Primary Invasive Lobular Carcinoma Research Articles

Abstract PO5-06-14: Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma

Abstract Background: Triple-negative (TN) invasive lobular carcinoma (ILC) of the breast is a rare entity, comprising roughly 2% of primary ILC. Clinicopathologic studies have shown that TN ILC is associated with more pleomorphism, higher nuclear grade, older patient age, and worse disease-related outcomes compared to estrogen receptor-positive (ER+) ILC. Intrinsic subtyping indicates that TN ILC less frequently expresses basal markers than TN invasive ductal carcinoma (IDC). This prompted investigation of the genomic landscape and clinical management of TN ILC to provide insight into potential therapeutic approaches. Methods: Twenty patients with TN ILC were included in this study. Three patients were treated at a single academic center and underwent comprehensive genomic profiling (CGP) of breast or metastatic tissue; clinicopathologic details were obtained from their electronic health records. Seventeen patients with TN ILC who underwent CGP of breast or metastatic biopsies were included from the Foundation Medicine, Inc. database. PD-L1 testing for all patients was determined by immunohistochemistry (IHC) using the VENTANA SP142 or Dako 22C3 commercial assays. HER2 expression was determined by IHC. Negative HER2 expression was defined as an IHC score of 0, or IHC 1+ or 2+ with non-amplified fluorescence in situ hybridization (FISH). Results: In the Foundation Medicine database of patients with TN ILC, the most frequent genomic alterations were in CDH1 (15/17 patients [88.2%]), TP53 (10/17 patients [58.8%]), and PIK3CA (10/17 patients [58.8%]). Short variant mutations in ERBB2 were found in 3/17 patients (17.6%). Fourteen patients were HER2-low (HER2 IHC 1+ or 2+ with non-amplified FISH). No cases were MSI high or expressed PD-L1 (8 of 17 cases underwent PD-L1 assessment by SP142). Median tumor mutational burden (TMB) was 5 muts/Mb. Among the three patients with TN ILC treated at a single center, the most common somatic mutations were in CDH1 and TP53, and no cases were MSI high or expressed PD-L1 (by SP142 or 22C3). All three patients had pleomorphic ILC, histologic grade 2 or 3, and HER2 IHC 0. One patient with mpT1cN1a(sn) ER+/HER2- ILC of the right breast in 2011 received adjuvant TAC (docetaxel, doxorubicin, cyclophosphamide), breast/axillary radiation, and ten years of adjuvant endocrine therapy. She was diagnosed with TN ILC of the left supraclavicular nodes in 2022; she did not exhibit a response to docetaxel and carboplatin with pembrolizumab, and she subsequently received palliative radiation followed by capecitabine. Another patient with locally advanced TN ILC progressed on the KEYNOTE-522 regimen, immediately developed metastases, and is currently receiving palliative sacituzumab govitecan. The third patient with cT3N1 TN ILC progressed on neoadjuvant therapy (through the I-SPY 2 trial) with oral paclitaxel, encequidar, and dostarlimab, followed by dostarlimab with dose-dense doxorubicin and cyclophosphamide. Her surgical pathology demonstrated ypT3N3a disease with 10 × 7 cm of tumor, and she developed skin metastases on the chest wall within weeks post-operatively. She received comprehensive chest wall and nodal radiation and is currently receiving palliative capecitabine with neratinib given a high variant allele frequency driver somatic ERBB2 mutation. Conclusions: The most common somatic mutations among TN ILC patients included in this study were in CDH1, TP53, and PIK3CA. Currently the management of TN ILC is extrapolated from that of TN IDC, however our patients did not respond to chemoimmunotherapy. The presence of HER2-low status or somatic ERBB2 mutations may provide opportunities for treatment with an anti-HER2 antibody-drug conjugate or tyrosine kinase inhibitor. Studies with larger sample sizes of TN ILC are needed for molecular profiling and assessment of outcomes with specific therapeutic approaches. Citation Format: Hemali Batra-Sharma, Smruthy Sivakumar, Prashanth Ashok Kumar, Ethan Sokol, Rebecca Shatsky, Jeffrey Ross. Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-14.

68Ga-FAPI PET/CT as an Alternative to 18F-FDG PET/CT in the Imaging of Invasive Lobular Breast Carcinoma.

Accurate staging of invasive lobular carcinoma (ILC), a subtype of breast cancer, is vital for effective clinical management. Although 18F-FDG PET/CT is a commonly used tool, its efficacy varies across different histologic subtypes. To mitigate this challenge, our investigation delves into the potential utility of 68Ga-fibroblast activation protein inhibitor (FAPI) PET/CT as an alternative for staging ILC, aiming to address a significant research gap using a more expansive patient cohort than the smaller samples commonly found in the existing literature. Methods: In this retrospective analysis, women diagnosed with primary ILC of the breast underwent both 18F-FDG PET/CT and 68Ga-FAPI PET/CT. Both modalities were compared across all lesion locations with the used reference standard. The interval between scans was 1 wk, without any intervening treatments. Lesions were categorized visually, and tracer activity was analyzed using SUVmax, tumor-to-background uptake ratio, and uptake ratios. Both modalities were compared across various parameters, and statistical analysis was performed using SPSS 22.0. A P value of less than 0.05 was chosen to determine statistical significance. Results: The study included 23 female ILC patients (mean age, 51 y) with hormone-positive, human epidermal growth factor receptor type 2-negative tumors. Most (65%) had the luminal A subtype. 68Ga-FAPI PET/CT outperformed 18F-FDG PET/CT, with higher tumoral activity and tumor-to-background uptake ratios (P < 0.001). Primary tumors showed significantly increased uptake with 68Ga-FAPI PET/CT (P < 0.001), detecting additional foci, including multicentric cancer. Axillary lymph node metastases were more frequent and had higher uptake values with 68Ga-FAPI PET/CT (P = 0.012). Moreover, 68Ga-FAPI PET/CT identified more lesions, including bone and liver metastases. Pathologic features did not significantly correlate with imaging modalities, but a positive correlation was observed between peritumoral lymphocyte ratio and 68Ga-FAPI PET/CT-to-18F-FDG PET/CT uptake ratios (P = 0.026). Conclusion: This study underscores 68Ga-FAPI PET/CT's superiority over 18F-FDG PET/CT for ILC. 68Ga-FAPI PET/CT excels in detecting primary breast masses, axillary lymph nodes, and distant metastases; can complement 18F-FDG PET/CT in ILC; and holds potential as an alternative imaging method in future studies.

Genomic and epigenomic basis of breast invasive lobular carcinomas lacking CDH1 genetic alterations

CDH1 (E-cadherin) bi-allelic inactivation is the hallmark alteration of breast invasive lobular carcinoma (ILC), resulting in its discohesive phenotype. A subset of ILCs, however, lack CDH1 genetic/epigenetic inactivation, and their genetic underpinning is unknown. Through clinical targeted sequencing data reanalysis of 364 primary ILCs, we identified 25 ILCs lacking CDH1 bi-allelic genetic alterations. CDH1 promoter methylation was frequent (63%) in these cases. Targeted sequencing reanalysis revealed 3 ILCs harboring AXIN2 deleterious fusions (n = 2) or loss-of-function mutation (n = 1). Whole-genome sequencing of 3 cases lacking bi-allelic CDH1 genetic/epigenetic inactivation confirmed the AXIN2 mutation and no other cell-cell adhesion genetic alterations but revealed a new CTNND1 (p120) deleterious fusion. AXIN2 knock-out in MCF7 cells resulted in lobular-like features, including increased cellular migration and resistance to anoikis. Taken together, ILCs lacking CDH1 genetic/epigenetic alterations are driven by inactivating alterations in other cell adhesion genes (CTNND1 or AXIN2), endorsing a convergent phenotype in ILC.

Abstract 5283: Glucocorticoid receptor (GR) activity alters invasive lobular breast cancer (ILC) oncogenic properties

Abstract Invasive breast cancer (BC) is commonly divided into two subtypes, invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC). ILC makes up 10-15% of all BC cases. Expression of nuclear hormone receptors further define clinical histology and directs treatment; receptors include estrogen (ER), progesterone (PR), androgen (AR), and glucocorticoid receptors (GR). Tumor GR expression activation is associated with good prognosis in ER+ but poor prognosis in ER- BC. The majority of ILC tumors are ER+, and we have found that GR expression in primary ILC tumors correlates with improved overall survival. We hypothesized that GR activity in ILC may play a role in ILC’s indolent natural history and could alter its metastatic tendency. We used two isogenic ILC cell lines, each with and without GR expression (MM134-GR+/GR- and SUM44PE-GR+/GR-) to study GR-mediated ILC associated phenotypes. Specifically, we describe GR’s effect on proliferation in vitro and tumor formation in the in vivo MIND model. We also measured GR-mediated ILC adherence to major components of the extra cellular matrix (collagen I, collagen II, laminin, fibronectin, vitronectin, and tenascin). To recapitulate early metastasis, we examined ILC cell movement through a 10 uM microchannel device. We found that GR expression and activation in ILC cell lines cause decreased cell proliferation and decreased adherence to laminin. Preliminary experiments suggest that GR activation altered ILC cell’s ability to enter and navigate through restrictive 10 uM microchannels. Given our preliminary findings, we will investigate the effect of selective GR modulators (SGRMs) on ILC phenotypes and gene expression. We hypothesize that the gene expression changes following GR activation will be different in ILC compared to IDC. In addition, we are examining the genes required for survival of ILC cells on collagen using an in vitro CRISPR-Cas9 screen. In vivo experiments examining the association of GR expression with tumor growth in vivo are ongoing. Citation Format: Ishrat Durdana, Candace Frerich, Muriel Laine, Smita Rindhe, Lynda Bennett, Robert Bachoo, Geoffrey Greene, Suzanne Conzen. Glucocorticoid receptor (GR) activity alters invasive lobular breast cancer (ILC) oncogenic properties. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5283.

Timing evolution of lobular breast cancer through phylogenetic analysis.

SummaryBackgroundLate distant recurrence is a challenge for the treatment of invasive lobular carcinoma (ILC) of the breast. Despite in-depth characterisation of primary ILC, the molecular landscape of metastatic ILC is still only partially understood.MethodsWe retrospectively identified 38 ILC patients from the tissue banks of six European institutions. DNA extracted from patient matched primary and metastatic FFPE tissue blocks was whole genome sequenced to compute somatic copy number aberrations. This, in turn, was used to infer the evolutionary history of these patients.FindingsThe data show different metastatic seeding patterns, with both an early and late divergence of the metastatic lineage observed in ILC. Additionally, cascading dissemination from a metastatic precursor was a dominant rule. Alterations in key cancer driver genes, such as TP53 or CCND1, were acquired early while additional aberrations were present only in the metastatic branch. In about 30% of the patients, the metastatic lineage harboured less aberrations than the primary tumour suggesting a period of tumour dormancy or prolonged adaptation at the distant site. This phenomenon was mostly observed in tumours from de novo metastatic patients.InterpretationOur results provide insights into ILC evolution and offer potential paths for optimised ILC care.FundingThis work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF) and the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS).

Abstract PD14-03: Genetic and epigenetic basis of invasive lobular carcinomas lacking CDH1-alterations

Abstract Introduction: Invasive lobular carcinoma (ILC) is the most prevalent histologic special type of breast cancer, representing ~15% of invasive breast cancers. ILCs are mostly hormone receptor-positive and diagnosed histologically based on their distinctive discohesive growth pattern, dispersed single cells or line of cells invading the stroma. ILCs commonly show biallelic inactivation of a CDH1 (~65%), a tumor suppressor gene that is mapped to 16q22.1 and encodes for E-cadherin, a critical component of the epithelial adhesion complex. Massively parallel sequencing and pathologic studies have shown that a subset of ILCs (up to 15%) may lack CDH1 loss-of-function (LOF) mutations/deletions and retain E-cadherin expression, despite their distinctive lobular phenotype. The genetic and epigenetic underpinning of ILCs lacking CDH1 alterations has yet to be defined. Here we sought to define the mechanistic basis of the lobular phenotype in ILCs lacking CDH1 LOF genetic alterations or CDH1 gene promoter methylation and to determine the repertoire of epigenetic and genetic alterations of CDH1-wildtype ILCs. Materials and methods: Reanalysis of the CDH1 gene status in 364 primary ILCs, previously subjected to clinical massively parallel sequencing, was performed to identify all primary ILCs lacking bi-allelic CDH1 alterations. The hematoxylin and eosin stained slides of all identified ILCs lacking bi-allelic CDH1 alterations were reviewed by two pathologists and primary pure ILCs lacking bi-allelic CDH1 alterations were curated. We evaluated the presence of genetic alterations in genes playing essential roles in epithelial adhesion included in the clinical sequencing panel of up to 505 genes. The presence of CDH1 promoter methylation in 18 ILCs lacking bi-allelic CDH1 alterations with available formalin-fixed, paraffin-embedded (FFPE) material was assessed using methylation-specific PCR (MSP) and bisulfite sequencing. Results: We identified 23/364 (6.3%) primary ILCs lacking bi-allelic CDH1 alterations, of which 65.3% and 34.7% were classic and pleomorphic lobular variants, respectively. In 18 cases with available FFPE material, our analyses revealed that 67% (12/18) of ILCs lacking bi-allelic CDH1 alterations displayed biallelic CDH1 inactivation via promoter methylation and 16q loss. Furthermore, we observed that 1/23 ILC lacking bi-allelic CDH1 alterations had bi-allelic inactivation of AXIN2. We then extended our query to all invasive breast carcinomas subjected to clinical sequencing, including the ones initially categorized as invasive breast carcinoma “type unknown”, and observed that the three additional cases with pathogenic LOF AXIN2 alterations were ILCs lacking bi-allelic CDH1 alterations. Conclusions: The lobular phenotype in ILCs can be due to CDH1 promoter methylation or genetic alterations affecting other genes related to epithelial cell adhesion, such as AXIN2 LOF mutations. Whole-genome sequencing analyses of ILCs whose molecular basis has not been identified by targeted sequencing are warranted. Citation Format: Fatemeh Derakhshan, Higinio Dopeso, Arnaud Da Cruz Paula, Pier Selenica, Antonio Marra, Edaise M da Silva, Andrea Gazzo, Shirin Issa Bhaloo, Dara S Ross, Anne Grabenstetter, Sarat Chandarlapaty, Pedram Razavi, Hannah Y. Wen, Hong Zhang, Edi Brogi, Britta Weigelt, Fresia Pareja, Jorge S. Reis-Filho. Genetic and epigenetic basis of invasive lobular carcinomas lacking CDH1-alterations [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 PD14-03.

Abstract PD14-01: Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity

Abstract Background: ILC is the second most common type of breast cancer and accounts for approximately 10% of all invasive breast cancers. A hallmark of ILC is the lack of E-cadherin (CDH1) expression, which is frequently used to discriminate between lesions with borderline ductal and lobular histologies. While the genomic landscape of primary ILCs has been well described, less is known about patients (pts) with metastatic-ILC (mILC). Better characterization of the genomic landscape associated with mILC is critical for identifying biomarkers that may provide new insight into ILC tumor biology and ultimately improve long-term outcomes in pts with mILC. Methods: We retrospectively analyzed de-identified next-generation sequencing (NGS) data from 150 advanced/metastatic pts with ILC and 51 with mixed lobular/ductal histology, defined using the histology of the sequenced biopsy. Diagnoses were abstracted from pathology reports submitted at the time of sequencing. We used the stage documented closest in time to biopsy collection, and samples were excluded if the staging date was unknown or exceeded 180 days after the biopsy date. Our dataset consisted of samples that were molecularly profiled using the Tempus xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage, full-transcriptome RNA-seq). The mutations identified for this study include somatic single-nucleotide variants and insertions/deletions. Furthermore, we examined the co-mutational landscape of CDH1-mutant disease and investigated transcript-level expression variation. Results: Mutations in CDH1 occurred in 65.3% of all mILC samples (98/150). CDH1 expression was similar between CDH1-mutant and WT mILC samples (Wilcoxon rank sum test, p=0.8). The median tumor mutational burden (TMB) score was significantly higher in CDH1-mutant samples (Wilcoxon rank-sum test, p=0.010). CDH1-mutant samples were more likely to have a high TMB (≥10 mutations/MB) when compared with the wild-type CDH1 cohort (10% vs. 6.2%), but this difference was not statistically significant (Fisher’s Exact test, p=0.5). Additionally, we observed that the ER+ subtype was more frequent in CDH1-mutant samples, although this difference was not statistically significant (97% vs 88%; Fisher’s exact test, p=0.063). PIK3CA mutations were enriched in CDH1-mutant mILC (Table 1). TBX3 and NCOR1 mutations were also mildly enriched in CDH1-mutant mILC, but these results were not significant when correcting for multiple testing (Table 1). CDH1-mutant mixed histology pts had lower CDH1 expression than WT pts (p&amp;lt;0.001, Wilcoxon rank sum exact test). PIK3CA mutations were enriched in CDH1-mutant mixed histology pts, but this difference was not statistically significant (50% vs. 31%; p=0.3, Fisher’s exact test). Log10 CDH1 expression across all mILC pts was lower than in mixed histology pts (3.01 vs 3.53; p&amp;lt;0.001, Wilcoxon rank-sum test). Conclusions: Our real-world dataset illustrates that the molecular landscape of CDH1-mutant mILC pts is distinct from CDH1-WT mILC pts. Additionally, mILC differs from mixed histology at a transcriptional level, with lower CDH1 expression regardless of CDH1 mutational status. Our findings suggest a use for CDH1 RNA expression levels in reclassifying mixed histology samples as mILC. Additionally, therapies targeting PIK3CA may be further investigated for their actionability in CDH1-mutant mILC cases. Table 1.Frequency of co-mutations in CDH1-mutant vs. WT mILC cohortsGenesCDH1-mutant (n=98)CDH1 WT (n=52)p-value1q-value2n (%)n (%)PIK3CA53 (54%)6 (12%)&amp;lt;0.001&amp;lt;0.001TBX313 (13%)0 (0%)0.0040.13NCOR111 (11%)0 (0%)0.0090.21Pearson’s Chi-squared test; Fisher’sexact test. 2Falsediscovery rate correction for multiple testing Citation Format: Andrew A Davis, Amir Behdad, Kayla Viets Layng, Firas Wehbe, Lorenzo Gerratana, Elizabeth Mauer, Alex Barrett, Ami N Shah, Paolo D’Amico, Lisa Flaum, William J Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity [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 PD14-01.

Aberrant Lymphatic Drainage of Primary Invasive Lobular Carcinoma with Concurrent Primary Lung Adenocarcinoma: A Case Report

Background: Lymphatic spread of breast cancer is currently well understood and can be assessed in breast cancer patients through the use of lymphatic mapping with sentinel node biopsy, CT, PET.&#x0D; The Case: Here we present a case report of a 73-year-old female with two distinct primary carcinomas, right-sided invasive lobular carcinoma of the breast and left-sided adenocarcinoma of the lung. We also discuss the predictable and unpredictable lymphatic drainage of the lobular carcinoma, including ipsilateral drainage to the axillary chain and suspected contralateral hilar and mediastinal lymph node metastasis.&#x0D; Conclusion: The unique lymphatic spread of breast cancer, in this case, emphasizes the use of lymphatic mapping for the staging of disease and staining biopsied tissue samples for tumor markers to guide treatment. Additional anatomic research in this patient or supporting reported cases are needed to determine the frequency and cause of aberrant lymphatic drainage of primary invasive lobular carcinoma of the breast.

Characterization of Stromal Tumor-infiltrating Lymphocytes and Genomic Alterations in Metastatic Lobular Breast Cancer.

Invasive lobular carcinoma (ILC) represents the second most common histologic breast cancer subtype after invasive ductal carcinoma (IDC). While primary ILC has been extensively studied, metastatic ILC has been poorly characterized at the genomic and immune level. We retrospectively assembled the multicentric EuroILC series of matched primary and metastatic samples from 94 patients with estrogen receptor (ER)-positive ILC. Stromal tumor-infiltrating lymphocytes (sTILs) were assessed by experienced pathologists. Targeted sequencing and low pass whole-genome sequencing were conducted to detect mutations and copy-number aberrations (CNAs). We compared the frequencies of the alterations in EuroILC with those from patients with ER-positive metastatic ILC (n = 135) and IDC (n = 563) from MSK-IMPACT. Low sTIL levels were observed in ILC metastases, with higher levels in the mixed nonclassic histology. Considering ILC metastases from EuroILC and MSK-IMPACT, we observed that >50% of tumors harbor genomic alterations that have previously been associated with endocrine resistance. A matched primary/metastasis comparison in EuroILC revealed mutations (AKT1, ARID1A, ESR1, ERBB2, or NF1) and CNAs (PTEN or NF1 deletion, CYP19A1 amplification) associated with endocrine resistance that were private to the metastasis in 22% (7/32) and 19% (4/21) of patients, respectively. An increase in CDH1, ERBB2, FOXA1, and TBX3 mutations, in CDH1 deletions and a decrease in TP53 mutations was observed in ILC as compared with IDC metastases. ILC metastases harbor genomic alterations that may potentially explain endocrine resistance in a large proportion of patients, and present genomic differences as compared with IDC metastases.

Frequent amplifications of ESR1, ERBB2 and MDM4 in primary invasive lobular breast carcinoma.

Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer following invasive ductal carcinoma (IDC). To identify potential genetic drivers of ILC progression, we used NanoString nCounter technology to investigate the DNA copy number (CN) in 70 well-curated primary ILC samples. We confirmed prior observations of frequent amplification of CCND1 (33%), and MYC (17%) in ILC, but additionally identified a substantial subset of ILCs with ESR1 and ERBB2 (19%) amplifications. Of interest, tumors with ESR1 CN gains (14%) and amplification (10%) were more likely to recur compared to those with normal CN. Finally, we observed that MDM4 (MDMX) was amplified in 17% of ILC samples. MDM4 knockdown in TP53 wild-type ILC cell lines caused increased apoptosis, decreased proliferation associated with cell cycle arrest, and concomitant activation of TP53 target genes. Similar effects were seen in TP53 mutant cells, indicting a TP53-independent role for MDM4 in ILC. To conclude, amplification of ESR1 and MDM4 are potential genetic drivers of ILC. These amplifications may represent actionable, targetable tumor dependencies, and thus have potential clinical implications and warrant further study.

Abstract P3-06-03: Copy number analysis identifies ESR1 and MDM4 as drivers of progression in invasive lobular breast carcinoma

Abstract Background: Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer after invasive ductal carcinoma (IDC). While specific clinical and pathological features differ between ILC and IDC, both histologies are treated the same, due to a lack of knowledge of targetable pathways underlying the observed differences. To identify potential genetic drivers of ILC progression, we set out to identify genes with copy number (CN) alterations, comparing tumors with good outcome to those with poor outcome. Method: We designed probes for a total of 67 genes known to be frequently altered in breast cancer and used sensitive nanoString technology to comprehensively investigate CN alterations of these genes in 70 well-curated primary ILCs. ILC cell lines MDA-MB-134-VI, SUM44PE, and BCK4 were used for functional studies including proliferation, apoptosis, colony formation, and analysis of gene expression. Results: Our studies reveal that ESR1 is frequently amplified in primary ILC (14% gains and 10% amplification), and that tumors with amplified ESR1 are more likely to recur compared to those with normal CN. Our analysis also identified a subset of ILCs with HER2 amplification (19%) despite a negative clinical IHC score, and these tumors expressed high HER2 mRNA, protein, and demonstrated enrichment of a molecular HER2 signature. The other most frequently amplified genes included CCND1 (33%), MDM4 (17%), and MYC (17%), and most frequently lost genes were NCOR2 (7%), FGFR4 (6%) and TP53 (6%). MDM4, a negative regulator of p53, has previously been reported to play a role in breast cancer, though little is known about its role in ILC. We demonstrate that decreasing MDM4 levels in p53 wild type ILC cell lines results in increased apoptosis, decreased proliferation associated with cell cycle arrest, and activation of p53 target genes. Intriguingly, a similar induction of G0/G1 cell cycle arrest and increase in apoptosis was observed in p53 mutant ILC cells after MDM4 downregulation, suggesting a p53-independent function of MDM4. Conclusion: Sensitive detection of CN changes identified amplifications of ESR1 and MDM4 as potential drivers of ILC. Functional studies demonstrate that MDM4 has both p53 dependent and independent functions that warrant further study. Citation Format: Atkinson JM, Cao L, Basudan A, Sikora MJ, Bahreini A, Tasdemir N, Jankowitz RC, McAuliffe PF, Dabbs D, Haupt S, Haupt Y, Peter Lucas PC, Lee AV, Oesterreich S. Copy number analysis identifies ESR1 and MDM4 as drivers of progression in invasive lobular breast carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-03.

Abstract P5-04-21: FGFR4 is a novel druggable target for recurrent ER-positive breast cancers

Abstract Background Breast cancer recurrence is a major clinical problem for estrogen receptor positive (ER+) disease, even decades after initial surgery. These long-term recurrences are a challenge for invasive ductal carcinoma (IDC), and are particularly frequent for the histological subtype of invasive lobular carcinoma (ILC). To study the long-term endocrine resistance seen in ILC patients, our lab recently generated six long-term estrogen deprivation (LTED) models of ILC cells and performed RNA-Sequencing to identify differentially expressed genes that ostensibly allow these cells to grow in the absence of estrogen. We overlapped these results with a previously published microarray dataset of tamoxifen-resistant cells, and found that FGFR4 is the most consistently overexpressed gene in the setting of acquired resistance to endocrine therapy in ILC cells. From a recent publication of RNA-Seq from other LTED models, FGFR4 RNA overexpression is also seen in all five IDC cell lines. Hypothesis FGFR4 is an important mediator of acquired endocrine resistance in breast cancer. Methods To study the role of FGFR4 in vitro, we used multiple shRNAs and specific small molecule inhibition for growth assays. To study the role of FGFR4 in de novo resistance to endocrine therapy, we collected 129 well curated ER+ ILC tumor specimens and performed gene expression analysis on the pre-treatment samples using a custom NanoString panel. To study the role of FGFR4 in acquired resistance, we collected over 50 pairs of primary-metastatic ER+ tumors and performed exon capture based RNA-Sequencing. Results FGFR4 inhibition decreases parental and LTED cell growth in classic 2D conditions and in colony formation assays. The LTED cells, with higher FGFR4 expression, are more sensitive to its inhibition. For the parental cells, combination FGFR4 and ER-targeting drugs results in synergistic decreases in growth. In our database of primary ILC clinical samples, increased expression of FGFR4 is predictive of shorter time to distant recurrence. Among primary-recurrent tumor pairs, FGFR4 is an outlier expression gain in 20/50 (40%), spanning all recurrence sites studied (i.e. local recurrences, and metastases to the brain, bone, ovaries, and GI tract). Finally, in analyzing large cohorts of metastatic tumors, there is a significant enrichment of hotspot FGFR4 mutations in tumors originating in the breast, with &amp;gt;2% of metastatic ILC tumors containing such a mutation. Conclusion/Future studies FGFR4 may play an important role in de novo resistance to endocrine therapy in ILC and acquired resistance in both ILC and IDC. Ongoing studies include overexpression of wild-type and FGFR4 hotspot mutations in ILC and IDC cell lines to determine growth and metastatic phenotypes. Citation Format: Levine KM, Ding K, Priedigkeit N, Sikora MJ, Tasdemir N, Zhu L, Tseng GC, Jankowitz RC, Dabbs DJ, McAuliffe PF, Lee AV, Oesterreich S. FGFR4 is a novel druggable target for recurrent ER-positive breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-21.

Abstract P1-07-05: AIB1 is a new putative prognostic biomarker in the luminal A and B-like (HER2-negative) classification of invasive lobular carcinoma

Abstract Background: Estrogen receptor (ER) positive HER2-negative breast cancer comprises 75–80% of all breast cancer. This fraction is even higher (&amp;gt;90%) in invasive lobular carcinoma (ILC). According to the St Gallen surrogate definitions of the intrinsic subtypes, Ki67 and progesterone receptor (PgR) are used to classify these tumors as luminal A- and luminal B-like (HER2-negative). These guidelines are based on information derived from patient materials with mixed histological types, where the vast majority of the patients have invasive ductal carcinoma. The `luminal-like classification´ together with histological grade, tumor size and lymph node status is widely used in the clinic for prognostication. The aim of the present study was to investigate if the same markers are applicable for ILC, and furthermore, if additional biomarkers involved in the endocrine signaling system, e.g. Amplified in breast cancer 1 (AIB1) and the putative G protein-coupled estrogen receptor (GPER), might provide complementary prognostic information. Patients: Two hundred and thirty-three (N = 233) well-characterized patients with primary ILC, diagnosed between 1980 and 1991 were included. Forty-two percent of the patients received adjuvant endocrine treatment and 2 % received adjuvant chemotherapy. All biomarkers were analyzed immunohistochemically on tissue microarray, whereas histological grade was evaluated on whole sections according to Elston and Ellis (NHG). The primary endpoint was breast cancer mortality (BCM). Results: In univariable analyses with 10-year follow-up, Ki67 (high vs. low), NHG (3 vs. 1+2) and AIB1 (high vs. low) were significantly associated to BCM (Hazard Ratio: 4.7, 95% CI: 2.1–10.4, p &amp;lt;0.001; HR: 3.1, 95% CI: 1.5–6.4, p = 0.003; HR: 3.2, 95% CI: 1.4–7.2, p = 0.005 respectively), whereas PgR (&amp;lt;1% vs ≥1%) and GPER (linear 0-4) were not (p = 0.25; p = 0.31 respectively). Essentially the same effect was seen after multivariable adjustment for lymph node status (+ vs. -), tumor size (&amp;gt;20 mm vs. &amp;lt;20 mm), adjuvant treatment and age (continuous). Subgrouping the tumors into luminal A- and B-like (HER2-negative) according to St Gallen surrogate definitions did not show significant prognostic differences between the two groups (p = 0.12). Patients with &amp;lt;20 mm, lymph node negative breast cancer and favorable tumor characteristics (low Ki67, NHG 1+2, and low AIB1) had a 10-year BCM of 4.2% (95% CI: 1.4–12%). This group constituted 34% of the patients included in the present study. Conclusions: In contrast to other previous studies, where breast cancers of mixed histological types were included, PgR was not significantly associated to prognosis in the ER-positive HER2-negative subgroup in the present study, consisting only of ILC. The prognostic role of PgR and the clinical usefulness of the luminal A and B-like (HER2-negative) classification (using only Ki67 and PgR) in ILC is still to be further investigated. The prognostic importance of Ki67 and NHG in this subgroup was, however, confirmed also in ILC, and AIB1 might be a new putative prognostic factor. By combining Ki67, NHG, and AIB1, together with lymph node status and tumor size, a group of patients with an excellent prognosis could be identified. Citation Format: Narbe U, Forsare C, Bendahl P-O, Lövgren K, Alkner S, Sjöström M, Rydén L, Leeb-Lundberg F, Ingvar C, Fernö M. AIB1 is a new putative prognostic biomarker in the luminal A and B-like (HER2-negative) classification of invasive lobular carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-07-05.

Lobular Metastatic Breast Cancer Patients With Gastrointestinal Involvement: Features and Outcomes

IntroductionMetastatic breast cancer typically involves the lungs, bones, brain, and liver and only occasionally affects the gastrointestinal (GI) tract. The relevant published data have been limited to case reports and small series of patients. Patients and MethodsThe present study focused on the treatment and outcomes of breast cancer patients with GI involvement diagnosed at the European Institute of Oncology. We analyzed the clinicopathologic features of the GI metastases and compared them with those of the primary tumors according to their histologic type (ductal or lobular carcinoma). ResultsFrom the database of the Department of Pathology, 40 patients who had undergone endoscopy or GI surgery with a final diagnosis of metastatic breast cancer from 2000 to 2014 were identified. The greatest proportion of patients (75%) had had primary invasive lobular carcinoma. Of the 40 patients, 82% had hormone receptor-positive disease in the metastatic lesion; 34 patients were candidates for systemic therapy. The median length of observation after GI metastasis was 18 months (range, 0.6-79 months). The overall survival from the diagnosis of GI involvement was 33 months (95% confidence interval, 16.8-38.3 months). ConclusionLobular breast carcinoma has a greater propensity to metastasize to the GI tract compared with other breast cancer subtypes. In the presence of GI symptoms, even if nonspecific, the GI tract should be thoroughly studied. Systemic treatment, including hormonal therapy, should be considered.

Abstract P6-09-43: Bcl2 as a long-term prognostic factor in invasive lobular carcinoma of the breast

Abstract Background: B-cell lymphoma 2 (Bcl2) is an anti-apoptotic protein with known tumor suppressor effect in breast cancer. Multiple studies have shown that high Bcl2-expression is associated with a better prognosis in breast cancer, but its clinical usefulness as a prognostic factor is still not fully elucidated. Invasive lobular carcinoma (ILC) of the breast is the second most common histologic subtype and comprises 5 to 15 % of all breast cancers. ILC has distinct clinico-pathological features and a specific pattern of recurrence, but treatment strategies are often similar as for the whole group of breast cancers. ILC´s are typically estrogen receptor positive (ER+), progesterone receptor positive (PgR+), HER2 negative, low proliferative and histological grade 2. According to the St Gallen surrogate definitions of the intrinsic subtypes of breast cancer most ILC´s are classified as 'luminal disease' and predominantly as low-risk 'luminal A-like'. Patients with 'luminal disease' in general and 'luminal A-like' in particular falls into prognostic categories where uncertainty about optimal treatment strategy often arises, as clinicians seek to avoid both over- and under-treatment. Additional prognostic factors are needed in order to further improve risk stratification. The use of multigene assays to better predict recurrence is increasing, but the availability is still limited and the cost is high. Measurement of apoptosis-related factors, such as Bcl2 gene expression is included as a prognosticator in a majority of these tests. The aim of this retrospective study was to evaluate the long-term prognostic effect of Bcl2 in a subset of patients with ILC. Patients and methods: One hundred and ninety-two well-characterized patients with primary ILC were included in the present study. Bcl2 was evaluated together with ER, PgR, HER2, Ki67, histological grade, tumor size, nodal status and age at diagnosis. Ninety-two percent of the patients were ER and/or PgR positive and 41% were node positive. Forty-one percent of the patients received adjuvant endocrine treatment and 3% received adjuvant chemotherapy. The median follow-up of the 52 patients still alive was 26 years. Bcl2 was analyzed immunohistochemically on whole sections of tumor tissue and Bcl2-positivity (Bcl2+) was defined as more than 10% cells with stained cytoplasma. Eighty-six percent of the patients (165/192) were Bcl2+. The primary endpoint was breast cancer mortality (BCM). Results: Overall, 32% (61/192) of the patients have died from breast cancer. Bcl2 (positive vs negative) was a significant prognostic factor for BCM in univariable Cox regression analysis (HR 0.44, 95% CI: 0.23-0.85). Essentially the same Bcl2 effect was seen after multivariable adjustment for ER, Ki67, histological grade, tumor size, nodal status, age at diagnosis and adjuvant therapy (HR 0.33, 95% CI: 0.15-0.74). Conclusions: Bcl2 is an independent long-term prognostic factor in this subset of patients with ILC. Bcl2 might add new prognostic information based on apoptotic tumor features that could be useful in the clinical treatment decision-making. Citation Format: Narbe U, Forsare C, Bendahl P-O, Rydén L, Ingvar C, Fernö M. Bcl2 as a long-term prognostic factor in invasive lobular carcinoma of the breast [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-43.

Contralateral breast cancer: incidence according to ductal or lobular phenotype of the primary

To identify differences in the incidence of contralateral breast cancer between patients with a primary tumour diagnosis of invasive ductal carcinoma (IDC) and those with a diagnosis of invasive lobular carcinoma (ILC). Data from two large cancer registries (registry A & B) the Northern and Yorkshire Cancer Registry Information Service (NYCRIS) and the West Midlands Cancer Intelligence Unit (WMCIU) from 1998-2003 for all cases of invasive breast cancer of either pure ductal or pure lobular reported histology were obtained. The invasive status of the contralateral tumour diagnosis and tumour morphology was collected. Chi-square tests were undertaken to examine the differences in contralateral rates for both registries and univariate analysis to ascertain which predictors affected contralateral breast cancer risk for registry A the WMCIU cases. A total of 38,132 patients were studied, 32,735 patients with IDC and 5397 (14.2%) patients with ILC over the 6-year period. There was no significant difference between the occurrence and time to occurrence of contralateral breast cancer according to original cancer histology, 901 (2.8%) patients with IDC versus 166 (3.1%) patients with ILC (p=0.169). The analysis of registry A cases showed no association between original histology (ductal versus lobular), age at diagnosis, tumour grade, use of radiotherapy for the primary cancer or use of systemic therapy (chemotherapy and/or endocrine therapy), and development of a contralateral breast cancer. There is no apparent increase in risk of developing a contralateral breast cancer according to the primary cancer histology either IDC or ILC. Standard mammographic follow-up does not need to take account of original tumour pathology. Increased intervention or post-treatment surveillance for the contralateral breast is not indicated in the context of ILC. The role of MRI should be restricted to those patients with ILC who are planning breast-conservation surgery, but not for assessing the contralateral breast.

Abstract 2394: Cancer-associated fibroblasts in invasive lobular breast carcinoma

Abstract Cancer-associated fibroblasts (CAFs) can promote breast cancer progression and therapy response through paracrine and juxtacrine signaling pathways. CAFs are abundantly observed in especially the invasive lobular carcinoma (ILC) subtype of breast cancer. Surprisingly, there is nothing known about the function of these CAFs in ILC. Given the fact that human ILC is generally resistant to standard chemotherapy, we believe that the cause of this therapy resistance lies into the active crosstalk between CAFs and tumor cells leading to a more progressive and resistant phenotype. Therefore, we aim to discover the exact function of CAFs in ILC as well as their drugable targets. In order to reliably study the role of CAFs in ILC, we generated a genetically engineered mouse model for ILC based on the tissue-specific loss of E-cadherin and PI3K signaling pathway activation, two hallmarks of human ILC. This clinically relevant mouse model spontaneously developed tumors that grow as typically invasive ‘indian files’ into a rigid extracellular matrix (ECM) that is very rich of CAFs. Uniquely, these tumors closely resemble human classical ILC, including its growth rate. Based on a mammary gland transplantation setting using fluorescent labeled hosts, we have shown that fibroblasts are recruited by ILC, both in vivo and in vitro, suggesting ILC-secreted mediators. Interestingly, in vitro inhibition of PDGFRβ (expressed by fibroblasts) can at least in part inhibit this recruitment. We are currently investigating whether inhibition of PDGFRβ in vivo can prevent the recruitment of fibroblasts and subsequently tumor development or progression. In parallel, we have isolated fibroblasts and epithelial cells from primary mouse ILCs and wild-type mammary glands to screen for other important players in the tumor-stroma crosstalk using RNA-seq and secretome proteomics. We will subsequently assess their functional significance during ILC development, progression and therapy response. Citation Format: Mirjam C. Boelens, Ellen Wientjens, Eva Schut, Sjoerd Klarenbeek, Karin E. de Visser, Jos Jonkers. Cancer-associated fibroblasts in invasive lobular breast carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2394. doi:10.1158/1538-7445.AM2015-2394

Abstract B77: The role of fibroblasts in invasive lobular breast carcinoma

Abstract Cancer-associated fibroblasts (CAFs) can promote breast cancer growth and metastasis, but the underlying mechanisms are still largely unknown. In order to study the role of CAFs in breast cancer development in an in vivo setting, we make use of a spontaneous mammary tumor mouse model for invasive lobular carcinoma (ILC). ILC is the second most common type of breast cancer, and is characterized by loss of E-cadherin, a rich stromal compartment, infiltrative growth, and treatment resistance. Often, the PI3K pathway is observed activated in the ILC subtype. Based on these characteristics, we have generated mouse models with mammary epithelial-specific conditional knockout of E-cadherin and PTEN that develop classical ILCs with a strong resemblance to human ILC. Using PDGFRβ as a fibroblast marker, we have observed an abundant presence of stromal fibroblasts in these ILCs, similar to human ILC. Based on a mammary gland transplantation setting using fluorescent labeled recipient mice, we have shown that fibroblasts get recruited from the host during tumor development. In vitro, we have also shown that tumor cells recruit fibroblasts. Inhibition of PDGFRβ in this in vitro model can at least in part inhibit this recruitment. We are currently investigating whether inhibition of PDGFRβ in vivo can prevent the recruitment of fibroblasts and subsequently tumor development or progression. In parallel, we have isolated fibroblasts from primary mouse ILCs and wild-type mammary glands to screen for other important players in the tumor-stroma crosstalk using RNA-seq and cytokine profiling. We will subsequently assess their functional significance during ILC development and progression. This research is supported by a fellowship of the Dutch Cancer Society Citation Format: Mirjam C. Boelens, Ellen Wientjens, Eva Schut, Sjoerd Klarenbeek, Karin E. de Visser, Jos Jonkers. The role of fibroblasts in invasive lobular breast carcinoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B77. doi:10.1158/1538-7445.CHTME14-B77

Invasive lobular carcinoma with metastasis to the lymph nodes. clinical pathological and immunohistochemical analysis

This study will focus on the comparative analysis of the clinical pathological characteristics of the patients with invasive lobu-lar carcinoma (ILC) that have axillary lymph node metastasis (ALNM) at the time of the diagnosis and those whose ILC remains confined to the breast. We further analysed the differences at the immunohistochemical (IHC) level between primary ILCs and their paired nodal metastases. Methods Pathology review and IHC analysis, on tissue microar-rays, was performed on 191 ILC cases. Results It was found that larger tumour size, higher histological grade, HER2 over-expression and moderate levels of tumour infil-trating lymphocytes were more frequently observed in ILCs with ALNM. Interestingly, proliferation, as assessed by Ki-67 staining, was found to be significantly increased in the nodal metastases when compared with their matched primary tumours. A significant heterogeneity for progesterone receptor PgR was also identified among primary ILCs and their metastases. This study contributed with some novel findings with respect to the clinical pathological features of ILC and its metastatic behaviour.

Cytosolic p120-catenin regulates growth of metastatic lobular carcinoma through Rock1-mediated anoikis resistance

Metastatic breast cancer is the major cause of cancer-related death among women in the Western world. Invasive carcinoma cells are able to counteract apoptotic signals in the absence of anchorage, enabling cell survival during invasion and dissemination. Although loss of E-cadherin is a cardinal event in the development and progression of invasive lobular carcinoma (ILC), little is known about the underlying mechanisms that govern these processes. Using a mouse model of human ILC, we show here that cytosolic p120-catenin (p120) regulates tumor growth upon loss of E-cadherin through the induction of anoikis resistance. p120 conferred anchorage independence by indirect activation of Rho/Rock signaling through interaction and inhibition of myosin phosphatase Rho-interacting protein (Mrip), an antagonist of Rho/Rock function. Consistent with these data, primary human ILC samples expressed hallmarks of active Rock signaling, and Rock controlled the anoikis resistance of human ILC cells. Thus, we have linked loss of E-cadherin - an initiating event in ILC development - to Rho/Rock-mediated control of anchorage-independent survival. Because activation of Rho and Rock are strongly linked to cancer progression and are susceptible to pharmacological inhibition, these insights may have clinical implications for the development of tailor-made intervention strategies to better treat invasive and metastatic lobular breast cancer.