Multi-Omics Marker Analysis Enables Early Prediction of Breast Tumor Progression

Ductal carcinoma in situ (DCIS) is a preinvasive form of breast cancer with a highly variable potential of becoming invasive and affecting mortality of the patients. Due to the lack of accurate markers of disease progression, many women with detected DCIS are currently overtreated. To distinguish those DCIS cases who are likely to require therapy from those who should be left untreated, there is a need for robust and predictive biomarkers extracted from molecular or genetic profiles. We developed a supervised machine learning approach that implements multi-omics feature selection and model regularization for the identification of biomarker combinations that could be used to distinguish low-risk DCIS lesions from those with a higher likelihood of progression. To investigate the genetic heterogeneity of disease progression, we applied this approach to 40 pure DCIS and 259 invasive breast cancer (IBC) samples profiled with genome-wide transcriptomics, DNA methylation, and DNA copy number variation. Feature selection using the multi-omics Lasso-regularized algorithm identified both known genes involved in breast cancer development, as well as novel markers for early detection. Even though the gene expression-based model features led to the highest classification accuracy alone, methylation data provided a complementary source of features and improved especially the sensitivity of correctly classifying DCIS cases. We also identified a number of repeatedly misclassified DCIS cases when using either the expression or methylation markers. A small panel of 10 gene markers was able to distinguish DCIS and IBC cases with high accuracy in nested cross-validation (AU-ROC = 0.99). The marker panel was not specific to any of the established breast cancer subtypes, suggesting that the 10-gene signature may provide a subtype-agnostic and cost-effective approach for breast cancer detection and patient stratification. We further confirmed high accuracy of the 10-gene signature in an external validation cohort (AU-ROC = 0.95), profiled using distinct transcriptomic assay, hence demonstrating robustness of the risk signature.Peer reviewe

Caractérisation clinico-pathologique, moléculaire et biologique de l'hétérogénéité tumorale et stromale du carcinome mammaire infiltrant de type lobulaire

Le carcinome lobulaire infiltrant (CLI) est le deuxième type le plus fréquent de cancer du sein, représentant approximativement 8000 cas par an en France. Il est caractérisé par des cellules tumorales dyscohesives, en lien avec une inactivation de la E-cadhérine (molécule d’adhésion intercellulaire codée par le gène CDH1). Les CLI présentent des caractéristiques clinico-pathologiques distinctes de celles des carcinomes infiltrants de type non spécifique (CI-TNS) (type le plus fréquent de cancer du sein), notamment des amplifications plus rares d’HER2 (<5%), ainsi que des caractéristiques stromales spécifiques (faible réaction stromale, lymphocytes infiltrant la tumeur (TILs) associés à un mauvais pronostique). Les enjeux médico-scientifiques actuels pour ce cancer sont (1) l’amélioration de la reproductibilité du diagnostic, restant suboptimale en raison de l’hétérogénéité des méthodes diagnostiques et de la maladie lobulaire elle-même, et (2) la découverte de stratégies thérapeutiques ciblant spécifiquement certaines particularités histo-biologiques des CLI ; ces deux objectifs nécessitant d’avoir (3) une meilleure compréhension de l’hétérogénéité tumorale des CLI, incluant celle du micro-environnement tumoral (MET). Dans ce contexte, nous avons réalisé une première étude de corrélation clinico-histomoléculaire des mutations de la E-cadhérine (visant à affiner les critères diagnostiques des CLI), une seconde étude centrée sur les CLI avec faible expression d’HER2, dits « HER2-low » (pouvant représenter une opportunité thérapeutique), et enfin, une étude de caractérisation du MET des CLI. Pour cela, nous nous sommes appuyés sur une série rétrospective de 251 patientes de l’institut curie, opérées pour un CLI entre 2005 et 2008. Un profilage mutationnel a été réalisé à partir d'un séquençage RNAseq. Des TMA ont été constitués pour 158 CLI et 77 CI-TNS ER+ de notre institut, pour réaliser des immunohistochimies comparatives de marqueurs de CAF et de cellules immunes. Pour étudier le MET avec une haute résolution, six CLI classiques ont été analysés par scRNAseq. Un atlas single cell composé de CLI et de CI-TNS venant de données publiques a également été constitué et utilisé pour déconvoluer de larges séries rétrospectives de CLI et de CI-TNS avec données RNAseq (TCGA). Concernant la corrélation histomoléculaire des mutations de la E-cadhérine, nous retrouvions une association entre les mutations non tronquantes et une expression diffuse de la E-cadhérine, ainsi qu’un mauvais pronostic (survenue de métastase et mortalité spécifique) associé aux mutations tronquantes en analyse multivarié. Concernant HER2, les CLI étaient moins fréquemment HER2-low que les CI-TNS (40.6% vs 46.3%) et cette catégorie était exclusivement associée à la présence de (rares) mutations ERBB3. Sur le plan du MET des CLI, nous avons mis en évidence que (1) le contenu en CAF des CLI était hétérogène, dépendant de certaines caractéristiques histopathologiques des tumeurs, (2) les CLI avaient plus de CAF inflammatoires et moins de CAF myofibroblastiques que les CI-TNS ER+, en lien avec (3) l'altération d'un mécanisme précédemment non décrit impliquant la E-cadhérine dans la plasticité des CAF, (4) le contenu en CAF dans les CLI impactait le mode d’organisation spatiale des TILs, lui-même impactant le pronostic, et enfin, (5) l'absence de bon pronostic associé aux TILs dans les CLI pourrait être partiellement lié à un mécanisme d’échappement immunitaire induit par l'inactivation de la E-cadhérine. En conclusion, l’inactivation de la E-cadhérine façonne les spécificités du MET des CLI, ainsi que certaines caractéristiques des cellules tumorales telle que la biologie d’HER2. La caractérisation précise du statut mutationnel de CDH1 peut par ailleurs rendre compte d’une partie de l’hétérogénéité clinique et histo-phénotypique des CLI. Nos résultats pourraient contribuer, in fine, à améliorer l’efficacité diagnostique ainsi que le ciblage thérapeutique des CLI.Invasive lobular carcinoma (ILC) is the second most common type of breast cancer, accounting for approximately 8,000 cases per year in France. It is characterized by dyscohesive tumor cells, linked to inactivation of E-cadherin (an intercellular adhesion molecule encoded by the CDH1 gene). ILC presents clinicopathological features distinct from invasive breast carcinoma of no special type (IC-NST) (the most common type of breast cancer), including rarer HER2 amplifications (<5%), as well as specific stromal features (weak stromal reaction, tumor-infiltrating lymphocytes (TILs) associated with poor prognosis). Current medico-scientific challenges for this cancer are (1) the improvement of diagnostic reproducibility, which remains suboptimal due to the heterogeneity of diagnostic methods and lobular disease itself, and (2) the discovery of therapeutic strategies specifically targeting histobiological features of ILCs; these two objectives requiring (3) a better understanding of the tumor heterogeneity of ILCs, including that of the tumor microenvironment (TME). In this context, we carried out an initial clinico-histo-molecular correlation study of E-cadherin mutations (aimed at refining diagnostic criteria for ILCs), a second study focusing on HER2 expression in ILCs (tumors with low HER2 expression, known as "HER2-low", being poorly described in ILCs and representing potential therapeutic opportunities), and finally, a study characterizing the TME of ILCs. To do this, we leveraged a retrospective series of 251 patients who underwent surgery at Institut Curie for primary ILC between 2005 and 2008, and who had frozen material available for RNA sequencing. Mutational profiling was performed using RNAseq data. TMAs were set up for 158 ER+ ILCs and 77 ER+ IC-NSTs from our institute, to perform comparative immunohistochemistry of CAF and immune cell markers. For a high-resolution TME study, six classical ILCs were analyzed by single cell RNAseq. A single-cell atlas of ILCs and IC-NSTs from public dataset was also constructed and used to deconvolute large retrospective series of ILCs and IC-NSTs with RNAseq data (TCGA). With regard to E-cadherin mutations, we found that the type of mutation had a major impact on E-cadherin expression (non-truncating mutations were associated with diffuse E-cadherin expression), but also on survival (truncating mutations being an independent prognostic factor associated with metastatic risk and cancer-related mortality). With regard to HER2, ILCs were less frequently HER2-low than IC-NSTs (40.6% vs. 46.3%), and this category was exclusively associated with the presence of (rare) ERBB3 mutations. Regarding the TME of ILCs, we found that (1) the CAF content of ILCs was heterogeneous, depending in particular on two histopathological features of the tumor (the histological subtype of ILCs and tumor cellularity), (2) ILCs had more inflammatory CAFs and fewer myofibroblastic CAFs than ER+ IC-NSTs, (3) this particular characteristic depended on a previously undescribed mechanism involving E-cadherin in CAF plasticity, (4) CAF content in ILCs impacted the spatial organization of TILs, itself impacting prognosis, and finally, (5) the lack of good prognosis associated with TILs in ILCs could be partly linked to an immune escape mechanism related to E-cadherin inactivation in tumor cells. In conclusion, E-cadherin inactivation shapes the TME specificities of ILCs, as well as some tumor cell characteristics such as HER2 biology. Precise characterization of CDH1 mutational status may also account for some of the clinical and histo-phenotypic heterogeneity of ILCs. The findings of our work could ultimately contribute to improve the diagnostic efficacy and therapeutic targeting of ILCs

Invasive Lobular Carcinoma of the Breast: Toward Tailoring Therapy?

International audienc

Upper‐limb acute superficial lymphatic thrombosis: A case report

Abstract Upper‐limb acute superficial lymphatic is a rare phenomenon that has received little attention in the medical literature to date, yet it mimics superficial venous thrombosis and may also complicate a skin punch biopsy

Lobular Breast Cancer: Histomorphology and Different Concepts of a Special Spectrum of Tumors

Invasive lobular breast cancer (ILC) is the most common special histological type of breast cancer (BC). This review recapitulates developments in the histomorphologic assessment of ILC from its beginnings with the seminal work of Foote and Stewart, which was published in 1941, until today. We discuss different concepts of ILC and their implications. These concepts include (i) BC arising from mammary lobules, (ii) BC growing in dissociated cells and single files, and (iii) BC defined as a morpho-molecular spectrum of tumors with distinct histological and molecular characteristics related to impaired cell adhesion. This review also provides a comprehensive overview of ILC variants, their histomorphology, and differential diagnosis. Furthermore, this review highlights recent advances which have contributed to a better understanding of the histomorphology of ILC, such as the role of the basal lamina component laminin, the molecular specificities of triple-negative ILC, and E-cadherin to P-cadherin expression switching as the molecular determinant of tubular elements in CDH1-deficient ILC. Last but not least, we provide a detailed account of the tumor microenvironment in ILC, including tumor infiltrating lymphocyte (TIL) levels, which are comparatively low in ILC compared to other BCs, but correlate with clinical outcome. The distinct histomorphology of ILC clearly reflects a special tumor biology. In the clinic, special treatment strategies have been established for triple-negative, HER2-positive, and ER-positive BC. Treatment specialization for patients diagnosed with ILC is just in its beginnings. Accordingly, ILC deserves greater attention as a special tumor entity in BC diagnostics, patient care, and cancer research.status: publishe

Lobular Breast Cancer: Histomorphology and Different Concepts of a Special Spectrum of Tumors

Invasive lobular breast cancer (ILC) is the most common special histological type of breast cancer (BC). This review recapitulates developments in the histomorphologic assessment of ILC from its beginnings with the seminal work of Foote and Stewart, which was published in 1941, until today. We discuss different concepts of ILC and their implications. These concepts include (i) BC arising from mammary lobules, (ii) BC growing in dissociated cells and single files, and (iii) BC defined as a morpho-molecular spectrum of tumors with distinct histological and molecular characteristics related to impaired cell adhesion. This review also provides a comprehensive overview of ILC variants, their histomorphology, and differential diagnosis. Furthermore, this review highlights recent advances which have contributed to a better understanding of the histomorphology of ILC, such as the role of the basal lamina component laminin, the molecular specificities of triple-negative ILC, and E-cadherin to P-cadherin expression switching as the molecular determinant of tubular elements in CDH1-deficient ILC. Last but not least, we provide a detailed account of the tumor microenvironment in ILC, including tumor infiltrating lymphocyte (TIL) levels, which are comparatively low in ILC compared to other BCs, but correlate with clinical outcome. The distinct histomorphology of ILC clearly reflects a special tumor biology. In the clinic, special treatment strategies have been established for triple-negative, HER2-positive, and ER-positive BC. Treatment specialization for patients diagnosed with ILC is just in its beginnings. Accordingly, ILC deserves greater attention as a special tumor entity in BC diagnostics, patient care, and cancer research

The prognosis of patients treated with everolimus for advanced ER‐positive, HER2‐negative breast cancer is driven by molecular features

Abstract Everolimus is widely used in patients with advanced ER‐positive, HER2‐negative breast cancer. We looked at alterations in the PIK3CA/AKT/mTOR pathway in a multicenter cohort as potential biomarkers of efficacy. Patients with advanced ER‐positive, HER2‐negative breast cancer treated with everolimus and endocrine therapy between 2012 and 2014 in two cancer centers were included. Targeted sequencing examined mutations in PIK3CA, ESR1, and AKT1 genes. An immunochemical analysis was conducted to evaluate expression of PTEN, INPP4B, STK11, p4EBP1, and pS6. We analyzed 71 patients (44 primary tumors; 27 metastatic tissues). Median age was 63 years [58–69]. All patients had heavily pretreated advanced disease. A mutation in the PIK3CA pathway was observed in 32 samples (PIK3CA exons 10 and 21 and AKT1 exon 4 in 15.5%, 24.0%, and 5.6% of samples), and in ESR1 in 5 samples (7.0%), respectively. Most samples showed cytoplasmic expression of the PIK3CA pathway proteins. Progression‐free survival was longer in patients with a pS6 or p4EBP1 histoscore ≥ median value (6.6 versus 3.7 months, p = 0.037), and in patients with a PTEN histoscore ≤ median value (7.1 versus 5.3 months, p = 0.02). Overall survival was longer in patients with pS6 ≥ 3rd quartile (27.6 versus 19.3 months, p = 0.038) and in patients with any mutation in the PIK3CA/AKT/mTOR pathway (27.6 versus 19.3 months, p = 0.011). The prognosis of patients treated with everolimus for advanced ER‐positive, HER2‐negative breast cancer appears primarily driven by molecular features associated with the activation of the PIK3CA/AKT/mTOR pathway

Reconstitution of HIV-1 reservoir following high-dose chemotherapy/autologous stem cell transplantation for lymphoma.

ObjectivesAutologous stem cell transplantation following high-dose chemotherapy (HDC/ASCT) is the prime model to study the impact of HDC in HIV-1-infected participants. We analyzed the impact of HDC/ASCT on the resurgent reservoir composition and origin.DesignWe included retrospectively a homogenous group of HIV-1-infected patients treated for high-risk lymphoma in a reference center with similar chemotherapy regimens.MethodsThirteen participants treated with HDC/ASCT from 2012 to 2015 were included. A median seven longitudinal blood samples per participant were available. Total HIV-1 DNA levels in peripheral blood mononuclear cells (PBMCs) were quantified by quantitative PCR. In six participants with sustained viral suppression, the highly variable C2V3 viral region was subjected to next-generation sequencing. Maximum-likelihood phylogeny trees were generated from the reconstructed viral haplotypes. Lymphocyte subsets were studied by flow cytometry.ResultsPBMC-associated HIV-1 DNA levels were stable over time. Viral diversity decreased along lymphoma treatment, but increased promptly back to prechemotherapy numbers after HDC/ASCT. Blood viral populations from all time-points were intermingled in phylogeny trees: the resurgent reservoir was similar to pre-HDC circulating proviruses. Memory subsets were the main contributor to the early restoration of the CD4+ T-cell pool, with a delayed increase in naïve cell counts.ConclusionsThe characterization of HIV-1 reservoir in blood revealed a fast and consistent replenishment from memory CD4+ T cells after HDC/ASCT. As HDC/ASCT is increasingly involved in HIV cure trials with gene-modified hematopoietic stem cells, the management of infected T cells in HIV-positive autologous transplants will be critical

Emerging Role of IL-4–Induced Gene 1 as a Prognostic Biomarker Affecting the Local T-Cell Response in Human Cutaneous Melanoma

International audienc

Tumor-infiltrating lymphocytes are associated with poor prognosis in invasive lobular breast carcinoma

International audienceThe prognostic impact of tumor-infiltrating lymphocytes (TILs) within invasive lobular carcinoma (ILC) remains to be better characterized. In estrogen receptor (ER)-negative invasive ductal carcinomas of no special type (IDC-NST), TILs are associated with good prognosis. The aim of this study was to examine TILs in ILC, with particular focus on prognostic and clinicopathologic features. A cohort comprising 459 consecutive ILCs diagnosed in a single institution from 2005 to 2008 met the eligibility criteria for this study. The percentage of tumor area occupied by TILs was quantified by two breast pathologists and categorized into three groups: no TILs, ≤5%, &gt;5%. Clinicopathologic features were tested by Fisher’s exact tests or Chi2 tests. Overall survival (OS) and invasive disease-free survival (iDFS) were estimated by Kaplan–Meier and Cox proportional hazard statistics. There were 239 TIL-negative cases, 185 cases with ≤5% TILs, and 35 cases with &gt;5% TILs. TILs were associated with younger age, larger tumors, lymph node involvement, poor Nottingham prognostic index, HER2 amplification, multinucleation, and prominent nucleoli (p &lt; 0.05). Poor OS was significantly associated with increasing TILs in the univariate Cox proportional hazards model (p &lt; 0.001) and Kaplan–Meier estimator (p &lt; 0.05, log-rank test). Similar results were observed for iDFS (p = 0.004 for Cox univariate and p = 0.005 for log-rank test). Notably, TILs can identify a subset of ILC patients with poor OS independently of molecular subtype and lymph node metastases (multivariate Cox, p &lt; 0.001, OS hazard ratio (HR) = 4.38 and HR = 6.15, for ≤5% and &gt;5% TILs, respectively, vs. absence of TILs). Prominent nucleoli was the only nuclear feature associated with poor OS (p = 0.05) and iDFS (p = 0.05) in univariate Cox survival analysis. TILs represent a promising new morphologic biomarker associated with poor outcome of ILC, in contrast with that observed in ER-negative IDC-NST