Clinical, pathological, and PAM50 gene expression features of HER2-low breast cancer

Càncer de mama; Genòmica del càncer; Recerca translacionalCáncer de mama; Genómica del cáncer; Investigación traslacionalBreast cancer; Cancer genomics; Translational researchNovel antibody-drug conjugates against HER2 are showing high activity in HER2-negative breast cancer (BC) with low HER2 expression (i.e., 1+ or 2+ and lack of ERBB2 amplification). However, the clinical and molecular features of HER2-low BC are yet to be elucidated. Here, we collected retrospective clinicopathological and PAM50 data from 3,689 patients with HER2-negative disease and made the following observations. First, the proportion of HER2-low was higher in HR-positive disease (65.4%) than triple-negative BC (TNBC, 36.6%). Second, within HR-positive disease, ERBB2 and luminal-related genes were more expressed in HER2-low than HER2 0. In contrast, no gene was found differentially expressed in TNBC according to HER2 expression. Third, within HER2-low, ERBB2 levels were higher in HR-positive disease than TNBC. Fourth, HER2-low was not associated with overall survival in HR-positive disease and TNBC. Finally, the reproducibility of HER2-low among pathologists was suboptimal. This study emphasizes the large biological heterogeneity of HER2-low BC, and the need to implement reproducible and sensitive assays to measure low HER2 expression.This work was supported by the grants from the European Union’s Horizon 2020 Research and Innovation Programme under Grant agreement No. 847912 (to A.P.), the Instituto de Salud Carlos III-PI16/00904 (to A.P.), Pas a Pas (to A.P.), Save the Mama (to A.P.), Breast Cancer Now-2018NOVPCC1294 (to A.P.), Fundación Científica Asociación Española Contra el Cáncer-Ayuda Postdoctoral AECC 2017 (to F.B.-M.), Fundación SEOM, Becas FSEOM para Formación en Investigación en Centros de Referencia en el Extranjero 2018 (to T.P.) and PhD4MD - Departament de Salut expedient SLT008/18/00122 (to N.C.)

Epstein–Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report

B cells; Epstein–Barr virus; Breast cancerCélulas B; Virus de Epstein-Barr; Cáncer de mamaLimfòcits B; Virus d'Epstein-Barr; Càncer de mamaEBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.This project was funded by Roche Farma, S.A. grant SP181123001 and the Spanish Ministry of Science, Innovation and Universities grant RTI2018-097414-B-I00. Partial financial support was received from the “El Paseíco de la Mama” 2015. This study received partial funding from Roche Farma, S.A. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication

Prognostic Implications of the Residual Tumor Microenvironment after Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer Patients without Pathological Complete Response

Neoadjuvant therapy; Relapse; Triple-negative breast cancerTerapia neoadyuvante; Recaída; Cáncer de mama triple negativoTeràpia neoadjuvant; Recaiguda; Càncer de mama triple negatiuWith a high risk of relapse and death, and a poor or absent response to therapeutics, the triple-negative breast cancer (TNBC) subtype is particularly challenging, especially in patients who cannot achieve a pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Although the tumor microenvironment (TME) is known to influence disease progression and the effectiveness of therapeutics, its predictive and prognostic potential remains uncertain. This work aimed to define the residual TME profile after NAC of a retrospective cohort with 96 TNBC patients by immunohistochemical staining (cell markers) and chromogenic in situ hybridization (genetic markers). Kaplan–Meier curves were used to estimate the influence of the selected TME markers on five-year overall survival (OS) and relapse-free survival (RFS) probabilities. The risks of each variable being associated with relapse and death were determined through univariate and multivariate Cox analyses. We describe a unique tumor-infiltrating immune profile with high levels of lymphocytes (CD4, FOXP3) and dendritic cells (CD21, CD1a and CD83) that are valuable prognostic factors in post-NAC TNBC patients. Our study also demonstrates the value of considering not only cellular but also genetic TME markers such as MUC-1 and CXCL13 in routine clinical diagnosis to refine prognosis modelling.This research was supported by grants from the Instituto de Salud Carlos III (PI13/02501 and PI11/0488) co-financed by the European Regional Development Fund (ERDF). ML acknowledges support from the “PATH-IMAGE” project, which was funded by ERDF (agreement 2903/335-41)

Case Report: A Case Study Documenting the Activity of Atezolizumab in a PD-L1-Negative Triple-Negative Breast Cancer

Biomarcadores; Cáncer de mama; InmunoterapiaBiomarcadors; Càncer de mama; ImmunoteràpiaBiomarkers; Breast cancer; ImmunotherapyThe immune checkpoint inhibitor atezolizumab is approved for PD-L1-positive triple-negative breast cancer (TNBC). However, no activity of atezolizumab in PD-L1-negative TNBC has been reported to date. Here, we present the case study of a woman with TNBC with low tumor infiltrating lymphocytes and PD-L1-negative disease, which achieved a significant response to atezolizumab monotherapy and durable response after the combination of atezolizumab and nab-paclitaxel. The comprehensive genomic analysis that we performed in her tumor and plasma samples revealed high tumor mutational burden (TMB), presence of the APOBEC genetic signatures, high expression of the tumor inflammation signature, and a HER2-enriched subtype by the PAM50 assay. Some of these biomarkers have been shown to independently predict response to immunotherapy in other tumors and may explain the durable response in our patient. Our work warrants further translational studies to identify biomarkers of response to immune checkpoint inhibitors in TNBC beyond PD-L1 expression and to better select patients that will benefit from immunotherapy.This study has received funding from Instituto de Salud Carlos III—PI19/01846 (to AP), Breast Cancer Now—2018NOVPCC1294 (to AP), Breast Cancer Research Foundation-AACR Career Development Awards for Translational Breast Cancer Research 19-20-26-PRAT (to AP), Fundació La Marató TV3 201935-30 (to AP), the European Union’s Horizon 2020 research and innovation programme H2020-SC1-BHC-2018-2020 (to AP), Asociación de Cáncer de Mama Metastásico CMM_CHIARAG19_001 (to AP), Pas a Pas (to AP), Save the Mama (to AP), Fundación Científica Asociación Española Contra el Cáncer AECC_Postdoctoral17-1062 (to FB-M) and INVES19056SANS (to MiS), FERO-ghd 2020 breast cancer award (MS), and Generalitat de Catalunya Peris PhD4MD 2019 SLT008/18/00122 (to NC)

Genetic and functional homologous repair deficiency as biomarkers for platinum sensitivity in TNBC: A case report

HRD-biomarkers; Pathological complete response; Triple-negative breast cancerBiomarcadores HRD; Respuesta patológica completa; Cáncer de mama triple negativoBiomarcadors HRD; Resposta patològica completa; Càncer de mama triple negatiuTriple-negative breast cancer is the most aggressive subtype of mammary carcinoma. In the early stage, neoadjuvant chemotherapy (NAC) is the standard of care for prognostic stratification and the best adjuvant treatment strategy. A 30-year-old female presented in the emergency room because of a gigantic right breast associated with an ulcerated lump at the upper quadrants. The right axillary nodes were palpable. An ultrasound was performed, showing the ulcerated neoformation with enlarged right axillary lymph nodes observed to level III. A core biopsy of the breast lesion was performed, and the pathological examination revealed a nonspecial type, grade 3, invasive, triple-negative breast cancer. No distant disease was found in the PET-CT scan. A germline genetic panel by next-generation sequencing identified a likely pathogenic variant in RAD51D (c.898C>T). Assessment of the functionality of the DNA homologous recombination repair pathway by RAD51 foci in the tumor revealed a profile of homologous recombination deficiency. NAC consisting of weekly carboplatin and paclitaxel followed by dose-dense doxorubicin/cyclophosphamide was performed with a complete metabolic response achieved in the PET-CT scan. The patient underwent a modified radical mastectomy plus axillary lymphadenectomy with a pathological complete response in the breast and axilla and remains disease-free after 2 years of follow-up. We report a young female with a triple-negative breast cancer stage cT4bN3M0 and a hereditary pathogenic mutation in RAD51D. The tumor was highly proliferative and homologous recombination-deficient by RAD51. The patient received platinum-based NAC, achieving a pathologic complete response. More effort should be made to identify predictive functional biomarkers of treatment response, such as RAD51 foci, for platinum sensitivity

A retrospective validation of CanAssist Breast in European early-stage breast cancer patient cohort

Hormone-receptor positive; Chemotherapy; Early-stage breast cancerReceptor de hormonas positivo; Quimioterapia; Cáncer de mama en fase inicialReceptor d'hormones positiu; Quimioteràpia; Càncer de mama en fase inicialCanAssist Breast (CAB), a prognostic test uses immunohistochemistry (IHC) approach coupled with artificial intelligence-based machine learning algorithm for prognosis of early-stage hormone-receptor positive, HER2/neu negative breast cancer patients. It was developed and validated in an Indian cohort. Here we report the first blinded validation of CAB in a multi-country European patient cohort. FFPE tumor samples from 864 patients were obtained from-Spain, Italy, Austria, and Germany. IHC was performed on these samples, followed by recurrence risk score prediction. The outcomes were obtained from medical records. The performance of CAB was analyzed by hazard ratios (HR) and Kaplan Meier curves. CAB stratified European cohort (n = 864) into distinct low- and high-risk groups for recurrence (P 50 years (HR: 2.93 (1.44–5.96), P = 0.0002). CAB had an HR of 2.57 (1.26–5.26), P = 0.01) in women with N1 disease. CAB stratified significantly higher proportions (77%) as low-risk over IHC4 (55%) (P < 0.0001). Additionally, 82% of IHC4 intermediate-risk patients were stratified as low-risk by CAB. Accurate risk stratification of European patients by CAB coupled with its similar performance inIndian patients shows that CAB is robust and functions independent of ethnic differences. CAB can potentially prevent overtreatment in a greater number of patients compared to IHC4 demonstrating its usefulness for adjuvant systemic therapy planning in European breast cancer patients

The Genomic and Immune Landscapes of Lethal Metastatic Breast Cancer

TCR repertoire; Breast cancer; Clade mutationsRepertori TCR; Càncer de mama; Mutacions cladeRepertorio TCR; Cáncer de mama; Mutaciones cladoThe detailed molecular characterization of lethal cancers is a prerequisite to understanding resistance to therapy and escape from cancer immunoediting. We performed extensive multi-platform profiling of multi-regional metastases in autopsies from 10 patients with therapy-resistant breast cancer. The integrated genomic and immune landscapes show that metastases propagate and evolve as communities of clones, reveal their predicted neo-antigen landscapes, and show that they can accumulate HLA loss of heterozygosity (LOH). The data further identify variable tumor microenvironments and reveal, through analyses of T cell receptor repertoires, that adaptive immune responses appear to co-evolve with the metastatic genomes. These findings reveal in fine detail the landscapes of lethal metastatic breast cancer

Predictive and prognostic value of total tumor load in sentinel lymph nodes in breast cancer patients after neoadjuvant treatment using one-step nucleic acid amplification: the NEOVATTL study

Càncer de mama; Gangli limfàtic sentinella; Càrrega tumoral totalBreast cancer; Sentinel lymph node; Total tumor loadCáncer de mama; Ganglio linfático centinela; Carga tumoral totalObjective To evaluate the predictive and prognostic value of total tumor load (TTL) in sentinel lymph nodes (SLNs) in patients with infiltrating breast cancer after neoadjuvant systemic therapy (NST). Methods This retrospective multicenter study used data from a Spanish Sentinel Lymph Node database. Patients underwent intraoperative SLN biopsy after NST. TTL was determined from whole nodes using a one-step nucleic acid amplification (OSNA) assay and defined as the total sum of CK19 mRNA copies in all positive SLNs. Cox-regression models identified independent predictive variables, which were incorporated into a nomogram to predict axillary non-SLN metastasis, and identified prognostic variables for incorporation into a disease-free survival (DFS) prognostic score. Results A total of 314 patients were included; most had no lymph node involvement prior to NST (cN0; 75.0% of patients). Most received chemotherapy with or without biologic therapy (91.7%), and 81 patients had a pathologic complete response. TTL was predictive of non-SLN involvement (area under the concentration curve = 0.87), and at a cut-off of 15,000 copies/µL had a negative predictive value of 90.5%. Nomogram parameters included log (TTL + 1), maximum tumor diameter and study-defined NST response. TTL was prognostic of disease recurrence and DFS at a cut-off of 25,000 copies/µL. After a 5-year follow-up, DFS was higher in patients with ≤ 25,000 copies/µL than those with > 25,000 (89.9% vs. 70.0%; p = 0.0017). Conclusions TTL > 15,000 mRNA copies/µL was predictive of non-SLN involvement and TTL > 25,000 mRNA copies/µL was associated with a higher risk of disease recurrence in breast cancer patients who had received NST.This study was supported by a grant from Sysmex España S.L. The sponsor coordinated data collection from study centers, and funded the statistical analysis and medical writing assistance

The Second Generation Antibody-Drug Conjugate SYD985 Overcomes Resistances to T-DM1

T-DM1; Conjugat anticòs-fàrmac; Càncer de mamaT-DM1; Conjugado anticuerpo-fármaco; Cáncer de mamaT-DM1; Antibody-drug conjugate; Breast cancerTrastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer. T-DM1 consists of trastuzumab covalently linked to the cytotoxic maytansinoid DM1 via a non-cleavable linker. Despite its efficacy, primary or acquired resistance frequently develops, particularly in advanced stages of the disease. Second generation ADCs targeting HER2 are meant to supersede T-DM1 by using a cleavable linker and a more potent payload with a different mechanism of action. To determine the effect of one of these novel ADCs, SYD985, on tumors resistant to T-DM1, we developed several patient-derived models of resistance to T-DM1. Characterization of these models showed that previously described mechanisms—HER2 downmodulation, impairment of lysosomal function and upregulation of drug efflux pumps—account for the resistances observed, arguing that mechanisms of resistance to T-DM1 are limited, and most of them have already been described. Importantly, SYD985 was effective in these models, showing that the resistance to first generation ADCs can be overcome with an improved design.This work was supported by Breast Cancer Research Foundation (BCRF-19-08), Fundación Mutua Madrileña, Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer. BM is supported by a fellowship from PERIS (Departament de Salut, Generalitat de Catalunya). JZ is supported by a fellowship from China Scholarship Council (CSC). JA is supported by Institució Catalana de Recerca i Estudis Avançats

Sequential immunohistochemistry and virtual image reconstruction using a single slide for quantitative KI67 measurement in breast cancer

Càncer de mama; Anàlisi d'imatges digitals; Quantificació de Ki67Breast cancer; Digital image analysis; Ki67 quantificationCáncer de mama; Análisis de imágenes digitales; Cuantificación de Ki67Objective Ki67 is a prognostic and predictive marker in breast cancer (BC). However, manual scoring (MS) by visual assessment suffers from high inter-observer variability which limits its clinical use. Here, we developed a new digital image analysis (DIA) workflow, named KiQuant for automated scoring of Ki67 and investigated its equivalence with standard pathologist's assessment. Methods Sequential immunohistochemistry of Ki67 and cytokeratin, for precise tumor cell recognition, were performed in the same section of 5 tissue microarrays containing 329 tumor cores from different breast cancer subtypes. Slides were digitalized and subjected to DIA and MS for Ki67 assessment. The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate inter-observer reproducibility. The Kaplan-Meier analysis was used to determine the prognostic potential. Results KiQuant showed an excellent correlation with MS (ICC:0.905,95%CI:0.878–0.926) with satisfactory inter-run (ICC:0.917,95%CI:0.884–0.942) and inter-antibody reproducibilities (ICC:0.886,95%CI:0.820–0.929). The distance between KiQuant and MS increased with the magnitude of Ki67 measurement and positively correlated with analyzed tumor area and breast cancer subtype. Agreement rates between KiQuant and MS within the clinically relevant 14% and 30% cut-off points ranged from 33% to 44% with modest interobserver reproducibility below the 20% cut-off (0.606, 95%CI:0.467–0.727). High Ki67 by KiQuant correlated with worse outcome in all BC and in the luminal subtype ( P = 0.028 and P = 0.043, respectively). For MS, the association with survival was significant only in 1 out of 3 observers. Conclusions KiQuant represents an easy and accurate methodology for Ki67 measurement providing a step toward utilizing Ki67 in the clinical setting