No Special Type Research Articles

Transcriptomic analysis identifies enrichment of cAMP/PKA/CREB signaling in invasive lobular breast cancer

ObjectiveInvasive lobular breast cancer (ILC) is the most common special type of breast cancer and has unique clinicopathological and molecular hallmarks that differentiate it from the more common invasive carcinoma—no special type (NST). Despite these differences, ILC and NST are treated as a single entity and there is a lack of ILC-targeted therapies. To fill this gap, we sought to identify novel molecular alterations in ILC that could be exploited for targeted therapies.MethodsDifferential gene expression and Geneset Enrichment and Variation analyses were performed on RNA-seq data from three large public breast cancer databases—the Sweden Cancerome Analysis Network-Breast (SCAN-B; luminal A ILC N = 263, luminal A NST N = 1162), The Cancer Genome Atlas (TCGA; luminal A ILC N = 157, luminal A NST N = 307) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; luminal A ILC N = 65, luminal A NST N = 533). Pathways enriched in overlapping differentially expressed genes from these datasets were clustered using Jaccard similarity to identify pathways enriched in ILC. The cAMP/PKA/CREB signaling was studied in ILC, ILC-like and NST cell lines and patient-derived organoids (PDOs) using forskolin, an activator of the pathway.ResultsClinicopathological features of patients with ILC and NST in SCAN-B were similar to prior population-based studies. There was a consistent pattern of up-regulation of cAMP/PKA/CREB related signaling in ILC compared to NST in SCAN-B, TCGA and METABRIC. Treatment with forskolin resulted in a greater increase in phospho-CREB in ILC cell lines and organoids than NST. CRISPR deletion of CDH1 in NST cell lines did not alter response of cells to forskolin as measured by phospho-CREB. Forskolin treatment caused growth inhibition in ILC and NST, with ILC cell lines being more sensitive to forskolin-mediated growth inhibition.ConclusionIn three separate datasets, cAMP/PKA/CREB signaling was identified to be higher in ILC than NST. This in silico finding was validated in cell line and organoid models. Loss of CDH1 was not sufficient to mediate this phenotype. Future studies should investigate the mechanisms for differential cAMP/PKA/CREB signaling and the potential for therapeutic targeting in patients with ILC.Graphical abstract

Mammary Schistosomiasis: Malignancy Mimicker or More?

Abstract Introduction/Objective Schistosomiasis is a neglected tropical disease (NTD) which affects more than 200 million people worldwide, with approximately 85% of cases occurring in sub-Saharan Africa. It serves both as an important etiologic agent in the development of carcinomas of many organ systems, as well as a common incidental histological finding in various organs, including the prostate, lung, spine, and liver. Detection of Schistosoma ova in the breast is rare. Here, we present a case of schistosomiasis of the breast in a patient concurrently diagnosed with breast carcinoma of the contralateral breast. Methods/Case Report A 48-year-old Mozambican female presented with a right breast mass, with suspicious microcalcifications detected in the contralateral breast on mammography. Biopsy confirmed invasive ductal carcinoma of no special type (NST) in the right breast and identified Schistosoma ova in the left, notably lacking an associated inflammatory response. A Ziehl-Neelsen special histochemical stain was negative, favoring schistosoma Hematobium species. Results (if a Case Study enter NA) NA Conclusion Our case marks the first in English literature where schistosomiasis was identified in the context of contralateral breast carcinoma, posing the question as to whether there is an association between schistosomiasis and breast carcinoma in our patient. One possible theory suggests hyperestrogenism secondary to Schistosoma infection in the liver. Breast schistosomiasis presenting as microcalcifications poses a diagnostic challenge, as it may clinically and radiologically resemble malignant breast disease. Histopathological diagnosis is crucial to confirm the diagnosis and prevent unnecessary procedures. Contrary to other organ systems, our case highlighted that schistosomiasis in the breast may not be accompanied by any inflammatory response. Definitive conclusions regarding the exact species of schistosomiasis cannot be drawn histologically based solely on H&E findings due to the plane of sectioning; therefore, performing a Ziehl-Neelsen stain is beneficial. As screening mammography increases in endemic areas, a high index of suspicion and further research is warranted.

Method for co-registration of high-resolution specimen PET-CT with histopathology to improve insight into radiotracer distributions

BackgroundAs the spatial resolution of positron emission tomography (PET) scanners improves, understanding of radiotracer distributions in tissues at high resolutions is important. Hence, we propose a method for co-registration of high-resolution ex vivo specimen PET images, combined with computed tomography (CT) images, and the corresponding specimen histopathology.MethodsWe applied our co-registration method to breast cancer (BCa) specimens of patients who were preoperatively injected with 0.8 MBq/kg [18\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^{18}$$\\end{document}F]fluorodeoxyglucose ([18F]FDG). The method has two components. First, we used an image acquisition scheme that minimises and tracks tissue deformation: (1) We acquired sub-millimetre (micro)-PET-CT images of ±2 mm-thick lamellas of the fresh specimens, enclosed in tissue cassettes. (2) We acquired micro-CT images of the same lamellas after formalin fixation to visualise tissue deformation. (3) We obtained 1 hematoxylin and eosin (H&E) stained histopathology section per lamella of which we captured a digital whole slide image (WSI). Second, we developed an automatic co-registration algorithm to improve the alignment between the micro-PET-CT images and WSIs, guided by the micro-CT of the fixated lamellas. To estimate the spatial co-registration error, we calculated the distance between corresponding microcalcifications in the micro-CTs and WSIs. The co-registered images allowed to study standardised uptake values (SUVs) of different breast tissues, as identified on the WSIs by a pathologist.ResultsWe imaged 22 BCa specimens, 13 cases of invasive carcinoma of no special type (NST), 6 of invasive lobular carcinoma (ILC), and 3 of ductal carcinoma in situ (DCIS). While the cassette framework minimised tissue deformation, the best alignment between the micro-PET-CT images and WSIs was achieved after deformable co-registration. We found an overall average co-registration error of 0.74 ± 0.17 mm between the micro-PET images and WSIs. (Pre)malignant tissue (including NST, ILC, and DCIS) generally showed higher SUVs than healthy tissue (including healthy glandular, connective, and adipose tissue). As expected, inflamed tissue and skin also showed high uptake.ConclusionsWe developed a method to co-register micro-PET-CT images of surgical specimens and WSIs with an accuracy comparable to the spatial resolution of the micro-PET images. While currently, we only applied this method to BCa specimens, we believe this method is applicable to a wide range of specimens and radiotracers, providing insight into distributions of (new) radiotracers in human malignancies at a sub-millimetre resolution.

ESR1 Fusions Invoke Breast Cancer Subtype-Dependent Enrichment of Ligand-Independent Oncogenic Signatures and Phenotypes.

Breast cancer is a leading cause of female mortality and despite advancements in personalized therapeutics, metastatic disease largely remains incurable due to drug resistance. The estrogen receptor (ER, ESR1) is expressed in two-thirds of all breast cancer, and under endocrine stress, somatic ESR1 mutations arise in approximately 30% of cases that result in endocrine resistance. We and others reported ESR1 fusions as a mechanism of ER-mediated endocrine resistance. ER fusions, which retain the activation function 1- and DNA-binding domains, harbor ESR1 exons 1 to 6 fused to an in-frame gene partner resulting in loss of the ER ligand-binding domain (LBD). We demonstrate that in a no-special type (invasive ductal carcinoma [IDC]-NST) and an invasive lobular carcinoma (ILC) cell line, ER fusions exhibit robust hyperactivation of canonical ER signaling pathways independent of estradiol or antiendocrine therapies. We employ cell line models stably overexpressing ER fusions with concurrent endogenous ER knockdown to minimize endogenous ER influence. Cell lines exhibited shared transcriptomic enrichment in pathways known to be drivers of metastatic disease, notably MYC signaling. Cells expressing the 3' fusion partners SOX9 and YAP1 consistently demonstrated enhanced growth and cell survival. ILC cells expressing the DAB2 fusion led to enhanced growth, survival, and migration, phenotypes not appreciated in the IDC-NST DAB2 model. Herein, we report that cell line activity is subtype-, fusion-, and assay-specific, suggesting that LBD loss, the fusion partner, and the cellular landscape all influence fusion activities. Therefore, it will be critical to assess fusion frequency in the context of the clinicopathology.

MRI features of breast cancer immunophenotypes with a focus on luminal estrogen receptor low positive invasive carcinomas

To compare the magnetic resonance imaging (MRI) features of different immunophenotypes of breast carcinoma of no special type (NST), with special attention to estrogen receptor (ER)-low-positive breast cancer. This retrospective, single-centre, Institutional Review Board (IRB)-approved study included 398 patients with invasive breast carcinoma. Breast carcinomas were classified as ER-low-positive when there was ER staining in 1–10% of tumour cells. Pretreatment MRI was reviewed to assess the tumour imaging features according to the 5th edition of the Breast Imaging Reporting and Data System (BI-RADS) lexicon. Of the 398 cases, 50 (12.6%) were luminal A, 191 (48.0%) were luminal B, 26 (6.5%) were luminal ER-low positive, 64 (16.1%) were HER2-overexpressing, and 67 (16.8%) were triple negative. Correlation analysis between MRI features and tumour immunophenotype showed statistically significant differences in mass shape, margins, internal enhancement and the delayed phase of the kinetic curve. An oval or round shape and rim enhancement were most frequently observed in triple-negative and luminal ER-low-positive tumours. Spiculated margins were most common in luminal A and luminal B tumours. A persistent kinetic curve was more frequent in luminal A tumours, while a washout curve was more common in the triple-negative, HER2-overexpressing and luminal ER-low-positive immunophenotypes. Multinomial regression analysis showed that luminal ER-low-positive tumours had similar results to triple-negative tumours for almost all variables. Luminal ER-low-positive tumours present with similar MRI findings to triple-negative tumours, which suggests that MRI can play a fundamental role in adequate radiopathological correlation and therapeutic planning in these patients.

Long-term outcomes by lobular versus ductal histology in four NSABP adjuvant breast cancer trials.

We evaluated differences in long-term outcomes of invasive lobular carcinoma (ILC) vs breast cancers of no special type (NST) treated with anthracycline-based adjuvant chemotherapy using 4 National Surgical Adjuvant Breast and Bowel Project (NSABP) randomized phase III trials (B-22, B-25, B-28, B-30). Our cohort included 11,251 patients with NST and 1,231 with ILC. Patients with ILC were older, had larger and more often estrogen receptor-positive tumors, and more positive lymph nodes. During early follow-up (0-5 years), patients with ILC had fewer recurrences (HR: 0.797; 95% confidence interval [CI] 0.685-0.929) and deaths (HR: 0.756; 95% CI 0.623-0.917). After 5 years patients with ILC had more recurrences (HR: 1.30; 95% CI 1.085-1.558) and deaths (HR: 1.044; 95% CI 0.898-1.214). Conditional probability analysis showed significant interactions between time-period and histologic type for recurrences (p < .001) and deaths (p < .001). Patients with ILC have elevated risk of late recurrence and death compared to patients with NST.

Prognostic impact of histological subtyping in triple-negative breast cancer

The impact of special histological types (ST) in triple-negative breast cancer (TNBC) and its association with overall outcome has gained increasing relevance as survival has been linked to specific histological TNBC subtypes. We evaluated the clinicopathological and survival data of 598 patients with 613 TNBCs, including 464 TNBCs of no special type (NST) and 149 TNBCs ST (low-grade, n = 12, 8.1%; high-grade, n = 112, 75.2%; apocrine and androgen receptor-positive [APO AR], n = 25, 16.8%). Patients with low-grade TNBC ST and TNBC ST APO AR were significantly older (P < 0.001) and had a lower Ki67 index (P < 0.001) than those with TNBC NST. Patients with high-grade TNBC ST were significantly older (P = 0.006) and had poorer pathological responses to neoadjuvant chemotherapy (NAC) (P < 0.001) than those with TNBC NST. Significant survival differences were observed between low-grade TNBC ST, TNBC ST APO AR, high-grade TNBC ST, and TNBC NST in the entire study group (DFS, P = 0.002; DDFS, P = 0.001) and in the non-NAC subgroup (OS, P = 0.034; DFS, P = 0.001; DDFS, P < 0.001). Patients with low-grade TNBC ST had the best survival outcomes. Patients with high-grade TNBC ST showed significantly worse outcomes than those with TNBC NST (entire study group: OS, P = 0.049; DFS, P < 0.001; DDFS, P = 0.001; non-NAC subgroup: OS, P = 0.014; DFS, P < 0.001; DDFS, P < 0.001). We conclude that prognostic stratification of TNBC ST is ultimately important for optimizing the therapeutic management of patients with these rare tumor entities.

International survey on invasive lobular breast cancer identifies priority research questions

There is growing awareness of the unique etiology, biology, and clinical presentation of invasive lobular breast cancer (ILC), but additional research is needed to ensure translation of findings into management and treatment guidelines. We conducted a survey with input from breast cancer physicians, laboratory-based researchers, and patients to analyze the current understanding of ILC, and identify consensus research questions. 1774 participants from 66 countries respondents self-identified as clinicians (N = 413), researchers (N = 376), and breast cancer patients and advocates (N = 1120), with some belonging to more than one category. The majority of physicians reported being very/extremely (41%) to moderately (42%) confident in describing the differences between ILC and invasive breast cancer of no special type (NST). Knowledge of histology was seen as important (73%) and as affecting treatment decisions (51%), and most agreed that refining treatment guidelines would be valuable (76%). 85% of clinicians have never powered a clinical trial to allow subset analysis for histological subtypes, but the majority would consider it, and would participate in an ILC clinical trials consortium. The majority of laboratory researchers, reported being and very/extremely (48%) to moderately (29%) confident in describing differences between ILC and NST. They reported that ILCs are inadequately presented in large genomic data sets, and that ILC models are insufficient. The majority have adequate access to tissue or blood from patients with ILC. The majority of patients and advocates (52%) thought that their health care providers did not sufficiently explain the unique features of ILC. They identified improvement of ILC screening/early detection, and identification of better imaging tools as top research priorities. In contrast, both researchers and clinicians identified understanding of endocrine resistance and identifying novel drugs that can be tested in clinical trials as top research priority. In summary, we have gathered information from an international community of physicians, researchers, and patients/advocates that we expect will lay the foundation for a community-informed collaborative research agenda, with the goal of improving management and personalizing treatment for patients with ILC.

Peningkatan Leukosit, Neutrofil, dan Prokalsitonin Persisten Tanpa Bukti Infeksi pada Kanker Stadium Akhir: Sebuah Laporan Kasus

Elevation of leukocyte, neutrophil, and procalcitonin (PCT) has been known as a marker of bacterial infection independent of the underlying disease. This article will describe a case of end-stage cancer with persistent elevation of leukocyte, neutrophil, and procalcitonin with no evidence of infection. A 45-year-old female presented with abdominal distension for three months prior to hospital admission. The patient was suspected to have breast cancer with hepatic metastasis based on diagnostic tests. Throughout treatment, the patient experienced persistent elevation of leukocytes, neutrophils, and PCT despite no signs or symptoms of infection from history, physical examination, and diagnostic tests. The patient received antibiotic therapy including ciprofloxacin escalated to meropenem, but leukocytes, neutrophils, and PCT did not decrease. A breast tumor biopsy was performed. The result of biopsy revealed after the patient had passed away. The histopathology was invasive carcinoma, No Special Type (NST) of right breast. The immunohistochemistry showed luminal B type, HER2 positive. The persistent increase in leukocytes and neutrophils in this patient indicates poor prognosis, disease progression, metastasis, and pro-cancer activity of neutrophils. The elevated PCT in this patient may be due to systemic inflammation and/or aberrant secretion from the cancer itself.

Transcriptomic analysis of cAMP/PKA/CREB signaling in invasive lobular breast cancer.

1109 Background: Invasive lobular breast cancer (ILC) is the most common special type of breast cancer and has unique clinicopathological and molecular hallmarks that differentiate it from the more common invasive carcinoma – no special type (NST). Despite these differences, ILC and NST are treated as a single entity and there is a lack of ILC-targeted therapies. To fill this gap, we sought to identify novel molecular alterations in ILC that could be exploited for targeted therapies. Methods: Differential gene expression and Geneset Enrichment and Variation analyses were performed on RNA-seq data from three large public breast cancer databases – the Sweden Cancerome Analysis Network-Breast (SCAN-B; luminal A ILC N=263, luminal A NST N=1162), The Cancer Genome Atlas (TCGA; luminal A ILC N=157, luminal A NST N=307) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; luminal A ILC N=65, luminal A NST N=533). Pathways enriched in overlapping differentially expressed genes from these datasets were clustered using Jaccard similarity to identify pathways enriched in ILC. The cAMP/PKA/CREB signaling was studied in ILC, ILC-like and NST cell lines and patient-derived organoids (PDOs) using forskolin, an activator of the pathway. Results: Clinicopathological features of patients with ILC and NST in SCAN-B were similar to prior population-based studies. There was a consistent pattern of up-regulation of cAMP/PKA/CREB related signaling in ILC compared to NST in SCAN-B, TCGA and METABRIC. Treatment with forskolin resulted in a greater increase in phospho-CREB in ILC cell lines and organoids than NST. CRISPR deletion of CDH1 in NST cell lines did not alter response of cells to forskolin as measured by phospho-CREB. Forskolin treatment caused growth inhibition in ILC and NST, with ILC cell lines being more sensitive to forskolin-mediated growth inhibition. Conclusions: In three separate datasets, cAMP/PKA/CREB signaling was identified to be higher in ILC than NST. This in silico finding was validated in cell line and organoid models. Loss of CDH1 was not sufficient to mediate this phenotype. Future studies should investigate the mechanisms for differential cAMP/PKA/CREB signaling and the potential for therapeutic targeting in patients with ILC.

Joint EANM-SNMMI guideline on the role of 2-[18F]FDG PET/CT in no special type breast cancer

IntroductionThere is much literature about the role of 2-[18F]FDG PET/CT in patients with breast cancer (BC). However, there exists no international guideline with involvement of the nuclear medicine societies about this subject.PurposeTo provide an organized, international, state-of-the-art, and multidisciplinary guideline, led by experts of two nuclear medicine societies (EANM and SNMMI) and representation of important societies in the field of BC (ACR, ESSO, ESTRO, EUSOBI/ESR, and EUSOMA).MethodsLiterature review and expert discussion were performed with the aim of collecting updated information regarding the role of 2-[18F]FDG PET/CT in patients with no special type (NST) BC and summarizing its indications according to scientific evidence. Recommendations were scored according to the National Institute for Health and Care Excellence (NICE) criteria.ResultsQuantitative PET features (SUV, MTV, TLG) are valuable prognostic parameters. In baseline staging, 2-[18F]FDG PET/CT plays a role from stage IIB through stage IV. When assessing response to therapy, 2-[18F]FDG PET/CT should be performed on certified scanners, and reported either according to PERCIST, EORTC PET, or EANM immunotherapy response criteria, as appropriate. 2-[18F]FDG PET/CT may be useful to assess early metabolic response, particularly in non-metastatic triple-negative and HER2+ tumours. 2-[18F]FDG PET/CT is useful to detect the site and extent of recurrence when conventional imaging methods are equivocal and when there is clinical and/or laboratorial suspicion of relapse. Recent developments are promising.Conclusion2-[18F]FDG PET/CT is extremely useful in BC management, as supported by extensive evidence of its utility compared to other imaging modalities in several clinical scenarios.

Comparison of clinical characteristics and outcomes in primary neuroendocrine breast carcinoma versus invasive ductal carcinoma.

Neuroendocrine breast carcinoma (NECB) is a rare, special histologic type of breast cancer. There are some small sample studies on the clinical outcomes of NECB patients, which are worthy of further discussion. We conducted a retrospective case-control study of clinical characteristics and outcomes among patients with primary NECB versus invasive carcinoma of no special type (NST) between November 2004 and November 2017 in the Peking Union Medical College Hospital, Beijing. NST patients were strictly matched 1:4 during the same period based on the TNM stage. Statistical comparisons were performed to determine the differences in survival between NST and NECB patients and to identify clinical factors that correlate with prognosis. A total of 121 participants affected by primary NECB were included in our analysis from November 2004 to November 2017. Elderly persons (>60 years of age) were more likely to have primary NECB than young persons (p=0.001). In addition, primary NECB patients had significantly higher odds of having tumors 2-5cm (36.5%) and >5cm (6.1%) in size than NST patients. Despite a significant difference in tumor size, the proportion of patients with lymph node metastases showed no difference between the two groups (p=0.021). In addition, the rate of patients with ER-negative tumors in the NECB group (4.2%) was significantly lower than that in the primary NST group (29.8%). Significant differences were noted in the PR-negative (13.3% versus 36.6%, P<0.001) and HER2-negative (90.5% versus 76.4%, P=0.001) expression statuses among these patients. Of 121 primary NECB patients, 11 (9.1%) experienced relapses during the follow-up period. We found that tumor size was an independent risk factor for relapse. For hormone receptors on tumor cells, ER-positive breast cancer patients had significantly lower odds of relapse than receptor-negative patients. Our data demonstrate no significant difference in mortality and relapse between the primary NECB and NST groups. The tumor size in the primary NECB group was significantly larger than that in the NST group. In addition, the absence of ER independently increased the relapse rate for breast carcinoma patients.

Abstract PO3-05-05: The effect of histological breast cancer subtype on progression free survival in patients with CDK4/6 inhibitors

Abstract Background: Multiple trials have proven that adding CDK4/6 inhibitors to endocrine treatment increases progression free (PFS) and overall survival (OS) of patients with metastatic estrogen receptor (ER)-positive HER2 negative breast cancer. However, only limited evidence is available of treatment efficacy of CDK4/6 inhibitors for invasive lobular carcinoma (ILC). This retrospective study aims to compare treatment duration of the three FDA/EMA approved CDK4/6 inhibitors in patients with no special type (NST) breast cancer versus patients with ILC. Methods: All patients with metastatic ER-positive HER2-negative breast cancer who were treated with a CDK4/6 inhibitor (1st, 2nd; 3rd line) in University Hospitals Leuven between December 2014 and February 2023, were included. A comparison of PFS and OS was made between patients with NST and ILC by use of the Kaplan Meier method. Other histological subtypes as well as mixed subtypes were excluded. Uni- and multivariable cox regression models were performed to quantify the association of histological subtype with PFS and OS. Results: A total of 418 patients were included of which 119 (28.5%) patients with ILC (median age at primary diagnosis 59 years, range 36 – 89 years) and 299 (71.5%) patients with NST (median age at primary diagnosis 55 years, range 23 – 90 years). Median follow-up was 26.8 months (range 1.1 – 88.1 months). Median PFS was 15.2 months (range 1.0 – 74.6 months) and 14.7 months (range 1.0 – 89.3 months) for patients with ILC and NST respectively. The OS rate after 60 months follow up was 39.2 % (CI 26.8 – 51.4) for ILC and 40.1% (CI 32.2 – 47.8) for NST. As shown in table 1, clear differences were observed in PFS rates after 12, 24 and 60 months between patients with NST that received CDK4/6 inhibitors in first line versus second or third line. These differences were less apparent for patients with ILC. For both NST and ILC, endocrine resistance at start of CDK4/6 inhibition impacted PFS rates negatively (table 2). In multivariable analyses, histological subtype was not predictive for CDK4/6 inhibition outcome (PFS: hazard ratio (HR) 0.996, CI 0.728 – 1.364, p-value 0.981; OS: HR 0.872, CI 0.602 – 1.263, p-value 0.468). Endocrine sensitivity vs. resistance at the time CDK4/6 inhibitor was started, was proven to be predictive for both PFS (HR 0.478, CI 0.337 – 0.678, p-value &amp;lt; 0.001) and OS (HR 0.528, CI 0.347 – 0.804, p-value 0.003). Conclusion: In our center, the histological subtype of breast cancer did not seem to impact PFS and OS significantly after treatment with CDK4/6 inhibition. Patients with NST seemed to have an increased benefit in PFS from treatment with CDK4/6 inhibition in first line as compared to later lines. While for patients with ILC, no difference between treatment lines were observed. Therefore, clinicians might be able to safely postpone CDK4/6 inhibitors to second- or third-line treatment in patients with metastatic ILC. Table 1: PFS rates by histological subtype and line of CDK4/6 treatment Table 2: PFS rates by histological subtype and endocrine sensitivity at start of CDK4/6 inhibition Citation Format: Cedric Bauters, Karen Van Baelen, Hans Wildiers, Giuseppe Floris, Sileny Han, Patrick Berteloot, Thaïs Baert, Ann Smeets, Ines Nevelsteen, Yannick Van Herck, Anne Deblander, Chantal Remmerie, Lieke Dullens, Maxime Van Houdt, Annouschka Laenen, Christine Desmedt, Patrick Neven. The effect of histological breast cancer subtype on progression free survival in patients with CDK4/6 inhibitors [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 PO3-05-05.

Abstract PO5-24-11: An integrated approach for comprehensive molecular and tumor microenvironment characterization of invasive lobular carcinoma

Abstract Introduction: Although recent studies have shown that invasive lobular carcinoma (ILC) is associated with worse long-term outcomes compared to invasive breast cancer of no special type (NST), they are treated in a similar manner. Therefore, classification approaches to help understand the molecular characteristics associated with the different BC subtypes are needed to personalize therapy. Here, we describe an integrated analytical approach to comprehensively characterize ILC samples. Methods: We collected genomic and transcriptomic data for 16,087 ILC and NST samples, with clinical and pathological annotation, from 59 datasets and used an internally developed algorithm based on clustering performed after gene expression analysis to classify them into 5 intrinsic subtypes: Basal, Luminal A (LumA), Luminal B (LumB), HER2 high, and HER2 low. We performed gene set enrichment analyses and utilized the DESeq2 algorithm to perform differential expression analysis of RNA-seq data. We performed molecular grade subtyping using methods described by Antysheva et al. (Cancer Res., 2022). Samples that were listed as ILC or had an inactivated CDH1 gene by mutation, deletion or low expression (&amp;lt; −2.5 MAD after median scaling) were defined as having an ILC histomolecular subtype. Using methods described by Bagaev et. al. (Cancer Cell, 2021), 29 functional gene expression signatures were selected, and unsupervised dense Louvain clustering was performed to identify TME subtypes. The Mann-Whitney U test was used for statistical analysis. The tertiary lymphoid structures (TLS) gene signature was evaluated using methods described by Sautès-Fridman et al. (Nat. Rev. Cancer, 2019). Results: In the ILC samples (n=1,887), we identified LumA (46%), LumB (40%), Basal (8%,), HER2 high (3%), and HER2 low (3%) subtypes. In contrast, the NST samples were classified as LumB (41%), LumA (25%), Basal (19%), HER2 high (9%), and HER2 low (5%). Molecular grade subtyping revealed that 67% of ILC samples were low grade and 33% were high grade, compared to NST where 55% were high grade and 45% were low grade. Notably, among high-grade ILC, LumB (64%) and Basal (23%) subtypes were predominant. Key differentially expressed genes included downregulated CDH1 and upregulated MMP23B, IGF1, and TFAP2B across LumA and LumB ILC samples. Further analysis revealed downregulation of TACSTD2 and CDH3 in the Basal subtype for ILC samples compared to NST samples. Transcriptomic-based TME subtyping revealed distinct TME distributions across the BC subtypes and molecular grades for ILC samples (Table 1). In high-grade ILC LumB samples, 38% had a Desert (D; predictive of poor immunotherapy response) and 39% had an Immune-Enriched (IE; predictive of favorable immunotherapy response) TME. In low-grade ILC LumB samples we found that while 51% had a D TME, only 14% had an IE TME. We found an overrepresentation of the TLS signature [Solid TLS] in ILC compared to NST samples, and higher matrix remodeling and CAF signatures in LumA compared to the LumB subtype among low-grade ILC samples. Samples with high CD274 expression levels had higher percentages of T cells and M1 macrophages, and LumA samples had significantly higher (p &amp;lt; 0.001) CD274 expression compared to the other BC subtypes. Conclusion: These results provide in-depth molecular and tumor microenvironment characterization of ILC. Further optimization of this analytical approach could lead to the development of more effective therapeutic strategies. Table 1. ILC subtypes classification Citation Format: Jason Mouabbi, Konstantin Chernyshov, Oleg Baranov, Vladimir Kushnarev, Polina Turova, Anna Butusova, Sofia Menshikova, Jessica Brown, Nikita Kotlov, Patrick Clayton, Krystle Nomie, Nathan Fowler, Debu Tripathy. An integrated approach for comprehensive molecular and tumor microenvironment characterization of 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 PO5-24-11.

Abstract PO1-14-11: Different molecular processes are associated with recurrence in the clinical high- genomic low risk (cH/gL) and clinical low- genomic high risk (cL/gH) groups of the MINDACT clinical trial

Abstract Background: Despite the growing understanding of estrogen receptor positive/HER2-negative (ER+/HER2-) breast cancer (BC) biology, many unknowns remain regarding the mechanisms of disease recurrence. While several multigene prognostic signatures have shown clinical utility in identifying those patients at high or low genomic risk of recurrence, they can present with some limitations. In this retrospective analysis of the MINDACT trial, we interrogated the available transcriptomic data to understand which biological characteristics of the tumor are associated with recurrence in patients from the clinical high- genomic low risk (cH/gL) and clinical low- genomic high risk (cL/gH) groups (where the clinical and genomic risk were defined by a modified version of Adjuvant Online! and the 70-gene signature, respectively). Methods: These analyses focused on the subset of patients with ER+/HER2- tumors of no special type (NST) who received endocrine therapy (which comprised the majority of this NST ER+/HER2- sub-cohort) and for which central pathology and gene expression data were available (n= 1245). Scores of gene expression modules associated with key biological processes in breast cancer defined in a collection of previous publications were computed from the gene expression matrix using the formula described in Desmedt et al. (Clin. Cancer Res. 2008). Gene Set Variation Analysis method (R package ‘GSVA’ – version 1.40.1) was used to derive the enrichment scores of fifty hallmark gene sets available in the ‘H’ collection of the MSigDB database (version 7.5.1). Survival analyses were performed for disease-free survival (DFS) and distant recurrence-free survival (DRFS). Univariable and multivariable regression models, implemented by Cox regression and Fine-Gray subdistribution hazard regression methods, were performed to evaluate the association of enrichment of hallmarks and gene modules with DFS and DRFS, respectively (covariates =age, nodal status, KI67, tumor size, chemotherapy, radiotherapy). Results: DFS event occurred in 150/913 (16.4%) and 51/332 (15.4%) NST ER+/HER2- patients in the cH/gL and cL/gH groups, respectively. DRFS event occurred in 70/913 (7.7%) and 21/332 (6.3%) NST ER+/HER2- patients in the cH/gL and cL/gH groups, respectively. In the cH/gL group, we observed an association of hallmarks related to cell cycle and downregulation of gene modules related to lymphocyte-centric immune activities with worse prognosis (Table). For the cL/gH, we did not observe an association between hallmarks related to cell cycle and worse prognosis but an enrichment of hallmarks related to PI3K/AKT/mTOR signaling as well as several metabolic hallmarks related to insulin activities (Table). Of interest, in this group of patients, higher scores of the HER2-associated gene expression signature were also associated with worse prognosis. Across the two groups effects were more pronounced for DRFS than DFS. Conclusion: Different molecular processes are associated with progression in these two distinct groups of patients. The association of hallmarks related to cell cycle with survival in the cH/gL group supports the observation that adjuvant chemotherapy could benefit a subset of these patients. In the cL/gH group, the association of signatures associated with PI3K pathway and HER2 can have potential clinical relevance given their current targetability in the metastatic setting. This retrospective study is funded by the Breast Cancer Research Foundation. Table. Representative hallmarks and gene expression modules independently associated with prognosis in NST ER+/HER2- patients in the cH/gL group and in the cL/gH group #p-values reported were not corrected for multiple testing Citation Format: Christine Desmedt, Ha-Linh Nguyen, François Richard, Marion Maetens, Coralie Poncet, Laura De Meulemeester, Kim Aalders, Mauro Delorenzi, Suzette Delaloge, Jean-Yves Pierga, Etienne Brain, Suzan Vrijaldenhoven, Peter A Neijenhuis, Emiel Rutgers, Florentine Hilbers, Laura Van ’t Veer, Giuseppe Viale, Christos Sotiriou, Martine Piccart, Fatima Cardoso. Different molecular processes are associated with recurrence in the clinical high- genomic low risk (cH/gL) and clinical low- genomic high risk (cL/gH) groups of the MINDACT clinical trial [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 PO1-14-11.

Abstract PO2-14-08: Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response

Abstract Organoids serve as an important preclinical model in multiple cancers including breast cancer, demonstrating preservation of biological features and feasibility of drug screening, with reasonable cost and high scalability. It has been reported that patient-derived organoids (PDOs) can be robustly derived from both primary and metastatic breast tumors, with high resemblance to source tissues in histological, genomic, and transcriptomic features, as well as consistency in drug response compared to xeno-transplantations or patients (Sachs et al, 2018). Since 2018, the Institute for Precision Medicine (IPM) has developed a panel of breast cancer organoids. The current inventory now consists of 128 PDOs from 94 primary tumors, 12 metastases, 12 rapid-autopsy tumors, and 10 normal breast tissues with a 60% success rate of culture; as well as 33 organoids from animal models including 11 patient-derived xenograft organoids (PDXOs), 15 rat (RDO) and 7 mouse (MDO) tumor organoids with 85% success rate. For PDOs, the protocol includes in-lab processing of fresh, deidentified tissue within 60 minutes of surgical operation, thanks to close collaboration with surgeons, consented patients, and an institutional biospecimen core. BC organoids have been comprehensively characterized in terms of morphology, histology, genomics, transcriptomics, and functional experiments. Key biological features are preserved. PDOs (n=56) exhibit a mutational spectrum consistent with human breast cancers. ER expression is detectable in a subset of cultures with robust estradiol response indicated by GREB1 expression; and PDOs from invasive lobular carcinoma (ILC) mostly showed more discohesive structures than PDOs from no special type (NST), with clear E-cadherin loss under immunofluorescence. We have also successfully constructed transcriptionally and genetic modified BC PDOs with shRNA and CRISPR, which showed replicable performance in proliferation assay, drug response, and signaling by western blot. Of note, longitudinal tracking of PDOs with single cell RNA-sequencing (scRNA-seq) revealed well-preserved heterogeneity compared to primary tumor, while transcriptomic clonality was mildly decreased in later passages from pilot data. Future work will include expansion of current collection from various sources, longitudinal characterization with scRNA-seq and DNA sequencing, as well as more functional studies. We aim to construct a robust resource of well-characterized and reliably-usable model bank for preclinical breast cancer research. Citation Format: Daniel Brown, Kai Ding, Fangyuan Chen, Jian Chen, Steffi Oesterreich, Peter Lucas, Priscilla McAuliffe, Jennifer Atkinson, ADRIAN LEE. Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response [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 PO2-14-08.

Abstract PO1-28-10: Tumor Infiltrating Lymphocytes identify patients with stage I TNBC with excellent outcome without chemotherapy

Abstract Introduction International guidelines recommend chemotherapy for most patients with stage I triple negative breast cancer (TNBC), except for special histological subtypes. However, the absolute benefit of systemic treatment for all stage I TNBC patients is not well known and no tools are yet available to select patients with an excellent prognosis for whom the chemotherapy can be safely omitted. High levels of stromal tumor infiltrating lymphocytes (sTILs) are strongly associated with favorable prognosis in TNBC, however, data solely focusing on patients with stage I TNBC who did not receive chemotherapy are lacking. Methods Patients with pT1N0M0 TNBC diagnosed between 2005 and 2015 not treated with (neo)adjuvant chemotherapy were identified from the nationwide Netherlands Cancer Registry (NCR) and linked to the national pathology database (PALGA). Newly sectioned hematoxylin &amp; eosin (H&amp;E) slides were used for central review of the histology and sTILs scoring following the International TIL working group guidelines (www.tilsinbreastcancer.org). Cause of death was provided by Statistics Netherlands (CBS). Primary endpoint was breast-cancer specific survival (BCSS) at 5-, 10- and 15-years for prespecified sTILs cut-offs of 30%, 50% and 75% sTILs. Results sTILs were evaluated for 1,041 patients with stage I TNBC who did not receive (neo)adjuvant chemotherapy. Mean age at diagnosis was 64.4 years [range 26 – 96] and median sTILs value was 5%. Most tumors (91.5%) were invasive ductal adenocarcinomas of no special type (NST) and 6.6% were of histological special types. In 52% of the patients the tumor was &amp;gt;1cm (pT1cN0). Median follow-up was 9.4 years in which 335 patients had died, of whom 107 due to breast cancer. Patients with pT1abN0 tumors had a favorable prognosis compared with patients with a pT1cN0 tumor (HR 0.47, CI 0.32- 0.72). 10-year BCSS in patients with pT1ab was 91.8% and 85.8% in patients with pT1c tumors. In the overall cohort, sTILs-levels of at least 30% were associated with better BCSS compared to sTILs &amp;lt; 30% (HR 0.45, CI 0.26 – 0.77), with 10year-BCSS of 96% and 87%, respectively (table 1). Patients with TNBC of special histological subtype had low sTILs (median 1%). A univariate cox model showed no association between sTILs (continuous) and BCSS in TNBC of special histological subtype (p = 0.56). Next, the prognostic value of sTILs was evaluated separately for pT1ab and pT1c. High sTILs ≥50% were not associated with improved BCSS compared to low sTILs &amp;lt; 50% in patients with pT1ab (HR 1.35, CI 0.65 – 2.82, p = 0.42). In contrast, high sTILs ≥50% showed a strong prognostic importance over low sTILs &amp;lt; 50% (HR 0.27, CI 0.10 – 0.74) in patients with pT1C tumors. We found excellent 10year-BCSS of 95% for patients if sTILs ≥50%, which further increases to 98% with sTILs ≥75% contrasting 10year-BCSS of 83% for patients with sTILs &amp;lt; 30%. Conclusions sTILs-levels provide important prognostic information in patients with stage I TNBC who did not receive (neo)adjuvant chemotherapy. The outcome of patients with pT1c TNBC and sTILs ≥50% was excellent, with 10year-BCSS of 95%, and 98% if sTILs &amp;gt;75%. This large registry study provides a foundation for clinical trials in patients with stage I TNBC with high TILs to prospectively confirm their excellent survival without chemotherapy. Citation Format: Veerle Geurts, Sara Balduzzi, Hugo Horlings, Tessa G Steenbruggen, Sabine Siesling, Sylvia Adams, Gabe Sonke, Roberto Salgado, Marleen Kok. Tumor Infiltrating Lymphocytes identify patients with stage I TNBC with excellent outcome without chemotherapy [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 PO1-28-10.

Abstract PO1-16-10: BRCA mutated Male Breast Cancer, hallmarks of a distinct disease. Data from the Spanish Male Breast Cancer Registry (GEICAM/2016-04)

Abstract Background: Male breast cancer (MBC) accounts for approximately 0.25% of all male cancers and &amp;lt; 1% of all BCs. Its incidence is increasing by 1.1% per year. Most population studies indicate that 10-15% of MBC patients (pts) have a germline BRCA2 mutation (BRCA2 mut). Around 5-10% of BRCA2mut male carriers will develop BC at their lives. Preliminary evidence suggests that BRCA2mut MBC pts tend to have a more aggressive disease. Here we present comparative series of MBC with and without BRCA to gain insight on differences between both groups. Methods: GEICAM/2016-04 (NCT03800355) is a retrospective, observational study which includes data from MBC pts diagnosed between 2000 and 2019 throughout Spain. This analysis includes the 1st unselected 186 pts included in the study, who had BRCA1/2 genes mutational status assessed within clinical practice. BC clinical subtypes are defined: luminal (HR+, HER2-), Triple Negative (TN) (HR-, HER2-), and HER2+ (HR+ or HR-); subtype is unknown in 6% pts. Results: 186 validated pts with BRCA1/2 testing were identified, representing the 24% of pts available in the database at the cut-off date (6-Mar-2023). 36 (19%) pts had germline BRCA1/2 mut (4 pts in BRCA1, 31 pts in BRCA 2 and 1 pt in both BRCA1 and BRCA2), and 150 (81%) pts had BRCA1/2 wild type (wt) or a variant of uncertain significance (VUS)/unknown (UK). Median age at BC diagnosis was 62 (34-87) years, 98% pts were Caucasian, and the median body mass index was 27 (18-46) Kg/m2 (overweight). Prior cancer history was reported in 6% BRCA1/2mut BC pts and 11% BRCA1/2wt or vus/uk, and family history included: 56% BC, 8% ovarian cancer, and 14% prostate cancer (PC). PC as part of prior medical history, cancer family history, and 2nd non-breast primary malignancy, was most frequent in BRCA1/2wt or vus/uk pts, with no statistical differences. At first diagnosis, stages III-IV were higher in BRCA1/2mut pts (28% vs. 17%, p=0.0093). Morphologically, invasive carcinoma of no special type (NST) was the most common pattern (92% in BRCA1/2mut vs. 86% in BRCA1/2wt or vus/uk), and lobular invasive carcinoma was not reported up-to-date. Histological grade 2 was the most frequent (54%), but grade 3 was present in 17/36 BRCA1/2mut pts. 88% pts received adjuvant treatment, 7% (neo-)adjuvant, 2% neoadjuvant and 3% did not receive any systemic therapy. Breast conserving surgery was performed in 4% (5/140) BRCA1/2wt or vus/uk EBC pts, and mastectomy was performed in 55% (n=6/11) de novo metastatic pts. In the advanced setting (n=38), visceral lesions were more frequent in BRCA1/2mut pts. Additionally, 92% BRCA1/2mut pts had ≤ 3 organs involved while one third of BRCA1/2wt or vus/uk pts had ≥ 4 metastatic locations. With a median follow-up of 64 months (mo.), median iDFS and DDFS were higher, but not statistically significant, in BRCA1/2wt or vus/uk pts vs. BRCA1/2mut pts. In advanced setting, median PFS to 1L-3L do not show statistically significant differences between both groups. Regarding OS, no differences were also observed, but the median values were not reached. Further detailed information according to BRCA1/2 mutational status and BC subtypes is included in the table below. Conclusions: In this subset analysis of GEICAM/2016-04, BRCA1/2mut pts had some features of worse prognosis, with a more prevalent de novo metastatic disease. No statistically significant differences were observed in outcomes in both early-stage and advanced setting vs. BRCA1/2wt or vus/uk pts. &amp;lt;graphic&amp;gt; Citation Format: Noelia Martínez-Jáñez, Purificación Martínez, Cristina Hernando, Sabela Recalde, Alfonso Sánchez, David Morales, Marta Santisteban, Isaura Fernández, Sonia Del Barco, Esther Zamora, Vega Iranzo, Tamara Martos, Eduardo Martínez-de Dueñas, Silvia Antolin Novoa, Severina Domínguez, Paula Domínguez, Mª José Echarri, Ana Santaballa Bertrán, Xavier Mira, Andrea Blasco, Susana Bezares, Ander Urriticoechea. BRCA mutated Male Breast Cancer, hallmarks of a distinct disease. Data from the Spanish Male Breast Cancer Registry (GEICAM/2016-04) [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 PO1-16-10.

Retrospective cohort study on the bilateral occurrence of invasive lobular breast cancer

ObjectiveInvasive lobular carcinoma (ILC) is the second most common histological subtype of invasive breast cancer, following the no special type (NST) invasive carcinoma. It has historically been assumed that ILC occurs bilaterally in 20–29 % of cases, which has influenced the inclusion of MRI in the standard workup of ILC according to European guidelines. However, challenging this long-held belief regarding the bilateral occurrence of ILC opens up the possibility of revising the guidelines and using MRI only for more specific indications. This study aims to evaluate whether the previously reported high percentage of bilaterality still holds true and to question the added value of MRI in the standard workup of ILC. Study designA retrospective cohort study was conducted following approval from the institutional review board (EC 21/18/249) at Antwerp University Hospital (UZA). The cohort comprised female patients of all ages who had been diagnosed with either ILC or NST invasive carcinoma and had sought consultation at the UZA breast clinic. A comprehensive database was established to collect information on patient characteristics, imaging, and pathology. ResultsA total of 271 patients with ILC were included in the study, with incidence dates ranging from 01/01/2007 to 01/01/2023. Among these patients, a synchronous bilateral ILC lesion was observed in 1.85 % (5/271) of cases. This proportion is significantly lower than the reported percentage of patients with a bilateral lesion in the literature population, which stands at 4.95 %. The reference group consisted of 809 patients with NST invasive carcinoma, with incidence dates ranging from 01/01/2017 to 01/01/2023. In the control group, a synchronous bilateral NST lesion was observed in 3.96 % (32/809) of cases. There is no significant difference in the bilaterality rates between the group of ILC patients and the group of NST patients. Furthermore, MRI did not detect any histopathologically confirmed contralateral ILC lesion that had not already been detected by mammography or ultrasound. ConclusionsThe study results indicate a lower occurrence of bilateral ILC than previously assumed. Additionally, the incidence of synchronous bilateral lesions in ILC patients is not higher compared to patients with NST invasive carcinoma. Performing an MRI does not provide additional value in detecting bilateral carcinomas in ILC. Consequently, it is recommended that the current European guidelines be reassessed, and the indications for undergoing an MRI should be adjusted accordingly.

Predictive Factors of Neoadjuvant Chemotherapy Response in Breast Cancer Validated by Three Anatomopathological Scores: Residual Cancer Burden, Chevallier System, and Tumor-Infiltrating Lymphocytes.

The aim of this retrospective, observational, descriptive study was to identify predictors of response to neoadjuvant therapy in breast cancer patients and to validate them using three anatomopathological scores, such as residual cancer burden (RCB) score, Chevallier system, and tumor-infiltrating lymphocytes (TIL) score. We conducted a study on 88 female patientsaged 37 to 78 years with confirmed breast cancerwho were indicated for neoadjuvant chemotherapy. We analyzed different individual variables (such as age, menarche, and menopause), clinical/imaging characteristics of the breast tumor and axillary nodes, immunohistochemical biomarkers (such as ER/PR/HER2 and Ki67), and histopathological features (such as subtype and grading) in relation to three anatomopathological scores calculated based on the surgical resection specimen. The percentage of patients who could have benefited from conservative surgery increased from 6% at the time of diagnosis to 20% post-primary systemic therapy (PST). Age under 49 (p = 0.01), premenopausal status (p < 0.01), no special type (NST) (p = 0.04), high Ki67 (p < 0.01), triple-negative breast cancer (TNBC) (p = 0.02) are positive predictive factors of neoadjuvant therapy, whilelobular/mixt carcinoma-type (p = 0.04), luminal A (p = 0.01), positive lymph node (p < 0.01), and low differentiation grade (p = 0.01) are negative predictive factors for the response to PST. There is a strong correlation between the RCB score and the Chevallier system for quantifying the response to PST, with most predictive factors being confirmed through appropriate statistical analysis for both.TIL score values correlated with only some of the predictors, most likely due to the importance of calculating this score on both biopsy specimens at diagnosis and resection specimens after chemotherapy.