Spatial molecular profiling of mixed invasive ductal and lobular breast cancers reveals heterogeneity in intrinsic molecular subtypes, oncogenic signatures, and mutations.

Mixed invasive ductal and lobular carcinoma (MDLC) is a rare histologic subtype of breast cancer displaying both E-cadherin positive ductal and E-cadherin negative lobular morphologies within the same tumor, posing challenges with regard to anticipated clinical management. It remains unclear whether these distinct morphologies also have distinct biology and risk of recurrence. Our spatially resolved transcriptomic, genomic, and single-cell profiling revealed clinically significant differences between ductal and lobular tumor regions including distinct intrinsic subtype heterogeneity - e.g., MDLC with triple-negative breast cancer (TNBC) or basal ductal and estrogen receptor positive (ER+) luminal lobular regions, distinct enrichment of cell cycle arrest/senescence and oncogenic (ER and MYC) signatures, genetic and epigenetic CDH1 inactivation in lobular but not ductal regions, and single-cell ductal and lobular subpopulations with unique oncogenic signatures further highlighting intraregional heterogeneity. Altogether, we demonstrated that the intratumoral morphological/histological heterogeneity within MDLC is underpinned by intrinsic subtype and oncogenic heterogeneity which may result in prognostic uncertainty and therapeutic dilemma.

Pathway-based signatures predict patient outcome, chemotherapy benefit and synthetic lethal dependencies in invasive lobular breast cancer.

BACKGROUND: Invasive Lobular Carcinoma (ILC) is a morphologically distinct breast cancer subtype that represents up to 15% of all breast cancers. Compared to Invasive Breast Carcinoma of No Special Type (IBC-NST), ILCs exhibit poorer long-term outcome and a unique pattern of metastasis. Despite these differences, the systematic discovery of robust prognostic biomarkers and therapeutically actionable molecular pathways in ILC remains limited. METHODS: Pathway-centric multivariable models using statistical machine learning were developed and tested in seven retrospective clinico-genomic cohorts (n = 996). Further external validation was performed using a new RNA-Seq clinical cohort of aggressive ILCs (n = 48). RESULTS AND CONCLUSIONS: mRNA dysregulation scores of 25 pathways were strongly prognostic in ILC (FDR-adjusted P < 0.05). Of these, three pathways including Cell-cell communication, Innate immune system and Smooth muscle contraction were also independent predictors of chemotherapy response. To aggregate these findings, a multivariable machine learning predictor called PSILC was developed and successfully validated for predicting overall and metastasis-free survival in ILC. Integration of PSILC with CRISPR-Cas9 screening data from breast cancer cell lines revealed 16 candidate therapeutic targets that were synthetic lethal with high-risk ILCs. This study provides interpretable prognostic and predictive biomarkers of ILC which could serve as the starting points for targeted drug discovery for this disease.

Integration of Pathological Criteria and Immunohistochemical Evaluation for Invasive Lobular Carcinoma Diagnosis: Recommendations From the European Lobular Breast Cancer Consortium.

Invasive lobular carcinoma (ILC) is the second most frequent type of breast cancer (BC) and its peculiar morphology is mainly driven by inactivation of CDH1, the gene coding for E-cadherin cell adhesion protein. ILC-specific therapeutic and disease-monitoring approaches are gaining momentum in the clinic, increasing the importance of accurate ILC diagnosis. Several essential and desirable morphologic diagnostic criteria are currently defined by the World Health Organization, the routine use of immunohistochemistry (IHC) for E-cadherin is not recommended. Disagreement in the diagnosis of ILC has been repeatedly reported, but interpathologist agreement increases with the use of E-cadherin IHC. In this study, we aimed to harmonize the pathological diagnosis of ILC by comparing 5 commonly used E-cadherin antibody clones (NCH-38, EP700Y, Clone 36, NCL-L-E-cad [Clone 36B5], and ECH-6). We determined their biochemical specificity for the E-cadherin protein and IHC staining performance according to type and location of mutation on the CDH1 gene. Western blot analysis on mouse cell lines with conditional E-cadherin expression revealed a reduced specificity of EP700Y and NCL-L-E-cad for E-cadherin, with cross-reactivity of Clone 36 to P-cadherin. The use of IHC improved interpathologist agreement for ILC, lobular carcinoma in situ, and atypical lobular hyperplasia. The E-cadherin IHC staining pattern was associated with variant allele frequency and likelihood of nonsense-mediated RNA decay but not with the type or position of CDH1 mutations. Based on these results, we recommend the indication for E-cadherin staining, choice of antibodies, and their interpretation to standardize ILC diagnosis in current pathology practice.

Molecular and Clinical Portrait of HER2-low Invasive Lobular Carcinomas.

Invasive lobular carcinomas (ILCs) have a low frequency of ERBB2 amplification, therefore restricting the use of conventional anti-HER2 therapies for this histologic special type. Conversely, ILCs with low HER2 overexpression may represent a broader target for the use of emerging antibody drug conjugate therapies targeting HER2, since these treatments have proven effective in HER2-low breast cancers. Very scarce data about HER2-low ILCs have been so far published, although these tumors could have different prevalence and histomolecular specificities compared with invasive breast carcinoma of no special type (IBC-NST). Our aims in that context were to decipher the clinicopathological and molecular features of a large series of HER2-low ILCs. Comparative evaluation of HER2-low prevalence was done based on a retrospective series of 7970 patients from Institut Curie, with either primary invasive lobular (N = 1103) or no special type (N = 6867) invasive carcinoma. Clinicopathological and molecular analyses of HER2-zero, HER2-low, and HER2-positive ILCs were performed on a subgroup of 251 patients who underwent surgery for a primary ILC between 2005 and 2008. The mutational profile of these 251 cases was determined from RNAseq data. Compared with HER2-negative IBC-NSTs, the HER2-negative ILCs were found to display a higher frequency of HER2-zero cases (59.4% vs 53.7%) and a lower frequency of HER2-low (40.6% vs 46.3%) (P < .001). Clinicopathological features associated with HER2-low status (vs HER2-zero) in ILC were older age, postmenopausal status, nonclassic ILC histological types, higher grade, proliferation, and estrogen receptor expression levels. Survival curve analysis showed a significantly lower risk of local recurrence for HER2-low (vs HER2-zero) ILCs, but no association was found between HER2 status and either breast cancer-specific survival or distant metastasis-free interval. ERBB3 was the unique mutated gene exclusively associated with HER2-low ILCs yet being mutated at a low frequency (7.1%) (false discovery rate < 0.05). In conclusion, HER2-low ILCs exhibit their own particularities, both on clinical-pathological and molecular levels. Our findings call for larger multicenter validation studies.

Outcomes in Nonmetastatic Hormone Receptor-Positive HER2-Negative Pure Mucinous Breast Cancer: A Multicenter Cohort Study.

BACKGROUND: Although considered a favorable subtype, pure mucinous breast cancer (PMBC) can recur, and evidence for adjuvant therapy is limited. We aimed to compare outcomes of nonmetastatic PMBC with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) to address these uncertainties. METHODS: Individual patient-level data from 6 centers on stage I-III hormone receptor-positive and HER2-negative PMBC, IDC, and ILC were used to analyze recurrence-free interval (RFI), recurrence-free survival (RFS), and overall survival (OS), and to identify prognostic factors for PMBC. RESULTS: Data from 20,684 IDC cases, 1,475 ILC cases, and 943 PMBC cases were used. Median follow-up was 6.6 years. Five-year RFI, RFS, and OS for PMBC were 96.1%, 94.9%, and 98.1%, respectively. On multivariable Cox regression, PMBC demonstrated superior RFI (hazard ratio [HR], 0.59; 95% CI, 0.43-0.80), RFS (HR, 0.70; 95% CI, 0.56-0.89), and OS (HR, 0.71; 95% CI, 0.53-0.96) compared with IDC. ILC showed comparable outcomes to IDC. Fewer than half (48.7%) of recurrences in PMBC were distant, which was a lower rate than for IDC (67.3%) and ILC (80.6%). In contrast to RFI, RFS events were driven more by non-breast cancer deaths in older patients. Significant prognostic factors for RFI among PMBC included positive lymph node(s) (HR, 2.42; 95% CI, 1.08-5.40), radiotherapy (HR, 0.44; 95% CI, 0.23-0.85), and endocrine therapy (HR, 0.25; 95% CI, 0.09-0.70). No differential chemotherapy associations with outcomes were detected across PMBC subgroups by nodal stage, tumor size, and age. A separate SEER database analysis also did not find any association of improved survival with adjuvant chemotherapy in these subgroups. CONCLUSIONS: Compared with IDC, PMBC demonstrated superior RFI, RFS, and OS. Lymph node negativity, adjuvant radiotherapy, and endocrine therapy were associated with superior RFI. Adjuvant chemotherapy was not associated with better outcomes.

CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation.

The proposed role of CDH1 (E-cadherin gene) methylation as a mechanism of gene inactivation in invasive lobular carcinoma (ILC) remains inconclusive. For many years, CDH1 promoter hypermethylation has been regarded as a mechanism for gene inactivation in ILC. However, this assumption has primarily relied on non-quantitative assays, which have reported CDH1 methylation frequencies ranging from 26 to 93% at CpG sites within the island region. Few studies employing quantitative methods and covering CpG island shores, regions of relatively low CpG density situated proximal to conventional promoter CpGs, have been conducted, revealing lower percentages of methylation ranging from 0 to 51%. Therefore, using the quantitative pyrosequencing method, we examined CDH1 methylation in the island region and shores in E-cadherin deficient ILC cases (15 with CDH1 mutation and 22 non-mutated), 19 cases of invasive breast carcinomas non-special type (IBC-NSTs), and five cases of usual ductal hyperplasia (UDH). Our analysis revealed CDH1 methylation frequencies ranging from 3 to 64%, with no significant increase in methylation levels in any group of ILCs (median = 12%) compared to IBC-NST (median = 15%). In addition, considering the poorly studied association between the number of tumor-infiltrating lymphocytes (TILs) and CDH1 methylation in breast cancer, we undertook a thorough analysis within our dataset. Our findings revealed a positive correlation between CDH1 methylation and the presence of TILs (r = 0.5; p-value < 0.05), shedding light on an aspect of breast cancer biology warranting further investigation. These findings challenge CDH1 methylation as a CDH1 inactivation mechanism in ILC and highlight TILs as a potential confounding factor in gene methylation.

HER2-positive mucinous cystadenocarcinoma of the breast coexisting with invasive lobular carcinoma: A case report and review of the literature.

Primary mucinous cystadenocarcinoma (MCA) of the breast is a rare variant of breast carcinoma. A 68-year-old female patient presented to the general surgery clinic with pain and swelling in the right breast. A mass was detected in the upper outer quadrant, and a fine-needle aspiration biopsy was performed. The May-Grünwald Giemsa stained slides showed aggregates of mucin-rich pleomorphic cells with large nuclei in a mucinous background containing discohesive single cells. The Papanicolaou stain revealed a papillary structure composed of malignant epithelial cells in a necrotic background. A modified radical mastectomy was performed, and upon gross examination, two tumors were discovered in the central and upper outer quadrants. The first tumor, located centrally, was identified as invasive lobular breast carcinoma. The second tumor was an MCA with cytokeratin 7(+) and cytokeratin 20(-), and was determined to be the primary MCA of the breast based on clinical and radiological information. Immunohistochemistry revealed that the tumor cells were negative for estrogen receptor and progesterone receptor, and HER2 was 2+. Fluorescence in situ hybridization analysis detected HER2 gene amplification. During the 72-month follow-up, there were no findings compatible with recurrence or new metastasis. Although primary MCA is rare, it causes differential diagnosis problems and has different biological behaviors.

Additional confirmation of successful cell suspension fractionation of metastatic axillary node tissue.

We present herein an extension to our recently developed and published method termed "Fractionation of Nodal Cell Suspension" (FNCS). The method enables efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic axillary lymph node (mALN) tissue, using the entire nodule. For the purpose of the present study, a case of invasive lobular breast cancer (BC) patient with pT2N3aMx status and defined primary tumor markers (ERα 8, PR-B 8, and HER2 score 0) was available. Initially, the mALN tissue of this patient was analyzed by immunohistochemistry (IHC), and a positive correlation of nodal ERα, PR-B and HER2 biomarkers to those of the primary tumor was obtained. Subsequently, the mALN was FNCS fractionated into N and C, and Western blot (WB) analysis demonstrated a single band for ERα, PR-B and nuclear loading control (HDAC1) in nuclear, but not in the cytosolic compartments, confirming the efficiency of our fractionation protocol. At the same time, HER2 bands were not observed in either compartment, in accordance with HER2 negativity determined by IHC in both primary tumor and mALN tissue. In conclusion, by confirming the nuclear expression of ERα and PR-B biomarkers in metastatic loci, we demonstrate the purity of the FNCS-generated compartments - the protocol that offers a reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of novel biomarkers in the whole mALN of BC patients.

Construction of immune score and its prognostic value in invasive lobular carcinoma of the breast using computational pathology analysis.

BACKGROUND: Previous studies have shown that high level of TILs in invasive lobular carcinoma (ILC) is associated with poor prognosis, contrary to that in TNBC and HER2-positive breast cancer. METHODS: The densities of six immune cell markers and three immune checkpoints in the ILC microenvironment were detected by computational pathology analysis. Then, the LASSO cox regression model was used to construct an immune score (IS) and further evaluate its prognostic value. RESULTS: In our ILC cohort, the low density of CD4, CD8, CD20, CD56, CD68, FOXP3, PD-1, and PD-L1 had significantly longer disease-free survival (DFS) and overall survival (OS); however, the low density of CTLA-4 was associated with shorter DFS and OS. Based on this, an IS was constructed, and patients with low-IS had significantly prolonged DFS (p < 0.0001) and OS (p < 0.0001). Multivariate analysis revealed that IS was an independent prognostic indicator for DFS and OS. Further analysis showed that IS may increase the prognostic value of TNM stage. We further explored the prognostic role of CD68 and FOXP3 in the transcriptional level and the corresponding ISm in the METABRIC dataset, and found that low proportion of CD68 and FOXP3 and their ISm were associated with longer OS, and ISm was also an independent prognostic factor for OS. CONCLUSION: IS was a promising biomarker to distinguish the prognosis in ILC patients.

Expression of Sarcosine Metabolism-Related Proteins in Invasive Lobular Carcinoma: Comparison to Invasive Ductal Carcinoma

PURPOSE: The aims of this study were to compare the expression of sarcosine metabolism-related proteins between invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) and to determine the implications of these results. MATERIALS AND METHODS: Tissue microarrays were constructed, containing 30 samples from normal breast tissue, 114 samples from patients with ILC, and 692 samples from patients with IDC. Immunohistochemical staining was performed to examine the expression of sarcosine metabolism-related proteins [glycine N-methyltransferase, sarcosine dehydrogenase, and l-pipecolic acid oxidase (PIPOX)]. RESULTS: The sarcosine metabolic phenotype differed between ILC and IDC (plow sarcosine type (30.4%)>high sarcosine type (5.0%)>intermediate type (2.9%). However, in ILC, the sarcosine metabolic phenotype was distributed as low sarcosine type (61.4%)>null type (32.5%)>intermediate type (5.3%)>high sarcosine type (0.9%). PIPOX showed higher expression in ILC than in IDC (p<0.001) and correlated with androgen receptor (AR) positivity (p=0.001) in ILC. CONCLUSION: Expression of sarcosine metabolism-related proteins differed between ILC and IDC. Low sarcosine type was the majority sarcosine metabolic phenotype of ILC. PIPOX expression was predominant in ILC and correlated with AR positivity.

Clinicopathlogic and Immunohistochemical Characteristics of Triple Negative Invasive Lobular Carcinoma

PURPOSE: Our study is performed to find out clinicopathlogic and immunohistochemical (IHC) characteristics of triple negative invasive lobular carcinoma (ILC), as has been demonstrated in their invasive ductal counterparts. MATERIALS AND METHODS: Retrospective analysis of variable clinicopathlogic parameters and IHC stains for androgen receptor, estrogen receptor, progesterone receptor, p53, c-kit, galectin-3, cytokeratin 5 (CK5), CK5/6, vimentin, E-cadherin, epidermal growth factor receptor, and HER2 were performed in 117 cases of ILC. RESULTS: Eight cases (6.8%) were triple negative carcinoma (TNC), which showed higher incidence of high histologic grade than non-TNC (p = 0.019). Galectin-3 was expressed with higher incidence in tumor cells of TNC (62.5%) than those of non-TNC (7.3%) (p = 0.000). In contrast, galectin-3 was expressed with higher incidence in stromal cells of non-TNC (53.2%) than those of TNC (12.5%) (p = 0.029). CK5 and CK5/6 were not expressed in all ILCs. CONCLUSION: TNC in ILC showed distinct clinicopathologic and IHC characteristics such as higher histologic grade and increased expression of galectin-3, compared to non-TNC in ILC. TNC in ILC was less frequent and did not show CK5 and CK5/6 expression when compared to TNC in invasive ductal carcinoma.

Retrospective study assesing the role of MRI in the diagnostic procedures for early breast carcinoma: a correlation of new foci in the MRI with tumor pathological features

BACKGROUND: Use of breast magnetic resonance imaging (MRI) to detect breast cancer has generated significant debate. We analyze the role of breast MRI in the detection of additional disease and the need to perform additional biopsies in early breast carcinoma patients. In addition, we correlate the detection of new foci with tumor pathological features. METHODS: Early breast carcinoma patients that had undergone an MRI as well as a mammography as diagnostic procedures were included in the study. The following pathologic features were studied: carcinoma type, histological grade, estrogen receptors (ER), progesterone receptors (PR), HER2 and Ki67. Univariate analysis was conducted to ascertain significant correlation among detection of new foci and each of the tumor pathological features. RESULTS: Data from 98 patients have been analyzed: median age 49 years (range 35-79); carcinoma type: (a) infiltrative ductal carcinoma (n = 73, 74 %), (b) infiltrative lobular cancer (n = 12, 12 %), (c) ductal carcinoma in situ (n = 6, 6 %); amplified HER2 (n = 18, 18 %); grade III (n = 33, 33 %); Ki67 ≥ 25 % (n = 33, 33.67 %); positive ER and PR (n = 79, 80 %); triple negative tumors (n = 8, 8 %). MRI detected additional disease in 38 cases (39.58 %), and 20 led to an additional biopsy (20.4 %). Thirty-eight patients (39 %) underwent mastectomy. We found a statistically significant correlation between new foci in MRI and high Ki67 ≥ 25 % (p < 0.005). No other statistically significant correlation was established. CONCLUSION: MRI detected additional disease in 39 % cases, requiring an additional biopsy 20 %. Tumors with high proliferative index were significantly correlated with the detection of new foci in MRI (AU)

Detección inmunohistoquímica de la expresión de la oncoproteína c-erbB-2 en tumores ductales y lobulillares de mama / Immunohistochemical detection of c-erbB-2 oncoprotein expression in human breast ductal and lobular tumors

El cáncer de mama es una de las enfermedades importantes en la mujer y se le dedican grandes esfuerzos científicos y económicos existiendo el propósito de la búsqueda constante de vías de mejor precocidad en el diagnóstico y con ello, mejoramiento del pronóstico. En los rangos etarios de mayor riesgo de presentar esta patología se han descrito varios factores desencadenantes como son los receptores de estrógenos (RE), progesterona (RP) y de la proteína c-erbB-2. Se utilizaron biopsias de cáncer mamario ductal y lobulillar de diagnóstico de rutina, sometidas a reacciones inmunohisto químicas con anticuerpos anti RE, anti RP y específicamente para anti c-erbB-2. El revelado fue a través de kit comercial HRP con AEC y DAB. Las imágenes fueron capturadas con microscopio Olympus CX31, cámara fotográfica digital incorporada y software Micrometrics SE Premium 2011. Los resultados permiten observar reacción positiva en color rojo intenso en las muestras reveladas con AEC y pardo cuando se utilizó DAB, en las células que expresan la marcación y que se distribuyen indistintamente en el sitio del tejido afectado por la patología. Estas técnicas son de uso clínico protocolizado en el diagnóstico de cáncer de mama, por lo cual su estandarización y visualización son de extrema importancia para el laboratorio de histopatología integrado a unidades de patologia mamaria.

Breast cancer is one of the major diseases in women and devote great economic and scientific eforts. The intention of continuing to find ways of better and early diagnosis, and improved prognosis. In the age ranges of increased risk for this disease have been described several triggers such as receptors: estrogen (ER), progesterone (PR), and protein c-erbB-2. Biopses were used ductal and lobular breast cancer diagnostic routine, subjected to immunohistochemical antibodies to ER, PR and specifically anti c-erbB-2. The development was through HRP AEC and DAB commercial kit. Images were captured with Olympus CX31 microscope and digital camera and software built Micrometrics SE Premium 2011. The results allow observed positive reaction in red color in the samples developed with AEC and brown when used DAB in cells expressing the bearing and which are distributed equally at the site of pathology tissue affected. These techniques are protocolized by clinical use in diagnosing breast cancer, so its standardization and visualization are of extreme importance to the histopathology laboratory of integrated units of breast disease.

Inmunodetección de FHIT y E-cadherina en fibroadenomas y cáncer ductal y lobulillar de mama / Inmunodetection of FHIT and E-cadherin in fibroadenomas and ductal and lobular human breast cancer

En Chile, el cáncer es la segunda causa de muerte después de las enfermedades cardiovasculares. Los principales cánceres asociados a muerte en mujeres fueron mama, estómago, vesícula biliar, broncopulmonar y cérvico uterino. Alteraciones de las E-cadherinas han sido relacionadas con varios tipos de cáncer, ya que uno de los principales eventos involucrados con su disfunción es el gatillar la invasión y metástasis del tumor. La inactivación del gen CDH1 ha sido demostrada en el cáncer gástrico difuso y el cáncer de mama lobulillar. Asimismo, la inactivación del gen FHIT parece estar asociado con la progresión a neoplasias más agresivas. Se realizaron determinaciones inmunohistoquímicas (IHQ) en fibroadenomas mamarios y cánceres previamente diagnosticados por RE, RPg y Her2, mostrando positividad en todos los casos. La detección (IHQ) de la expresión de FHIT y E-cadherina en tejidos con patologías benignas y malignizados, puede aportar una importante información diagnóstica y pronóstica en el cáncer de mama.

In Chile, cancer is the second leading cause of death after cardiovascular diseases. The major death-related cancers in women were breast, stomach, gallbladder, lung and cervical cancer. Alterations of E-cadherin have been linked to various cancers, as one of the main events involved in its dysfunction is the trigger of tumor invasion and metastasis. CDH1 gene inactivation has been demonstrated in diffuse gastric cancer and lobular breast cancer. Furthermore, inactivation of the FHIT gene to be associated with progression to more aggressive tumors. Immunohistochemistry (IHC) determinations were performed in fibroadenomas and breast cancers previously diagnosed by ER, PgR and Her2, showing positivity in all cases. Immunohistochemical detection of FHIT and E-cadherin expression in tissues with benign disease and malignant, may provide an important diagnostic and prognostic information in breast cancer.