER2 positivity, and shorter breast-cancer-specific survival (HR = 1.38, CI 1.13–1.68). This association remained significant after adjusting for confounding factors. The genes with expression levels that correlated with miR-21-3p were enriched in particular pathways, including the epithelial-to-mesenchymal transition and proliferation. Among the most significantly downregulated targets were MAT2A and the tumor suppressive genes STARD13 and ZNF132. The results from this study emphasize that both 3p- and 5p-arms from a MIR warrant independent study. The data show that miR-21-3p overexpression in breast tumors is a marker of worse breast cancer progression and it affects genes in pathways that drive breast cancer by down-regulating tumor suppressor genes. The results suggest miR-21-3p as a potential biomarker.

HER3 Is an Actionable Target in Advanced Prostate Cancer.

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials.

Risk factors at five-year survival in grade 3 breast cancer: a retrospective observational study of the New Zealand population

BackgroundBreast cancer is the most common cancer in New Zealand, with approximately 3000 new registrations annually, affecting one in nine women and resulting in more than 600 deaths. This study analyzed data of patients with selected prognostic factors of Nottingham grade 3 tumors over a specified five-year period. The study aimed to identify factors that result in differential survival in the female, New Zealand population.MethodThis is an observational, retrospective cohort study of prospectively collected data from New Zealand Breast Cancer Register. The selected period of 1st January 2011 to 31st, December 2015 allowed a consistent overlap for a national five-year data of grade 3 breast cancer in New Zealand. Mortality was carried out using univariate Fine-Gray competing risk statistical models.ResultsThis study showed that women in the older age group (> 70 years) had higher five-year mortality risk (HR: 1.7, 95% CI: 0.9–3.0, p = 0.053). Hormonal receptor analysis showed that ER positive, PR negative, and ER negative, PR negative subjects were at increased risk (HR = 3.5, 95% CI 2.3–5.4, p < 0.001) and (HR = 2.6, 95% CI, 1.8–3.9, p < 0.001) respectively. Molecular subtypes Triple Negative Breast Cancer and Luminal B subjects were at increased risk (HR = 3.0, 95% CI, 1.8–4.7, p < 0.001 and (HR = 3.3, 95% CI, 1.7–6.3, p < 0.001) respectively. HER2 enriched subjects were at a higher, but not significant, risk of five-year mortality compared to luminal A (HR = 1.6, 95% CI, 0.8–3.0, p = 0.10). NZ Europeans were at increased risk (HR = 1.7, 95% CI, 0.8–3.2, p = 0.11), with the highest Cumulative Incidence Function CIF, the largest proportion of HER2 enriched and TNBC across ethnicities.; however, Pacific Islanders experienced the highest HER2 CIF.ConclusionThe survival rates for grade 3 breast cancer vary across the selected prognostic factors and ethnicity. The results of this study make an initial contribution to the understanding of grade 3 breast cancer in the New Zealand population.

Circulating Natural Autoantibodies to HER2-Derived Peptides Performed Antitumor Effects on Oral Squamous Cell Carcinoma

Natural autoantibodies play a crucial role in destruction of malignant tumors due to immune surveillance function. Epidermal growth factor receptor 2 (HER2) has been found to be highly expressed in a variety of epithelial tumors including oral squamous cell carcinoma (OSCC). The present study was thus undertaken to investigate the effect of anti-HER2 natural autoantibodies on OSCC. Compared with cancer-adjacent tissues, cancer tissues from OSCC patients exhibited higher HER2 expression especially in those with middle & advanced stage OSCC. Plasma anti-HER2 IgG levels examined with an enzyme-linked immunosorbent assay (ELISA) developed in-house showed differences between control subjects, individuals with oral benign tumor and patients with OSCC. In addition, anti-HER2 IgG-abundant plasma was screened from healthy donors to treat OSCC cells and to prepare for anti-HER2 intravenous immunoglobulin (IVIg). Both anti-HER2 IgG-abundant plasma and anti-HER2 IVIg could significantly inhibit proliferation and invasion of OSCC cells by inducing the apoptosis, and also regulate apoptosis-associated factors and epithelial-mesenchymal transition (EMT), respectively. Besides, the complement-dependent cytotoxicity (CDC) pathway was likely to contribute to the anti-HER2 IgG mediated inhibition of OSCC cells. After the HER2 gene was knocked down with HER2-specific siRNAs, the inhibitory effects on OSCC cell proliferation and apoptotic induction faded away. In conclusion, human plasma IgG, or IVIg against HER2 may be a promising agent for anti-OSCC therapy.

Targeting HER2 Expressing Tumors with a Potent Drug Conjugate Based on an Albumin Binding Domain-Derived Affinity Protein.

Albumin binding domain derived affinity proteins (ADAPTs) are a class of small and folded engineered scaffold proteins that holds great promise for targeting cancer tumors. Here, we have extended the in vivo half-life of an ADAPT, targeting the human epidermal growth factor receptor 2 (HER2) by fusion with an albumin binding domain (ABD), and armed it with the highly cytotoxic payload mertansine (DM1) for an investigation of its properties in vitro and in vivo. The resulting drug conjugate, ADAPT6-ABD-mcDM1, retained binding to its intended targets, namely HER2 and serum albumins. Further, it was able to specifically bind to cells with high HER2 expression, get internalized, and showed potent toxicity, with IC50 values ranging from 5 to 80 nM. Conversely, no toxic effect was found for cells with low HER2 expression. In vivo, ADAPT6-ABD-mcDM1, radiolabeled with 99mTc, was characterized by low uptake in most normal organs, and the main excretion route was shown to be through the kidneys. The tumor uptake was 5.5% ID/g after 24 h, which was higher than the uptake in all normal organs at this time point except for the kidneys. The uptake in the tumors was blockable by pre-injection of an excess of the monoclonal antibody trastuzumab (having an overlapping epitope on the HER2 receptor). In conclusion, half-life extended drug conjugates based on the ADAPT platform of affinity proteins holds promise for further development towards targeted cancer therapy.

Evolution of HER2-positive mammary carcinoma: HER2 loss reveals claudin-low traits in cancer progression

HER2-positive breast cancers may lose HER2 expression in recurrences and metastases. In this work, we studied cell lines derived from two transgenic mammary tumors driven by human HER2 that showed different dynamics of HER2 status. MamBo89HER2stable cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2labile cell line gave rise to HER2-negative tumors from which MamBo38HER2loss cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2labile cells induced the loss of HER2 expression. MamBo38HER2loss cells showed a spindle-like morphology, high stemness and acquired in vivo malignancy. A comprehensive molecular profile confirmed the loss of addiction to HER2 signaling and acquisition of an EMT signature, together with increased angiogenesis and migration ability. We identified PDGFR-B among the newly expressed determinants of MamBo38HER2loss cell tumorigenic ability. Sunitinib inhibited MamBo38HER2loss tumor growth in vivo and reduced stemness and IL6 production in vitro. In conclusion, HER2-positive mammary tumors can evolve into tumors that display distinctive traits of claudin-low tumors. Our dynamic model of HER2 status can lead to the identification of new druggable targets, such as PDGFR-B, in order to counteract the resistance to HER2-targeted therapy that is caused by HER2 loss.

The percentage of residual DCIS in patients diagnosed with primary invasive breast cancer treated with neoadjuvant systemic therapy: A nationwide retrospective study

IntroductionNeoadjuvant systemic therapy (NST) is increasingly applied in breast cancer to improve surgical and oncological outcome. Approximately 21% of patients receiving NST achieve pathological complete response (pCR) of the breast. There is disagreement on the definition of pCR with respect to residual DCIS (ypT0 versus ypT0/is). The aim of this retrospective study was to determine the percentage of breast pCR (ypT0) and residual DCIS (ypTis), and its association with clinicopathological variables, in patients treated with NST and surgery. Materials and methodsPatients with invasive breast cancer treated with neoadjuvant chemotherapy, with or without targeted therapy, in the period of 2010–2019 were selected from the Netherlands Cancer Registry (NCR). Descriptive statistics and multivariable logistic regression analyses were used to analyse the percentage of ypT0 and ypTis and its association with clinicopathological variables. ResultsFrom the NCR database, 20495 patients were included, of whom 5847 (28.5%) achieved breast pCR (ypT0) and 881 (4.3%) showed residual DCIS (ypTis). The percentage of ypTis was highest in HER2+ tumour subtypes (ER+HER2+ 7.9%, ER-HER2+ 9.8%, ER+HER2- 2.1%, triple negative 3.3%, p < 0.001). Multivariable logistic regression analyses demonstrated high tumour grade (OR 2.00, p = 0.003) and HER2+ tumour subtype (ER+HER2+ OR 3.58, ER-HER2+ OR 4.37, p < 0.001) as independent predictors for ypTis. ConclusionpCR (ypT0) was achieved in 5847 (28.5%) patients receiving NST and residual DCIS (ypTis) was found in 881 (4.3%) patients. Consequently, the rate of pCR may be affected by ypTis when not excluded from the definition. The percentage of ypTis is highest in HER2+ subtypes.

Selected Articles from This Issue

In this First Disclosure, Smith and colleagues describe ASN004, a Dolaflexin-containing antibody drug conjugate targeting the 5T4 oncofetal antigen expressed on a wide range of tumors. ASN004 contains a scFv-Fc antibody and a Auristatin F Hydroxypropylamide payload with a drug-to-antibody ratio of 10-12. In a single dose study, 1 mg/kg achieved complete tumor regression in a cervical cancer model. ASN004 was also shown to have greater activity than the HER2-targeted trastuzumab-DM1 in a gastric model with high HER2 expression despite low 5T4 expression. Pharmacokinetics revealed low free payload drug and dose-dependent exposure. Their results support the continued clinical development of ASN004.In this First Disclosure, Duranti and colleagues outline a single-chain diabody targeting the hERG1 potassium channel and the β1 subunit of integrin receptors (scDb-hERG1-β1). The diabody bound with high affinity to the two proteins only when they were linked together and spared normal cells. The scDb-hERG1-β1 was antiproliferative against tumor spheroids through inhibition of AKT and HIF-1α signaling. The diabody also showed a favorable pharmacokinetic and toxicologic profile when injected into mice. Taken together, the disclosure provides evidence for the continued clinical development of scDb-hERG1-β1.VEGF inhibitors and immune therapies have continued to create favorable outcomes for renal cell carcinoma (RCC) patients. For those who don't respond to these therap. options, Yang and colleagues hypothesized mTOR inhibition will remain a salvage option either by itself or in combined therapy. A recent clinical trial demonstrated mTOR inhibition combined with the vascular agent BNC105P failed to show increased efficacy compared to mTOR inhibition alone. In this study, they demonstrate the transcriptomic gene signatures of patients from that clinical study that correlated with benefit with mTOR inhibition. Their findings were then validated using the phase III CheckMate 025 study.There remains an urgent need for new drug discovery in pediatric cancer with an added goal of reducing long-term toxicity. In this article, Vassal and colleagues underline the outcome of a pediatric cancer international multistakeholder meeting to develop a consensus set of requirements for preclinical drug development in pediatric cancers. The recommendations include the use of cell-based and mouse models as well as proof-of-concept data packages for clinical development. They are designed to represent the minimum guidelines necessary for the international development of drugs for pediatric cancer within the new regulatory environment set up by the RACE act.

HER2 Testing: Insights From Pathologists' Perspective on Technically Challenging HER2 FISH Cases.

College of American Pathologists and the American Society of Clinical Oncology guidelines provide straightforward criteria for HER2 interpretation in breast carcinomas; however, a subset of cases present unusual diagnostic dilemmas. Ten challenging HER2 fluorescence in situ hybridization (FISH) cases were selected for analysis. The study included a variety of problematic cases such as those with discordant immunohistochemistry (IHC) and FISH results, cases with high intratumoral variability in HER2 copy number, a case with a highly amplified clone in 5% to 10% of the tumor sample, and a case with tumor cells containing tightly clumped HER2 signals. Six high volume HER2 FISH laboratories performed and interpreted HER2 FISH (adding HER2 IHC if necessary). Interpretation strategies were discussed. There was 100% concordance between laboratories in 4/10 cases. Tumors with increased intratumoral variability (tumors with high variability in HER2 copy number per cell but which otherwise do not fulfill College of American Pathologists and the American Society of Clinical Oncology criteria for heterogeneity) exhibited 100% concordance in 3/4 cases, but 1 case had only 50% agreement. Low positive HER2 cases (group 1 cases with <6 average HER2 copies/cell) had 1 laboratory disagreeing with the majority in 4/4 cases, and this was the only category with discordance between IHC and FISH methodologies. All laboratories identified the case with heterogeneity and interpreted it as positive. Five of the 6 laboratories interpreted the case with tightly clustered HER2 signals as positive. This study offers specific observations and interpretation strategies that laboratories can use when confronted with difficult HER 2 cases. It then highlights communication strategies a laboratory may use to discuss these unusual HER2 results with the clinical team.

Validation of Potential Protein Markers Predicting Chemoradioresistance in Early Cervical Cancer by Immunohistochemistry.

BackgroundIn a previous study, a proteomic panel consisting of BCL-2, HER2, CD133, CAIX, and ERCC1 significantly predicted survival in patients with locally advanced cervical cancer. However, the prognostic significance of these proteins has not been assessed in early cervical cancer. The present study investigated the clinical significance and chemoradioresistance prediction power of these proteins in patients with early-stage cervical cancer.Materials and MethodsBCL-2, HER2, CD133, CAIX, and ERCC1 expression was determined by the immunohistochemical staining of 336 cervical cancer tissue microarrays. The associations of these proteins with clinicopathologic characteristics and disease progression were assessed.ResultsThere was a trend of low CAIX expression (p=0.082) and high ERCC1 expression (p=0.059) in patients with a favorable response to adjuvant radiation. High HER2 expression was significantly associated with shorter disease-free survival (DFS) in the total group (5-year DFS of 80.1% vs. 92.2%, p=0.004). A prognostic significance remained in multivariate analysis (Hazard ratio, HR=2.10, p=0.029). In the adjuvant radiation group, low CAIX and high ERCC1 expression indicated significantly unfavorable DFS (75.0% vs. 89.0%, p=0.026 and 76.8% vs. 88.6%, p=0.022, respectively). Low CAIX expression remained an independent prognostic marker in multivariate analysis (HR=0.45, p=0.037). The combined molecular-clinical model using random survival forest method predicted DFS with improved power compared with that of the clinical variable model (C-index 0.77 vs. 0.71, p=0.006).ConclusionHER2, CAIX, and ERCC1 expression can be predictive protein markers for clinical outcomes in early cervical cancer patients treated primarily with radical surgery with or without adjuvant radiation.

Abstract 3125: Analysis of intratumoral protein and gene expression heterogeneity in gastric cancer with a clustering analysis

Abstract Background/Aim: Intratumoral heterogeneity has been reported in various cancer types and may cause cancer metastasis and chemotherapy treatment resistance, including resistance to molecular targeted therapy. In gastric cancer, the intratumoral heterogeneity of cancer-associated molecules, such as HER2, has been reported and relates to the acquisition of chemotherapy resistance. To reveal whether intratumoral heterogeneity is related to cancer etiology, such as the mechanism of metastasis, and the development of new treatment strategies, such as combination therapy, we investigated intratumoral heterogeneity of gastric cancer with operative specimen and Cancer Genome Atlas (TGCA) data. Methods: The study included 503 patients who underwent gastrectomy and immunohistochemical staining for gastric cancer between 2001 and 2006 and TGCA data. We conducted clustering analysis based on protein and gene expression data for FGFR2IIIb (FGFR2), HER2(ERBB2), CXCR2(CXCR2), CXCR4(CXCR4), cMet(MET), TGFβ1(TGFB1), IGF1R(IGF1R), and p-smad2(SMAD2), and we analyzed the relation of FGFR2IIIb (FGFR2) and HER2(ERBB2) expression with the expression of other proteins and genes. Additionally, we investigated clinicopathological feature correlations. Result/Discussion: We divided gastric cancer specimens into nine clusters by immunohistochemical staining and 11 clusters using TCGA data. According to immunohistochemical staining, the high HER2 and FGFR2IIIb expression groups formed a cluster with high expression of various proteins. Analysis of stage 3-4 samples revealed more high protein expression clusters than the analysis of stage1-2, and this suggests that cancer acquires heterogeneity with cancer progression. In the TGCA data of stage 3-4, high FGFR2 and MET and high ERBB2 and SMAD2 or IGF1R expression formed clusters, which is consistent with the clustering analysis that was performed with immunohistochemical staining results. Conclusion: Our findings suggest that the existence of a combination of intratumoral heterogeneity and clusters may change with cancer progression. Citation Format: Gen Tsujio, Masakazu Yashiro, Yurie Yamamoto, Tomohiro Sera, Atsushi Sugimoto, Shuhei Kushiyama, Sadaaki Nishimura, Mami Yoshi, Tasuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Kazuya Muguruma, Masaichi Ohira. Analysis of intratumoral protein and gene expression heterogeneity in gastric cancer with a clustering analysis [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 3125.

Abstract 1373: Primary or iPSC-derived cell-based cytotoxicity assays to assess potential safety risks of engineered T cell therapies in vitro

Abstract Engineered T cell therapies, such as Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) T cells, but also bi-specific antibody therapies, play a major role in the field of immuno-oncology and offer great promise in becoming a new wave of highly specific therapies against solid tumors. Solid tumors generally lack the expression of tumor specific target antigens, posing a significant safety challenge. Selected solid tumor target antigens are often expressed at low levels in healthy tissues throughout the human body, risking the activation of the engineered T cells against these tissues. This can potentially result in life-threatening side effects, as such, assessing the safety of engineered T cell therapies is a critical step during early development and before filing for Investigational New Drug (IND) status. We have generated an in vitro safety profile for CAR-T cells targeting the ‘Human Epidermal growth factor Receptor 2' (HER2). The gene encoding this receptor is found to be amplified and/or overexpressed in 20-30% of invasive breast carcinomas and ovarian cancers. To determine which tissues are most at risk for unwanted reactivity by the CAR-T cells, in silico analysis for expression of the HER2 gene was performed. Subsequently, HER2 protein expression in various tissues was validated by staining cells with a HER2 antibody and determining the percentage of positive cells by flow cytometry. Primary tissues and iPSC-derived cell with high and low HER2 protein expression were selected, characterized and utilized for in vitro co-culture assays to evaluate on-target off-tumor and/or off-target cytotoxicity of the HER2 targeting CAR-T cells. Readouts for the in vitro cytotoxicity assays included measuring target cell viability by flow cytometry and/or HCA and T cell activation by cytokine release. Our study generated high quality data that provided insight into the safety of the HER2 targeting CAR-T cells. Moreover, we were able to demonstrate the value of using iPSC-derived cells in de-risking selected tissues against unwanted reactivity of engineered T cell therapies. Our strategy to generate a safety profile for T cell therapies and bi-specific antibodies is robust can be applied during both early stage development and late stage testing of the therapeutic product. Engineered T cell therapies have the capacity to fill tremendous unmet medical needs and are moving into the clinic at a high rate. This expedited timetable, although good news for patients with late stage disease, results in less time to conduct the rigorous safety tests required before FDA approval. The use of primary tissues and iPSC derived cells to test the safety of these therapies in vitro has been and will be essential in getting these novel drugs on the market. Citation Format: Sanne L. Holt, Sophie Vermond, Monique Hazenoot, Rene McLaughlin, Marco Guadagnoli, Marijn Vlaming. Primary or iPSC-derived cell-based cytotoxicity assays to assess potential safety risks of engineered T cell therapies in vitro [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 1373.

Abstract 1090: Overcoming trastuzumab resistance using trastuzumab deruxtecan (T-DXd), a HER2 targeting antibody drug conjugate, in HER2 amplified gastric cancer

Abstract HER2 is amplified in 7% to 20% of gastric cancer (GC) and is associated with poor clinical outcome. Trastuzumab, HER2-targeting antibody, has already been successfully employed for the treatment of HER2 amplified metastatic GC patients. However, most GC patients who achieved an initial response to the trastuzumab-based regimen developed resistance within 1 year after treatment. In this study, we aimed to investigate the efficacy and biological mechanism of trastuzumab deruxtecan (T-DXd) for overcoming trastuzumab resistance in HER2 amplified GC cell lines. We established four trastuzumab resistant cell lines from HER2 amplified GC cell lines by trastuzumab dose-escalation treatment; YCC-33TR, YCC-38TR, NCI-N87TR, and SNU-216TR. Next, we compared the anti-tumor efficacy of trastuzumab and T-DXd (Daiichi Sankyo, Japan), a HER2-targeting antibody-drug conjugate (ADC), in parental and trastuzumab resistant cell lines. Drug sensitivities were determined by the inhibition rates (IR) at 100μg/ml using cell viability assay. Both in baseline and in post-treatment drug responses, expression levels of proteins such as HER2, Erk, Akt, H2A.X and cPARP were detected by western blotting. The parental cell lines were sensitive to trastuzumab, while four trastuzumab resistant (TR) cell lines demonstrated significant resistance to trastuzumab (p&amp;lt;0.05). Compared with the parental cell lines, pHER2 expression was increased in trastuzumab resistant cell lines, and HER2 expression was decreased but still remained at a high level, suggesting that T-DXd can bind to HER2. As expected, T-DXd was sensitive in both parental and trastuzumab resistant cell lines. In detail, sensitivity to T-DXd in parental and resistant cells were as follows: YCC-33 vs YCC-33TR (79.6% vs 90.7%), YCC-38 vs YCC-38TR (94.4% vs 76.5%), NCI-N87 vs NCI-N87TR (83.8% vs 82.6%), and SNU-216 vs SNU-216TR (58.3% vs 42.6%). When treated with T-DXd to trastuzumab resistant cell lines, the expression levels of HER2 and HER2-related molecules showed various patterns for each cell line, and the expression levels of pH2A.X or cPARP were increased compared to trastuzumab treatment. It seems that T-DXd acts to cause DNA damage and induce apoptosis in parental and trastuzumab resistant cell lines. In summary, the established trastuzumab resistant cell lines retained high HER2 expression, and both HER2 amplified parental and trastuzumab resistant cell lines were sensitive to T-DXd. Our data indicated that T-DXd may have a potential therapeutic effect to overcome trastuzumab resistance in GC. Citation Format: Juin Park, Inhye Jeong, Sun Kyoung Kang, Seo Young Yu, Woo Sun Kwon, Tae Soo Kim, Jihyun Hwang, Hyun Cheol Chung, Sun Young Rha. Overcoming trastuzumab resistance using trastuzumab deruxtecan (T-DXd), a HER2 targeting antibody drug conjugate, in HER2 amplified gastric cancer [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 1090.