Classic Invasive Lobular Carcinoma Research Articles

Differences in Histological Subtypes of Invasive Lobular Breast Carcinoma According to Immunohistochemical Molecular Classification.

The aim of this study was to compare the differences between histological and molecular subtypes of invasive lobular carcinoma (ILC) of the breast, in order to be able to predict the behavior and prognosis of the disease, as well as to effectively determine therapy. This study included 263 cases of breast ILC diagnosed over a seven-year period. The diagnosis of invasive lobular carcinoma is based on the characteristic growth pattern and phenotype of cancer cells with the respective subtypes: classic, alveolar, solid, tubulolobular, pleomorphic and mixed lobular type. The examined cases were divided into five groups according to molecular classification based on the expression of ER, PR, HER2 and Ki67 immunohistochemical markers. It was found that the pleomorphic subtype occurred statistically significantly less often as the luminal A subtype compared to others (p = 0.00027), and the HER2-enriched subtype occurred statistically significantly more often in the pT4 stage (p = 0.024). The results of this study significantly singled out the luminal A subtype, and among them classic ILC, as the subtype with the most favorable expression ratio of the investigated predictive/prognostic immunohistochemical markers.

Exploration of cancer associated fibroblasts phenotypes in the tumor microenvironment of classical and pleomorphic Invasive Lobular Carcinoma.

Multiplex immunofluorescence were performed on formalin fixed paraffin embedded tissues using Opal-7 color kit. The antibodies used for phenotyping CAFs were Pan CK (AE1/AE3), CD45, A-SMA, FAP, S100, Thy-1 with optimized dilutions. The images were acquired and analyzed using Vectra 3.0 imaging system and InForm software respectively. We studied 19 different CAFs colocalized phenotypes in the tumor, stroma and overall tissue compartments between classic and pleomorphic ILC. Total A-SMA+, A-SMA+FAP+S100+ and A-SMA+S100+ CAFs demonstrated higher densities in classic ILC cases while FAP+S100+ and S-100+ CAFs were increased in the pleomorphic subtype samples. Our study explores multiple CAFs phenotypes between classical and pleomorphic ILC. We showed that CAFs subset differ between Classic ILC and Pleomorphic ILC. A-SMA CAFs are more prevalent in the TME of classic ILCs whereas Pleomorphic ILCs are dominated by CAFs without A-SMA expression. This also iterates the importance of exploring this particular type of breast carcinoma in more detail, paving the way for meaningful translational research.

The term “classic invasive lobular carcinoma” of the breast defines breast malignancies of vastly different nature

PurposeTo describe in detail the special features of a previously unappreciated “classic invasive lobular carcinoma” which is confined to the terminal ductal lobular units (TDLUs) and differs considerably from the extensive classic invasive lobular carcinoma, and to suggest specific terminology. MethodAll invasive breast cancer cases without associated microcalcifications diagnosed in our Institution with the histopathologic diagnosis of classic invasive lobular carcinoma during the years 1996–2019 (n = 560) formed the basis of this study. The cases were prospectively classified according to their imaging biomarkers (mammographic features) and followed up to Dec 31, 2021, to determine long-term patient outcome. An additional 2600 invasive breast cancer cases (diagnosed other than invasive lobular carcinoma) without associated microcalcifications served as a reference group. Detailed histopathologic analysis used large format (10x8 cm) thin section technique and staining methods including hematoxylin-eosin (H&E), E-cadherin, cytokeratin CK 5/6, a transmembrane glycoprotein (CD44) and anti-actin or anti-smooth muscle myosin heavy chain. ResultsThe imaging biomarkers differentiated two separate disease subgroups, having the same histopathologic diagnosis, classic invasive lobular carcinoma. One of these has the imaging biomarker of extensive architectural distortion with no central tumour mass, occupies the extralobular mesenchyme and has a long-term survival of 56%. The other subgroup forms stellate or circular non-calcified tumour masses usually smaller than 20 mm, which appear to arise in the intralobular mesenchyme, and has a significantly better long-term survival of 84%. ConclusionsThere is a striking difference between the subgross histopathology and the mammographic appearance (imaging biomarkers) of two breast malignancies having the same histopathologic diagnosis, “classic invasive lobular carcinoma”. The large difference in the long-term outcome of these two tumour types is even more striking. Using the same specific term, “classic invasive lobular carcinoma”, to describe these two separate entities can adversely affect management decisions.

Imaging biomarkers are underutilised but highly predictive prognostic factors for the more fatal breast cancer subtypes

PurposeThe development and refinement of breast imaging modalities offer a wealth of diagnostic information such as imaging biomarkers, which are primarily the mammographic appearance of the various breast cancer subtypes. These are readily available preoperatively at the time of diagnosis and can enhance the prognostic value of currently used molecular biomarkers. In this study, we investigated the relative utility of the molecular and imaging biomarkers, both jointly and independently, when predicting long-term patient outcome according to the site of tumour origin. MethodsWe evaluated the association of imaging biomarkers and conventional molecular biomarkers, (ER, PR, HER-2, Ki67), separately and combined, with long-term patient outcome in all breast cancer cases having complete data on both imaging and molecular biomarkers (n = 2236) diagnosed in our Institute during the period 2008–2019. Large format histopathology technique was used to document intra- and intertumoural heterogeneity and select the appropriate foci for evaluating molecular biomarkers. ResultsThe breast cancer imaging biomarkers were strongly predictive of long-term patient outcome. The molecular biomarkers were predictive of outcome only for unifocal acinar adenocarcinoma of the breast (AAB), but less reliable in the multifocal AAB cases due to variability of molecular biomarkers in the individual tumour foci. In breast cancer of mesenchymal origin (BCMO), conventionally termed classic invasive lobular carcinoma, and in cancers originating from the major lactiferous ducts (ductal adenocarcinoma of the breast, DAB), the molecular biomarkers misleadingly indicated favourable prognosis, whereas the imaging biomarkers in BCMO and DAB reliably indicated the high risk of breast cancer death. Among the 2236 breast cancer cases, BCMO and DAB comprised 21% of the breast cancer cases, but accounted for 45% of the breast cancer deaths. ConclusionsIntegration of imaging biomarkers into the diagnostic workup of breast cancer yields a more precise, comprehensive and prognostically accurate diagnostic report. This is particularly necessary in multifocal AAB cases having intertumoural heterogeneity, in diffuse carcinomas (DAB and BCMO), and in cases with combined DAB and AAB. In such cases, the imaging biomarkers should be prioritised over molecular biomarkers in planning treatment because the latter fail to predict the severity of the disease. In combination with the use of the large section histopathology technique, imaging biomarkers help alleviate some of the current problems in breast cancer management, such as over- and under-assessment of disease extent, which carry the risk of overtreatment and undertreatment.

Abstract P2-21-07: Exploring spatial correlations in Breast invasive Lobular Carcinoma subtypes using a novel CAF multiplex immunofluorescence panel

Abstract Background: Recent advances in immunotherapy led to development of new technologies to identify topographical distributions and correlations in the tumor microenvironment, enabling a better understanding of the tumor immune landscape. Based on the 2019 WHO classification Invasive lobular carcinoma (ILC) has various histological presentations. The pleomorphic variant has been reported to be biologically different to the classical counterpart. Cancer associated fibroblasts (CAFs) secrete cytokines and growth factors aiding in tumor growth. CAFs have been shown to express alpha-smooth muscle actin (α-SMA), Thy1, fibroblast activation protein (FAP), S-100. IHC based studies have shown differences in CAFs subpopulation between ILC and IDC. Here, we explored different CAFs subpopulations between Pleomorphic and Classical ILC using multiplex immunofluorescence (mIF). Study design: Multiplex immunofluorescence (mIF) was performed on formalin-fixed paraffin-embedded (FFPE) tissue sections [(n=6, classic ILC; n=6 pleomorphic ILC)] stained with Opal 7-color Kit using an automated system. Sections were stained consecutively with Pancytokeratin, CD45, A-SMA, FAP, S100, Thy-1. Slides were scanned using the Vectra 3.0 spectral imaging system (PerkinElmer). Five tumor ROIs were examined with Phenochart (Akoya/PerkinElmer) viewer and subsequent images were analyzed to explore spatial distances of CK+ cells to the CAFs. Data was generated using InForm and Phenoptr softwares (Akoya Labs) as a median value. Statistical analysis data correlation was performed using SPSS version 24.0 (IBM Corp) Results: Twelve invasive lobular carcinomas cases were evaluated. All cases with classical features were nuclear grade 2 and all pleomorphic cases (n=6) were nuclear grade 3. Six were Luminal B, five were Luminal A and one case was triple negative invasive lobular carcinoma. We studied nineteen different phenotypes of CAFs in the tumor, stroma, and overall tissue compartments. We found statistically significant differences (p-value< 0.05) between the distances of CAFs from the tumor cells in classic and pleomorphic ILC in three phenotypes. The A-SMA+, A-SMA+/S100+ phenotypes were closer to the tumor cells in classic subtype and the S100 only phenotype was closer to the tumor cells in pleomorphic carcinomas. In addition, there were two other phenotypes namely A-SMA+/Thy-1+(closer to tumor cells in classic ILC) and FAP+/S-100+ (closer to tumor cells in pleomorphic ILC), which showed a trend of significance (Table 1) Discussion: TME studies in invasive breast lobular carcinoma have been primarily based on chromogenic IHC. Multiplex immunofluorescence quantifies cell phenotype’s densities and positions in different tissue compartments (tumor vs stroma vs total tissue). Knowing the proximity of an individual TME cells to the tumor cells, aids in pinpointing possible therapeutic targets. Fibroblast dysregulation in cancers, enhances their pro-tumorigenic and anti-tumorigenic potential. Due to their heterogeneity, they can express a wide array of markers. Identifying the full possibility of CAF subsets is one of the keys to discovering actionable targets. Our results showed Alpha- SMA positive, Alpha-SMA/S-100 positive, Alpha SMA/Thy-1 positive CAFs in closer proximity to the classic ILC tumor cells as compared to pleomorphic ILC. Additionally, S-100 positive and FAP/S-100 positive CAFs showed a closer proximity to tumor cells in the pleomorphic subtype. Based on these results we hypothesize that pleomorphic ILCs are closely associated with pro-tumorigenic CAFs as compared to classic ILCs. Larger datasets are needed to confirm this. In conclusion we utilized an objective approach to quantify, phenotype and spatially correlate each cell in the tumor microenvironment. This has helped identifying CAFs subsets that differs in the spatial correlation to tumor cells in ILC subtypes, which can be potential actionable targets. Table 1 Phenotypes of Cancer associated fibroblasts and their statistical correlation between classic vs pleomorphic invasive Lobular carcinomas. Citation Format: Harsh Batra, Renganayaki K. Pandurengan, Heladio P. Ibarguen, Salome A McAllen, Qingqing Ding, Aysegul Sahin, Ignacio Wistuba, Edwin Roger Parra, Maria Gabriela Raso. Exploring spatial correlations in Breast invasive Lobular Carcinoma subtypes using a novel CAF multiplex immunofluorescence panel [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-07.

Abstract P2-23-15: Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas

Abstract Background: Invasive lobular carcinomas (ILCs) are the most frequent special histologic subtype of breast cancer, accounting for up to 15% of all breast cancer cases. ILCs are characterized by a distinctive discohesive growth pattern, with cells arranged in single cell infiltrative file and dispersed throughout the stroma, which stems from the loss of E-cadherin expression due to bi-allelic inactivation of the CDH1 gene. A subset of breast cancers display a similar single cell infiltrative growth pattern but, in contrast to classic ILC, display diffuse strong membranous E-cadherin reactivity and membranous p120 expression. We refer to such cases as “lobular-like invasive ductal carcinoma” (LLIDC), but it is unclear if this terminology is appropriate and if such cases show biallelic inactivation of CDH1, similarly to ILCs. Here, we sought to define whether LLIDCs would harbor bi-allelic alterations of CDH1 and to perform an exploratory, hypothesis generating analysis of the repertoire of somatic genetic alterations of LLIDCs and classic ILCs. Materials and methods: Representative H&Es, as well as sections subjected to E-cadherin and p120 immunohistochemistry from seven classic ILCs and seven bona fide “lobular-like invasive ductal carcinomas” were retrieved and independently reviewed by two pathologists with experience and expertise in breast pathology. DNA samples were extracted from representative sections from tumor and normal breast tissue from each patient and subjected to an FDA-approved targeted sequencing assay comprising the coding regions and selected regulatory elements of 515 genes. Somatic single nucleotide variants (SNVs) were detected with MuTect, indels with Strelka, Varscan2, Scalpel and Lancet. All mutations were manually inspected using the Integrative Genomics Viewer (IGV). The cancer cell fraction (CCF) of each mutation was inferred, as well as clonal probability, using ABSOLUTE. Copy number alterations and loss of heterozygosity were determined using FACETS. Mutational signatures were inferred using SigMA based on all synonymous and nonsynonymous somatic mutations. Results: Based on the histopathologic evaluation, of the 14 cases analyzed, seven were classified as ILC, and the other seven were classified as LLIDC. Sequencing analysis revealed that the classic ILCs harbored 16q LOH and CDH1 mutations (7/7), of which five were frameshift indel and two were splice site mutations consistently coupled with loss-of-heterozygosity (LOH) of the wild-type allele. Conversely, five of the seven LLIDCs did not harbor CDH1 mutations or genomic rearrangements. CDH1 mutations were identified in 2 LLIDCs: one harbored a subclonal CDH1 in-frame indel mutation coupled with LOH. This case displayed membranous E-cadherin and p120 expression with areas of aberrant expression. The other CDH1-mutated LLIDC harbored a complex in-frame indel with subclonal LOH. This case displayed membranous E-cadherin and p120 expression. The comparative analysis of the repertoire of somatic genetic alterations and mutational signatures present in LLIDCs and classic ILCs did not reveal any significant differences. Conclusion: Despite the histologic similarities, LLIDCs differ from classic lobular carcinomas based on the lack of CDH1 bi-allelic inactivation and the patterns of expression of E-cadherin and p120 catenin. Further whole-genome sequencing analyses are warranted to define the molecular basis of the discohesive cancer cells of LLIDC display. Citation Format: Edaise M. da Silva, Thais Basili, Jing Yu, Juan Blanco-Heredia, Pier Selenica, Qiqi Ye, Arnaud da Cruz Paula, Higinio Dopeso, Antonio Marra, Steffi Oesterreich, Jorge Reis-Filho, Rohit Bhargava. Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-15.

Abstract P5-14-01: Transcriptomic insights into lobular breast cancer biology: a retrospective analysis of the MINDACT clinical trial

Abstract Background: Invasive lobular carcinoma (ILC) represents the second most common subtype of breast cancer after invasive breast cancer of no special type (NST). In this retrospective analysis of the MINDACT trial, we aimed at identifying/refining the transcriptomic differences between: 1) estrogen receptor positive/HER2-negative (ER+/HER2-) ILC versus ER+/HER2- NST, 2) classic and non-classic ER+/HER2- ILC, and, 3) recurring and non-recurring ER+/HER2- ILC in the subgroup of patients with a low clinical and low genomic (cL/gL) risk (as defined by a modified version of Adjuvant Online! and the 70-gene signature). Patients and methods: Central pathology review was performed for histological subtype, grade and Ki67 (G.V.) for 5929/6693 (88.6%) of the patients included in the MINDACT trial (NCT00433589). Analysis of transcriptomic data adjusted for age and grade was performed using the R/Bioconductor package ‘limma’ to identify differentially expressed genes (DEGs). DEGs having absolute log-fold change (logFC)≥ 0.2 and FDR-adjusted p-value (q-value) < 0.05 were considered. Gene set enrichment analyses (GSEA) of MSigDB hallmark gene sets were performed. Adjusted Cox regression models were used to evaluate the association of these hallmarks with disease free survival (DFS) and distant recurrence free survival (DRFS). Results: After central pathological review, 464 patients with ER+/HER2- ILC and 3798 patients with ER+/HER2- NST were identified. Patients with ILC were significantly older at diagnosis, had larger tumors, less axillary nodal involvement, more grade 2 tumors than patients with NST. At the transcriptomic level, we observed a high number of DEGs between these 2 subgroups, confirming their distinct phenotype. CDH1, the gene coding for E-cadherin, was as expected the most highly overexpressed gene in NST versus ILC. We further observed an increased expression of leptin (LEP), leptin receptor (LEPR), lipoprotein lipase (LPL), and the fatty acid transporter CD36 in ILC. This could suggest that ILC relied on increased lipid uptake thanks to the increased contact of ILC tumor cells with the adipocytes. IGF1 was also overexpressed in ILC versus NST, as a potential consequence of high LEP and high LEPR expression. Differences were also evident with regard to the extracellular matrix (ECM), with many collagens, matrix metalloproteinases (MMPs) and other key enzymes (e.g. LOXL1) being differentially expressed. We confirmed a decreased ER-signaling and increased PI3K/Akt signaling in ILC versus NST. Out of the 464 ER+/HER2- ILC tumors, 253 (55%) were classic ILC and 211 (45%) non-classic ILC. There were more grade 3 tumors, more highly proliferative tumors and more nodal involvement in patients with non-classic versus classic ILC. At the transcriptomic level, differences were subtler than the differences seen above. Still, a significant enrichment of the hallmarks related to cell cycle in the non-classic ILC, and of the hallmarks related to epithelial-to-mesenchymal transition, hypoxia, adipogenesis and IL6/JAK/STAT3 signaling in classic ILC was observed. Finally, 216/464 patients with ER+/HER2- ILC (47%) were assigned to the cL/gL risk group and did not receive chemotherapy. 28/216 of these patients (13%) relapsed (DFS, median FU: 8.7 years). Enrichment of hallmarks related to apoptosis, inflammatory response, hypoxia and oncogenic signaling (PI3K/Akt, Ras, c-Myc) was associated with worse survival. Conclusion: This represents, to the best of our knowledge, the largest set of gene expression data for patients with ILC, issued from a clinical trial where histology was reviewed centrally. These results could be used to personalize treatment for patients with ILC. This project is funded by the Breast Cancer Research Foundation. Citation Format: Christine Desmedt, Ha-Linh Nguyen, François Richard, Sabine Linn, Otto Metzger, Coralie Poncet, Jelle Wesseling, Florentine Hilbers, Kim Aalders, Mauro Delorenzi, Suzette Delaloge, Jean-Yves Pierga, Etienne Brain, Suzan Vrijaldenhoven, Peter A Neijenhuis, Karen Van Baelen, Marion Maetens, Emiel Rutgers, Martine Piccart, Laura Van ’t Veer, Giuseppe Viale, Fatima Cardoso. Transcriptomic insights into lobular breast cancer biology: a retrospective analysis of the MINDACT clinical trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-01.

Abstract ED4-4: Lobular/pleomorphic Breast Cancer

Abstract Invasive lobular carcinoma (ILC) accounts for up to 15% of all invasive breast cancers. The general marker to identify lobular cancer is the loss or low expression of E-cadherin which is the result of genetic alterations of the E-cadherin gene which accounts for about 95%. The majority of classical ILCs are of low grade and express both hormone receptors strongly and rarely any HER2. Those not expressing the hormone-receptors are categorised as apocrine breast cancers which is a small group. Recently it has been shown that ILC is a breast cancer type which has unique genomic features. Desmedt and colleagues genomically profiled 430 ILCs, to characterize the genomic alterations present in these tumors compared to invasive ductal carcinoma (now cold unfortunately NST non-specific subtype). The most frequent altered gene was CDH1 which results in a mRNA or protein loss of the cell adhesion molecule E-cadherin. Amongst the genes found to be altered at a high frequency are ESR1 and PIK3CA/PTEN. Currently those have little or no clinical impact in early breast cancer there might be consequences for metastatic breast cancer. The molecular alterations result in a less cohesive tissue connection and influence imaging as well as surgical procedures and in the end the prognosis in those patients. The size and extend of ILC is often underestimated by imaging and clinical examination. MRI thought to be the optimal imaging turned out to have no influence on positive margins and is no longer considered standard for all ILCs. Several data suggest that ILCs have an improved outcome the first 5 years after primary diagnosis but thereafter the survival curves in general lie below those for non lobular breast cancer. ILC has a distinct metastatic pattern and often metastasise into the GI tract and the ovaries and less frequently to the lung. Breast conserving surgery as well as mastectomy can be used when indicated based on tumor size and patients preference. Because ILC more often presents at higher stage, mastectomy is more frequently performed. But even after neoadjuvant chemotherapy we could demonstrate, despite response even resulting in pCR, mastectomy was more frequently conducted in ILC patients compared to non-ILC. In this analysis 71% of the non-ILC did receive BCS compared to only 59% in ILC (p Systemic therapy follows the general recommendations based on stage and histopathological profile. Neoadjuvant chemotherapy results in significantly lower rates of pathological responses. All analyses showed response rate in single digit numbers (far below 10%) but the less classical ILCs with either grade 3 or hormone-receptor negativity have a higher chance of achieving a pCR. Those ILCs with grade 3 and hormone-receptor negativity (apocrine cancer) have a pCR rate comparable to invasive ductal carcinoma (IDC). It is not surprising that lobular breast cancer does not respond as well as IDC to neoadjuvant chemotherapy because their biological features are, as described above, more luminal A like (high hormone receptor positivity, low grade, low proliferation). Because many patients with ILC present with extended diseases, they might still benefit from neoadjuvant chemotherapy even if they do not achieve a pathologic complete response, downstaging is an option and would potentially lead to less mastectomies. Because of the luminal A like type of the majority of ILCs endocrine therapy seems to be the treatment of choice. Some data even suggested a better outcome for ILC breast cancer when treated with an aromatase inhibitor compared to tamoxifen. But other data are controversial in this regard and did not confirm the data from BIG 1-98. In conclusion the molecular profile of ILC seems to be distinct but does not result in different treatment paradigms. We should not underestimate the necessity of optimal treatment, including chemotherapy for invasive lobular breast cancer patients. Citation Format: Sibylle Loibl. Lobular/pleomorphic Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr ED4-4.

Invasive lobular carcinoma with extracellular mucin (ILCEM): clinicopathologic and molecular characterization of a rare entity

AbstractInvasive lobular carcinoma with extracellular mucin (ILCEM) is a rare histologic subtype of breast cancer. Little is known about the pathologic or genomic signatures that distinguish ILCEM from classic invasive lobular carcinoma (ILC) or mucinous carcinoma. We studied 17 breast cancers with lobular morphology and extracellular mucin. Thirteen tumors with sufficient tissue for DNA extraction were analyzed by a next generation sequencing (NGS) assay that interrogates 447 genes for mutations and copy number variations (CNVs). Median patient age was 66 yrs (range: 31–77 yrs). Sixteen patients presented with masses, 7 of which were >2 cm. Seven patients had lymph node metastases. The cases of ILCEM were moderately (n = 13) or poorly differentiated (n = 4), frequently exhibiting variant morphology that has not been previously described or emphasized, including grade 3 nuclei (n = 11), diffuse signet ring cells (n = 10), solid growth (n = 4), tumor necrosis (n = 3) or apocrine features (n = 2). All tumors showed absent or reduced membranous E-cadherin expression. Concurrent lobular carcinoma in situ (LCIS) was seen in 11/17 cases, 1 of which was a striking example of signet ring cell LCIS with extracellular mucin. Receptor profiles were ER+/HER2− (n = 15) and ER+/HER2+ (n = 2). With a median follow-up of 83.5 months (range: 3–171 months) in 12 patients with available information, 8 patients had recurrences resulting in 4 cancer-related deaths. The most common CNVs were 16q loss (n = 11) and 1q gain (n = 9). CDH1 gene-level alterations were detected in all but one case, including frameshift (n = 7), nonsense (n = 2), and donor splice site (n = 1) mutations and indels (n = 2). Recurrent mutations were also seen in PIK3CA (n = 3), POLQ (n = 3), TP53 (n = 3), ERBB3 (n = 3), ERBB2 (n = 2), and RUNX1 (n = 2). Genes with recurrent amplifications included GATA3 (n = 4), FOXA1 (n = 3), CCND1 (n = 2). Our data highlights ILCEM as a distinct variant of ILC that often presents with higher-grade and variant morphologic features and is associated with an aggressive clinical course. NGS data support an overall lobular-type molecular profile and reveal potentially targetable alterations in a subset of cases with recurrence.

Discordance of HER2 Positive Classic Invasive Lobular Carcinoma Between IHC/FISH and Oncotype DX: A Case Report and Literature Review

The classical-type of invasive lobular carcinoma (ILC) usually expresses high levels of estrogen receptor (ER) and progesterone receptor (PR) and does not over-express human epidermal growth factor receptor 2 (HER2) and has a low level of expression of the proliferative marker. Their prognosis is comparable with those of histologic grade 1-2 invasive ductal carcinoma (IDC). HER2 status is prognostically significant and has a huge impact on clinical management; thus, its correct testing and evaluation are critical. Discordant results for HER2 between immunohistochemistry (IHC)/fluorescence in situ hybridization (FISH) and Oncotype DX (ODX) testing can be occasionally seen and cause confusion among clinicians. Here, we present a case of classical ILC (cILC), which was HER2 positive by IHC/FISH but negative by ODX. We also review relevant literature and provide a possible explanation for this discordancy and suggestions on how to approach the discordant results and guide the clinical management.

Biomarker profile of invasive lobular carcinoma: pleomorphic versus classic subtypes, clinicopathological characteristics and prognosis analyses.

To compare the clinicopathologic features and prognosis of pleomorphic invasive lobular carcinoma (P-ILC) and classic ILC (C-ILC) according to the biomarker profile. A total of 667 C-ILCs and 133 P-ILCs between 2011 and 2021 were included. Clinicopathologic features and stromal tumor-infiltrating lymphocytes (sTILs) status were evaluated. P-ILCs were divided into subtypes based on ER/PR and HER2 expression. The overall survival and disease-free survival (DFS) of patients were compared among matched P-ILCs, C-ILCs, and invasive ductal carcinomas (IDCs) with biomarker subtypes. Compared to C-ILCs, P-ILCs had greater tumor sizes and stages, fewer ER-positive, more HER2-positive, triple negative (TN), and Ki-67 > 20% tumors (P < 0.05). P-ILCs were subdivided into ER+ (63.1%), HER2+ (21.1%) and TN (15.8%). ER+ P-ILCs were mainlyshowed trabecular and solidgrowth patterns. Apocrine and solid featureswere more strongly associated with HER2+ P-ILCs and TN-P-ILCs, respectively.The prognosis of each biomarker group (ER+, HER2+ and TN) differed by subtype. The P-ILC biomarker subtypes hadworse prognosis than the same subtypes in the IDC group, while there wasno difference between the P-ILC and the C-ILC counterparts. Solid variants of P-ILChad the worst prognosis. Bone was the most common metastatic site in ER+ P-ILCs and TN-P-ILCs. HER2+ P-ILCs tended to metastasize to the brain and liver. DFS of HER2+ P-ILCs and TN-P-ILCs were worse than that of ER+ P-ILCs. Lacking lobular carcinoma in situ and sTILs ≤ 10% were associated with worse survival of ER+ P-ILCs and TN-P-ILCs, respectively. For HER2+ P-ILCs, Ki-67 > 20% and sTILs ≤ 10% were significant factors for lower DFS. P-ILCs is an aggressive subtype of ILCs. Analyzing the prognostic factors of P-ILCs with heterogeneous morphological and biomarker characteristics is helpful for creating an individualized treatment.

Abstract P1-08-13: Comparison of gene expression profiling results and clinical outcomes among patients with pleomorphic ILC and classic ILC

Abstract Background Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer. Pleomorphic ILC (pILC) is a pathologically defined subtype of ILC that often presents at a higher stage compared to Classical ILC (cILC). pILC is traditionally thought to have poor outcomes compared with cILC. Utility and application of gene expression profiling, such as Oncotype Dx testing, for these patients is unknown. The purpose of this study is to compare Oncotype Dx recurrence scores, treatment patterns, and clinical outcomes for patients with cILC and pILC at our institution. Methods A retrospective analysis of a large institutional cancer database was performed to identify patients with ILC treated between 2004 and 2017. Patient and disease characteristics were collected, including subtype of ILC (classical vs pleomorphic), staging, treatment history, and presence of Oncotype Dx testing. Findings were analyzed for differences in clinical characteristics, Oncotype Dx results, progression free survival (PFS) and overall survival (OS) between patients who received endocrine therapy/chemotherapy (CET) versus endocrine therapy alone (ET) in both cILC and pILC groups. Results: 692 patients with ILC were identified with 100 (14.5%) categorized as pILC. The mean age at presentation was 62 for cILC and 60 for pILC. cILC was more frequently ER positive (98.5% vs. 94.0%, p=0.004). and less likely HER2 positive (7.0% vs 12.2%, p=0.07). pILC more commonly had more advanced tumor stage (Stages II-III 61% vs 41%, p&amp;lt;0.001) but lower nodal stage (N+ 48.5% vs 66%, p&amp;lt;0.001) 25% (25/100) of pILC patients had Oncotype Dx testing performed compared to 33% (198/592) of cILC patients. The majority of patients who had testing had T1cN0 or T2N0 disease (36% and 44% for pILC vs 34%, and 27% for cILC, respectively). A low risk recurrence score (0-10) accounted for 8% (2/25) of pILC cases and 18.2% (36/198) of cILC. Intermediate risk (11-25) accounted for 72% (18/25) of pILC and 73.2% (145/198) of cILC. High risk (26+) accounted for 20% (5/25) of pILC and 8.6% (17/198) of cILC. There was no statistically significant difference in Oncotype Dx recurrence score distribution between pILC and cILC (p=0.117). More patients with pILC received CET compared to cILC (62.0% vs 36.7%, p&amp;lt;0.001). Among pILC cases, 62% received CET and 34% received ET. There was no significant difference in RFS or OS between CET and ET among pILC cases (10-year RFS 74.7% (ET) vs. 76.6% (CET), p=0.52. 10-year OS 92.6% (ET) vs 78.0% (CET), p=0.40). Conclusions At our institution, patients with cILC more often had Oncotype Dx testing compared to those with pILC. Some of the difference may be related to more frequent HER2-positivity in pILC. When testing was done, pILC was twice as likely to have a high risk recurrence score compared to cILC, but this result was not statistically significant. Patients with pILC were more likely to receive CET than patients with cILC. There was no difference in survival between patients with pILC treated with CET versus those treated with ET alone although the sample size is small and this is worthy of further study in a larger population. These results suggest that gene expression testing may be underutilized in pILC. Citation Format: Marcus Dempster, Matthew Wright, Daniela Cocco, Ayat Elsherif, Stephanie A Valente, Hong Li, Megan L Kruse. Comparison of gene expression profiling results and clinical outcomes among patients with pleomorphic ILC and classic ILC [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 P1-08-13.

Clinicopathological features of and neoadjuvant therapy for human epidermal growth factor receptor 2-positive classic invasive lobular carcinoma

Human epidermal growth factor receptor 2 (HER2)-positive classic invasive lobular carcinoma (ILC) is a rare entity, and its specific pathological characteristics and clinical behavior are unclear. Therefore, we retrospectively investigated the clinicopathological features of HER2-positive classic ILC and described HER2-targeted neoadjuvant chemotherapy outcome. Fourteen HER2-positive classic ILC cases were identified, their clinicopathological data were collected, and their clinical management was investigated. In general, HER2 positivity is associated with higher grade carcinoma in ductal lesions; however, our 14 cases had typical morphologic features of classic ILC. We did not identify unique morphologic features in HER2-positive classic ILC with the clinicopathological parameters examined, including age, menopausal status, histological grade, and hormone receptor status. Four of six patients who received HER2-targeted neoadjuvant chemotherapy had a pathological complete or partial response. Based on our findings, clinicopathological features of classic ILC may not predict HER2 overexpression or amplification. HER2-targeted neoadjuvant chemotherapy was effective against some HER2-positive classic ILCs. Taken together, these results demonstrate that HER2 immunohistochemistry and fluorescence in situ hybridization should be considered for all ILC patients regardless of subtype.

Clinicopathological Features, Tumor Mutational Burden, and Tumour-Infiltrating Lymphocyte Interplay in ERBB2-Mutated Breast Cancer: In Silico Analysis.

Recent evidence suggests that somatic mutations in ERBB2 activate ERBB2 signaling. These mutations occur at a frequency of approximately 3% in breast cancer (BC). ERBB2 mutations indicate poor prognosis as they are associated with recurrence and metastasis. This study aimed to evaluate the clinicopathological features, immune infiltration levels, tumor mutational burden (TMB), and tumor-infiltrating lymphocytes (TILs) in ERBB2-mutated breast cancer (ERBB2-mutated BC) using a bioinformatic approach and publicly available datasets (i.e., TCGA-BRCA and TIMER2.0). ERBB2-mutated BCs were associated with a high histological grade. ERBB2-mutated BCs comprised invasive breast carcinoma of no special type (21/35, 60%), classic invasive lobular carcinoma (12/35, 34.3%), and pleomorphic invasive lobular carcinoma (2/35, 5.7%). A Kaplan-Meier survival curve demonstrated that ERBB2-mutated BC was associated with a significantly worse prognosis compared to ERBB2 non-mutated BC (p < 0.01). Furthermore, 40% (14/35) of the patients with ERBB2-mutated BC harbored CDH1 mutations. Mutations at L755 and V777 accounted for 30.5% of these mutations in ERBB2-mutated BC, suggesting that these sites are mutational hot spots in BC, particularly in invasive lobular carcinoma. Of the ERBB2-mutated BCs, 8.6% were classified as TIL-high, whereas 77.1% were TILs-low; TMB significantly correlated with TILs (p < 0.05). CD8+ T cell infiltration levels were significantly higher in ERBB2 non-mutated BC. Among ERBB2-mutated BCs, 22.9% were classified as TMB-high, which was significantly higher than the rate in the ERBB2 non-mutated BC (p < 0.01). These findings provide evidence for a link between ERBB2 mutations and high TMB in BC.

Mitotic score and pleomorphic histology in invasive lobular carcinoma of the breast: impact on disease-free survival.

Pleomorphic invasive lobular carcinoma (ILC) has long been thought to have worse outcomes than classic ILC and is therefore often treated with chemotherapy. However, recent data question the utility of the pleomorphic designation, as the poor outcomes seen may be related to other associated high-risk features. Importantly, mitotic count may better define a subset of ILC with high risk of recurrence. We sought to determine the impact of pleomorphic histology versus mitotic count on disease-free survival (DFS) in pure ILC. Additionally, we evaluated whether pleomorphic histology was associated with receipt of chemotherapy when adjusting for other factors. We analyzed a cohort of 475 patients with stage I-III pure ILC. We used Kaplan-Meier estimates, and Cox proportional hazards and logistic regression for multivariate analyses. Pleomorphic histology was confirmed by central pathology review. In a multivariate model, pleomorphic histology was not associated with reduced DFS. Only mitotic score, receptor subtype, and pathologic stage were independently and significantly associated with DFS. Patients with pleomorphic ILC were significantly more likely to receive chemotherapy than patients with classic ILC (adjusted odds ratio 2.96, p = 0.026). The pleomorphic designation in ILC does not have clinical utility and should not be used to determine therapy. Rather, mitotic count identified clear prognostic groups in this cohort of pure ILC.

Abstract P5-02-09: The prevalence of invasive lobular carcinomas with unconventional immunohistochemical phenotypes

Abstract Background The prognosis of invasive lobular carcinoma (ILC) is controversial. Some studies report equivalent or better outcomes than invasive ductal carcinoma, likely due to ILC’s favorable prognostic phenotype. Classic ILC (CL) is typically of low histologic grade, estrogen receptor (ER) positive and human epidermal growth factor receptor-2 (HER2) negative. However, worse long term outcomes have been observed because ILC tends to be multicentric, bilateral, and to show higher rates of late metastases in uncommon sites. Additionally, subtypes of ILC, including solid (SOL) and pleomorphic (PL), have been reported to display more aggressive behavior. Yet current treatment paradigms are distinguished by hormone receptor profile and stage, not by tumor morphology. This study aimed to evaluate the clinicopathologic and immunohistochemical (IHC) characteristics of ILC, particularly those with unusual patterns. Methods From 2009-2018, consecutive in-house biopsy and resection specimens with the diagnosis of ILC were identified by a retrospective review of our institutional database. Demographic, morphologic, and IHC data were obtained from pathology reports. Cases with equivocal HER2 IHC results are sent for fluorescence in situ hybridization analysis at our Center. CL ILC was defined by small cells with low nuclear grade and discohesive growth arranged in single file or cords. PL ILC is composed of larger, more atypical cells, which still exhibit discohesion typical of lobular phenotype. SOL ILC consists of tumors growing in large sheets with minimal intervening stroma. Results Over the nine year study period, a total of 3,028 ILCs from 2,322 patients were identified. Most (74%, 2229/3028) cases were classified as CL while 22% (665/3028) were PL and 4% (134/3028) SOL (Table 1). The median patient age was 60 years (range 32-95). The median tumor size was larger for PL (2.3 cm) and SOL (2.4 cm) subtypes than CL (1.1 cm). IHC results were available for 896 specimens. The majority of all ILCs showed an expected phenotype: ER positive and HER2 negative (99% CL, 84% PL, 97% SOL). A smaller subset of ER positive ILC were also HER2 positive (1% CL, 8% PL, 3% SOL). No CL or SOL ILC were ER negative; in contrast 8% (21/270) of PL ILC were ER negative. Twelve (5%) PL ER negative cases were also HER2 negative. Overall, 15% of ILC were HER2 positive, with the PL variant showing the highest incidence (11%, 30/270). Lymph node (LN) metastases were seen in 22% (66/291) of CL cases, 55% (53/96) PL and 29% (17/58) SOL. Metastases in 10 or more LN (N3) was seen only in PL and SOL cases (13% and 5%, respectively). Conclusions In this study, up to 8% of ILC cases were found to be ER negative, a finding unique to the PL variant. No CL or SOL ILC was ER negative. HER2 positivity, not typically expected in ILC, was seen in 15% of cases, most commonly in the PL type. ILC that was both negative for ER and HER2 was observed in 5% of cases, and only within the PL subgroup. PL ILC showed higher median tumor size at presentation than CL and nodal metastases were also more common in PL than in CL or SOL ILC (55% vs 22% and 29%, respectively). Our findings are in keeping with prior observations that compared to CL ILC. PL ILC exhibits features associated with unfavorable clinicopathologic features, such as higher tumor and nodal stage at presentation. Additionally, this is the first study to systematically examine the prevalence of unconventional IHC phenotypes of ILC (ER negativity and/or HER2 positivity) in a large series from one institution. Table 1. Summary of Clinicopathologic FeaturesClassicPleomorphicSolidILC types (n=3028)2229665134Median age, (range)60 yrs (32-94)61 yrs (31-95)66 yrs (44-93)Median size, cm 1.12.32.4Immunophenotype (n=896)ER+/HER2-99% (551/559)84% (228/270)97% (65/67)ER+/HER2+1% (8/559)8% (21/270)3% (2/67)ER-/HER2+0% (0/559)3% (9/270)0% (0/67)ER-/HER2-0% (0/559)5% (12/270)0% (0/67)Lymph node status (n=445)N077% (225/291)45% (43/96)71% (41/58)N120% (59/291)31% (30/96)19% (11/58)N22% (7/291)11% (11/96)5% (3/58)N30% (0/291)13% (12/96)5% (3/58) Citation Format: Matthew G Hanna, Anne Grabenstetter, Dara S Ross, Lee K Tan. The prevalence of invasive lobular carcinomas with unconventional immunohistochemical phenotypes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-09.

Invasive breast lobular carcinoma with extracellular mucin: a clinicopathological analysis

Objective: To study the clinicopathological features of invasive lobular carcinoma (ILC) of the breast with extracellular mucin and outcomes of patients. Method: Clinicopathological features and clinical follow-up (39-123 months and a median follow-up of 55 months) of seven ILC with extracellular mucin were obtained. Hematoxylin-and-eosin (H&E) and immunohistochemistry (IHC) stained sections were reviewed, and fluorescence in situ hybridization (FISH) assay was performed for tumors with HER2 IHC 2+. Patient prognosis was analyzed and literatures related to ILC with extracellular mucin were reviewed. Results: All seven patients were female, aged from 43 to 73 years (median age, 55 years). The tumors ranged in size from 1 to 5 cm (median size 2 cm). All seven cases were of histological grade 2. Most areas of the tumors presented with the morphology of classic ILC, and variable amount of extracellular mucin were observed focally. In six cases, part of the tumor cells contained intracellular mucin, and the nucleus were pushed to one side of the cells, creating the impression of signet-ring cells. Two patients had lymph node metastases at diagnosis, and developed liver and bone metastases at 38th and 48th month, respectively, after surgery, and died at 48th and 123th month, respectively. While the other five patients, except one lost to follow-up, had been disease-free during the follow-up period. IHC results showed estrogen receptor (ER) and progesterone receptor (PR) positivity in 7/7 and 6/7 cases, respectively. Tumors of six patients were HER2 IHC 0/1+. The remaining one was HER2 IHC 2+, while FISH assay revealed HER2 gene amplification in that tumor. The proportion of cases with HER2-positivity was 1/7. The proliferation index Ki-67 ranged from less than 5% to 30%, and Ki-67 less than or equal to 10% were in 5/7 cases. According to the 2013 St. Gallen International Expert Consensus on breast cancer, all tumors were of luminal types; of those, two were luminal A and five were luminal B. Conclusions: ILC with extracellular mucin tends to occur in women over 50 years old. All tumors in the study are grade 2 classic ILC, with signet-ring cells as a common feature. All seven tumors are classified as luminal types, with luminal B as the main molecular subtype.

Adaptive iterative learning control for MIMO nonlinear systems performing iteration-varying tasks

The present work aims to develop a novel adaptive iterative learning control(AILC) method for nonlinear multiple input multiple output (MIMO) systems that execute various control missions with iteration-varying magnitude-time scales. In order to reduce the variations of the systems, this work proposes a series of time scaling transformations to normalize the iteration-varying trial lengths. An AILC scheme is then developed for the transformed control systems on a uniform trial length, which is shown to be capable of ensuring the asymptotic convergence of the tracking error. In other words, the proposed AILC algorithm is able to relax the constraint in conventional ILC where the control task must remain the same in the iteration domain. Additionally, the basic assumption in classic ILC that the control system must repeat on a fixed finite period is also removed. The convergence analysis of the AILC is derived rigorously according to the composite energy function (CEF) methodology. It is shown that the newly developed learning control strategy works well for control plants with either time-invariant or time-varying parametric uncertainties. To show the effectiveness of the AILC, three examples are illustrated in the end. Meanwhile, the proposed learning method is also implemented to a traditional X–Y table system.

Exploring Collagen Parameters in Pure Special Types of Invasive Breast Cancer

One of the promising tools to evaluate collagen in the extracellular matrix is the second-harmonic generation microscopy (SHG). This approach may shed light on the biological behavior of cancers and their taxonomy, but has not yet been applied to characterize collagen fibers in cases diagnosed as invasive breast carcinoma (BC) of histological special types (IBC-ST). Tissue sections from 99 patients with IBC-ST and 21 of invasive breast carcinoma of no special type (IBC-NST) were submitted to evaluation of collagen parameters by SHG. Tissue microarray was performed to evaluate immunohistochemical-based molecular subtype. In intratumoral areas, fSHG and bSHG (forward-SHG and backward-SHG) collagen parameters achieved their lowest values in mucinous, papillary and medullary carcinomas, whereas the highest values were found in classic invasive lobular and tubular carcinomas. Unsupervised hierarchical cluster analysis and minimal spanning tree using intratumoral collagen parameters allowed the identification of three main groups of breast cancer: group A (classic invasive lobular and tubular carcinomas); group B (IBC-NST, metaplastic, invasive apocrine and micropapillary carcinomas); and group C (medullary, mucinous and papillary carcinomas). Our findings provide further characterization of the tumor microenvironment of IBC-ST. This understanding may add information to build more consistent tumor categorization and to refine prognostication.

High Frequency of ERBB2 Activating Mutations in Invasive Lobular Breast Carcinoma with Pleomorphic Features.

Background: Characterisation of molecular alterations of pleomorphic lobular carcinoma (PLC), an aggressive subtype of invasive lobular carcinoma (ILC), have not been yet completely accomplished. Methods: To investigate the molecular alterations of invasive lobular carcinoma with pleomorphic features, a total of 39 tumour samples (in situ and invasive lesions and lymph node metastases) from 27 patients with nuclear grade 3 invasive lobular carcinomas were subjected to morphological, immunohistochemical and massive parallel sequencing analyses. Results: Our observations indicated that invasive lobular carcinomas with pleomorphic features were morphologically and molecularly heterogeneous. All cases showed absence or aberrant expression of E-cadherin and abnormal expression of β-catenin and p120. CDH1 (89%), PIK3CA (33%) and ERRB2 (26%) were the most common mutated genes. ERBB2 mutations preferentially affected the tyrosine-kinase activity domain, being the most frequent the targetable mutation p.L755S (57%). We also observed higher frequency of mutations in ARID1B, KMT2C, MAP3K1, TP53 and ARID1A in PLC than previously reported in classic ILC. Alterations related to progression from in situ to invasive carcinoma and/or to lymph node metastases included TP53 mutation, amplification of PIK3CA and CCND1 and loss of ARID1A expression. Conclusions: The high frequency of ERBB2 mutations observed suggests that ERBB2 mutation testing should be considered in all invasive lobular carcinomas with nuclear grade 3.