Background: Invasive lobular carcinoma (ILC) is a distinct histologic type of breast cancer with unique features. The lack of well-characterized ILC organoid models has limited research and therapy development. We aim to establish a well-characterized ILC organoid biobank under the BCRF Legacy Project, creating a valuable resource for ILC research. Methods: We performed whole-genome (WGS) and RNA-sequencing (seq) on patient FFPE tumor tissues. ILC organoids are subjected to whole-exome (WES), bulk RNA-seq, single cell (sc)RNA-seq, scATAC-seq, and MSK-IMPACT targeted sequencing. STR profiling confirmed specimen relatedness. Data were analyzed using validated bioinformatics tools. Immunofluorescence (IF) analysis of E-cadherin and p120 was performed. Results: After establishing a total of 9 ILC organoids, WES was performed on all 9 organoids while WGS was performed on 2 patient samples corresponding to 2 of these organoids. WGS analysis of bilateral ILCs (TP19-M179), corresponding to LIO-046 organoid, revealed that both tumors shared a CDH1 p.L333Wfs23 frameshift mutation coupled with loss-of-heterozygosity (LOH), concurrent 1q gain and 16q loss, and dominant aging/clock mutational signatures. The left ILC was found to have fewer copy number alterations and somatic mutations than the right ILC. LIO-046 organoid was generated from right breast tumor tissue. Organoid IPM-BO-084 was generated from left ovarian metastasis of a mixed ILC (TP20-M130). Based on WGS analysis, the primary invasive carcinoma and ovarian metastases were found to be characterized by a somatic BRCA2 p.Q2157Ifs18 mutation associated with LOH, and displayed genomic features of homologous recombination deficiency (HRD). The levels of chromosomal instability were higher in ovarian metastases compared to the primary lesion. WES analysis of ILC organoid models identified genetic alterations affecting CDH1, which correlated with IF data where frameshift or nonsense mutations were associated with loss of E-cadherin and cytoplasmic p120 expression. Conclusions: We successfully established long-term ILC organoids. Our comprehensive genomic analysis of ILC organoids and patient samples underscores the genomic heterogeneity of ILC. Advanced sequencing techniques provide a framework for understanding ILC's molecular landscape. As we expand our biobank, these organoids and their data will be invaluable for developing targeted therapies and advancing ILC research. We are committed to making the genomic data and organoid models available to the research community. Our future goal is to foster collaboration and accelerate advancements in ILC research through this shared resource. Citation Format: Jagmohan Hooda, Jian Chen, Daniel D. Brown, Rohit Bhargava, David N Brown, Pier Selenica, Kaitlyn Gill, Hunter Green, Britta Weigelt, Adrian Lee, Steffi Oesterreich. Comprehensive Genomic Characterization of ILC Organoids: A BCRF Legacy Project Initiative [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-01-16.

To the Editor: Although small cell carcinoma is the most common neuroendocrine neoplasm of the cervix, rare cases of low-grade neuroendocrine tumors (NETs), or carcinoids, have been described. In a recent issue, Sah et al1 reported 3 cervical NETs, including a Grade 1 NET associated with the high-grade squamous intraepithelial lesion, adenocarcinoma in situ, and invasive adenocarcinoma. We add to the sparse literature with the report of another rare case. A 33-year-old woman underwent radical hysterectomy with bilateral salpingectomy and sentinel lymph node excision for a 2.7 cm fungating mass in the cervical canal. The mass was entirely submitted for histopathologic evaluation which revealed a tumor with 2 spatially and histologically distinct morphologies (Fig. 1A). The predominant component was an invasive mucinous adenocarcinoma with complex glandular and villoglandular architecture, comprising pseudostratified cells with hyperchromatic and elongated nuclei, intracytoplasmic mucin, abundant apical mitoses, and apoptotic bodies. In addition to prominent goblet cells, there were small cells with round, open nuclei and eosinophilic (neurosecretory-like) cytoplasmic granules, reminiscent of intestinal neuroendocrine cells, interspersed throughout the neoplastic glands (Fig. 1B). Adjacent to the adenocarcinoma was a smaller, distinct population of tumor cells with a nested, corded, and trabecular growth pattern (Fig. 1C). The cells had round, monotonous nuclei with stippled (salt and pepper) chromatin and eosinophilic granules in the cytoplasm. No necrosis or mitotic figures were identified in this component, which was morphologically consistent with Grade 1 NET.FIG. 1: Histopathologic features of a mixed endocervical adenocarcinoma and Grade 1 NET of the cervix. (A) The neoplasm is composed of spatially distinct adenocarcinoma and NET. (B) Adenocarcinoma shows intestinal differentiation with goblet cells and scattered neuroendocrine cells with bright eosinophilic granules (arrowheads). (C) The minor NET component shows the trabecular and corded architecture and monotonous nuclei with stippled chromatin (inset). Immunohistochemical analysis for (D) synaptophysin highlights tumor cells with neuroendocrine differentiation in both components. (E) Ki-67 index is 0% in the NET, and markedly elevated in the adenocarcinoma (inset). (F) High-risk HPV RNA in situ hybridization is positive in the NET, as well as adenocarcinoma (not shown) components. (G and H) Molecular analyses of adenocarcinoma and NET components, including somatic mutations (G) and copy number profiles (H). HPV, human papillomavirus; NET, neuroendocrine tumor.Immunohistochemistry for neuroendocrine markers, synaptophysin, chromogranin, and INSM1, revealed expression confined to the NET and highlighted scattered neuroendocrine cells admixed within the adenocarcinoma (Fig. 1D). Ki-67 proliferative index was 0% in the NET, and markedly elevated in the adenocarcinoma (Fig. 1E). Adenocarcinoma cells expressed CDX2 and CK20, consistent with intestinal differentiation, as well as CK7 and PAX8, in keeping with their Mullerian origin. Both the adenocarcinoma and the NET exhibited diffuse, strong p16 positivity, and positivity for RNA in situ hybridization targeting 18 different high-risk human papillomavirus (HPV) types (Fig. 1F). In addition, HPV16 and HPV18-specific in situ hybridization demonstrated the presence of both HPV types. Based on the overall features, a final diagnosis of mixed endocervical HPV-associated intestinal-type mucinous adenocarcinoma (Stage pT1a2, N0) and Grade 1 NET was rendered. Targeted tumor-normal panel sequencing was performed on separately microdissected NET and adenocarcinoma components. Shared ARID1A and SMAD4 pathogenic mutations, as well as chromosomal gains (1q, 17q) and losses (1p, 9p, 17p, 18), were identified in both components consistent with a clonal relationship (Fig. 1G, H). Immunohistochemistry confirmed the loss of ARID1A and SMAD4 expression in all tumor cells. Although ARID1A mutations are most commonly found in endometrial carcinomas, they have also been reported in mixed Mullerian and gastrointestinal-type mucinous tumors of the ovary2. SMAD4 mutations are prevalent in gastrointestinal (particularly, pancreatic) malignancies. Recent molecular analyses of 68 gastric-type cervical adenocarcinomas revealed mutations involving ARID1A in 6% and SMAD4 in 9% of cases, with one tumor harboring mutations in both genes3. The molecular features of our present case suggest a pathogenic origin from a Mullerian stem/progenitor cell capable of transdifferentiating into glandular and neuroendocrine cell lineages, recapitulating the spectrum of cell types typically found within the gastrointestinal tract.

AIMS:Activating somatic mutations or gene amplification of KIT result in constitutive activation of its receptor tyrosine kinase, which is targetable in various solid tumours. Here, we sought to investigate the presence of KIT genetic alterations in breast cancer (BC) and characterise the histological and genomic features of these tumours. METHODS:A retrospective analysis of 5,575 BCs previously subjected to targeted sequencing using the FDA-authorised Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Targets (MSK-IMPACT) assay was performed to identify BCs with KIT alterations. A histological assessment of KIT-altered BCs was conducted, and their repertoire of genetic alterations was compared with that of BCs lacking KIT genetic alterations, matched for age, histological type, oestrogen receptor/HER2 status and sample type. RESULTS:We identified 18 BCs (0.32%), including 9 primary and 9 metastatic BCs, with oncogenic/likely oncogenic genetic alterations affecting KIT, including activating somatic mutations (n=4) or gene amplification (n=14). All KIT-altered BCs were of high histological grade, although no distinctive histological features were observed. When compared with BCs lacking KIT genetic alterations, no distinctive genetic features were identified. In two metastatic KIT-altered BCs in which the matched primary BC had also been analysed by MSK-IMPACT, the KIT mutations were found to be restricted to the metastatic samples, suggesting that they were late events in the evolution of these cancers. CONCLUSIONS:KIT genetic alterations are vanishingly rare in BC. KIT-altered BCs are of high grade but lack distinctive histological features. Genetic alterations in KIT might be late events in the evolution and/or progression of BC.

Abstract ATP6AP1 and ATP6AP2, which we previously identified to be recurrently altered in granular cell tumors (GCTs), encode for accessory proteins of the vacuolar (V)-ATPase, which controls endosomal acidification. The mechanistic basis of how ATP6AP1/AP2 loss of function (LOF) leads to oncogenesis remains to be elucidated. Here, we sought to unravel the molecular mechanisms downstream of ATP6AP1/AP2 inactivation resulting in oncogenesis and identify associated therapeutic dependencies. Using CRISPR/Cas9 technologies, we generated ATP6AP1-knock out (KO), ATP6AP2-KO and non-target (NT) control immortalized Schwann cells, the likeliest cell of origin of GCTs. Compared to NT controls, ATP6AP1/AP2-KOs displayed increased cellular proliferation, migration, and anchorage independent growth. ATP6AP1/AP2-KOs, but not NT controls, demonstrated in vivo tumorigenesis upon subcutaneous implantation in NSG mice. In addition, ATP6AP1/AP2-KOs, compared to NT controls, displayed abnormal bulk endocytic function, evidenced by reduced endocytic cargo delivery to lysosomes in a DQ-Red BSA assay, and increased LC3BII/LC3BI ratio and expression of the autophagy marker GABARAP, indicating altered autophagic flux. ATAC-seq analysis of our cell modes revealed changes in chromatin accessibility upon ATP6AP1/AP2 inactivation, affecting transcription factors (TFs) associated to the Wnt pathway, including LEF/TCF (canonical Wnt) and NFAT (non-canonical Wnt), and TFs associated to autophagy pathways, among others. RNA-sequencing analysis revealed expression changes in key Wnt pathway components upon ATP6AP1/AP2 LOF, such as biphasic modulators (DKK1, DKK2 and SFRP1), secreted ligands (WNT2, WNT9a and WNT7B), transcriptional regulators (TLE1, TLE2, TLE3 and TLE4) and receptors (LGR5, FZD7 and FZD1). Consistent with these findings, ATP6AP1/AP2-KOs displayed upregulation of downstream canonical (MYC, Cyclin D1) and non-canonical (NFATC1, CALM1) Wnt targets at the mRNA and protein levels, and increased sensitivity to pharmacologic inhibition of the Wnt pathway, with significantly lower IC50s to IWR-1, compared to NT controls. Lastly, ATP6AP1/AP2-KOs displayed increased sensitivity to pharmacologic inhibition or induction of autophagy and/or endocytic processes, with lower IC50s to Chloroquine, N-Ethylmaleimide, Torin-1 and Bafilomycin-A1 compared to NT controls. Taken together, we demonstrate that ATP6AP1/AP2 inactivation leads to the acquisition of oncogenic properties in vitro and in vivo, altered endocytosis and autophagy. Furthermore, our findings suggest that ATP6AP1/AP2 LOF results in chromatin accessibility changes leading to activation of an oncogenic transcriptional program with increased signaling via the Wnt pathway. Notably, inactivation of these genes results in unique and previously unknown vulnerabilities, offering potential avenues for therapeutic exploration. Citation Format: Higinio Dopeso, Yingjie Zhu, Laxmi Gusain, Thais Basili, David Brown, Fatemeh Derakhshan, Edaise M. da Silva, Richard Koche, Pierre-Jacques Hamard, Xinyu Guo, Eneda Toska, Elisa DeStanchina, Hong Zhang, Jorge Reis-Filho, Britta Weigelt, Fresia Pareja. Altered endosomal pH regulation: molecular mechanisms of oncogenesis and therapeutic dependencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3936.

Abstract Background: Invasive lobular carcinoma (ILC) of the breast is the second most common histologic subtype of breast cancer (BC), following invasive ductal carcinoma of no special type (IDC-NST). The hallmark histologic feature of ILC is cellular discohesiveness, the result of bi-allelic inactivation of CDH1, and represents an important genotypic-phenotypic correlation in BC. Although most ILCs harbor CDH1 loss-of function mutations associated to loss-of-heterozygosity (LOH) of the wild type-allele, a subset of ILCs lack these alterations despite displaying a typical lobular phenotype. Here, we sought to identify alternative molecular mechanisms converging on CDH1 inactivation by employing an integrative artificial intelligence (AI) and genomics approach. Materials and Methods: A genomics-driven AI-based algorithm using hematoxylin and eosin (H&E) whole slide images (WSIs) as input, previously developed to detect bi-allelic CDH1 mutations (inactivating mutation associated to LOH) in BC was employed. WSIs of 1,057 BCs including ILCs (n=187) and non-lobular BCs (n=870) previously subjected to FDA-cleared tumor/normal targeted sequencing were subjected to analysis with the AI-based algorithm. Cases predicted to harbor CDH1 bi-allelic mutations by the AI-model but lacking CDH1 bi-allelic mutations by targeted sequencing were assessed through targeted sequencing data re-analysis, CDH1 gene promoter methylation evaluation and/or whole genome sequencing analysis. Results: AI-based analysis WSIs corresponding to 1,057 BCs resulted in the identification of 34 cases found to lack CDH1 bi-allelic mutations by targeted sequencing but predicted to harbor these genetic alterations by the AI-based model. CDH1 gene promoter methylation assessment revealed CDH1 promoter methylation in 18 cases. Targeted sequencing data reanalysis revealed other genetic mechanisms of CDH1 inactivation including CDH1 homozygous deletions (n=3), intragenic deletion with LOH (n=1), and likely pathogenic non-coding CDH1 alterations associated with LOH (n=2). WGS analysis of an ILC revealed a novel deleterious CDH1 fusion stemming from translocation t(13;16), resulting in loss of the 5’UTR, transcription start site and exons 1 and 2 of CDH1, associated with complete loss of E-cadherin protein expression. Taken together, we identified alternative/novel mechanisms of bi-allelic CDH1inactivation in 74% (25/34) cases analyzed. Conclusions: By applying an AI-based algorithm trained to detect a genetic alteration (i.e., CDH1 bi-allelic mutations), we were able to identify alternative epigenetic and genetic molecular mechanisms of CDH1 inactivation in ILCs, including novel non-coding CDH1 genetic alterations and a new inactivating CDH1 fusion gene. These findings indicate that molecular mechanisms affecting a single gene or process converging on the same phenotype can be unveiled by the integration of AI and genomics, highlighting the robustness of this approach for the discovery of novel biology. Citation Format: Fresia Pareja, Higinio Dopeso, Yikan Wang, Andrea Gazzo, David Brown, Pier Selenica, Jan Bernhard, Fatemeh Derakhshan, Edaise M. da Silva, Lorraine Colon-Cartagena, Thais Basili, Antonio Marra, Jillian Sue, Qiqi Ye, Arnaud Da Cruz Paula, Selma Yeni, Xin Pei, Hunter Green, Kaitlyn Gill, Yingjie Zhu, Matthew Lee, Ran Godrich, Adam Casson, Britta Weigelt, Nadeem Riaz, Hanna Y Wen, Edi Brogi, Matthew Hanna, Diana Mandelker, Jeremy Kunz, Brandon Rothrock, Sarat Chandarlapaty, Christopher Kanan, Gerard Oakley III, David Klimstra, Thomas Fuchs, Jorge Reis-Filho. Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics [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 GS03-04.