Assessment of mammary adiposity in breast cancer using digital pathology

L'obesit\ue0 \ue8 un fattore di rischio significativo per molti tumori, compreso il carcinoma della mammella. I pazienti con carcinoma della mammella che sono sovrappeso o obesi hanno un aumentato rischio di recidiva e di mortalit\ue0 correlata al tumore. Sebbene siano state proposte diverse ipotesi per descrivere i meccanismi biologici alla base dell\u2019interazione tra obesit\ue0 e carcinoma della mammella, i progressi nella comprensione del microambiente tumorale e del ruolo degli adipociti presenti in esso e della loro rilevanza per lo sviluppo e la progressione tumorale o per l'emergere di resistenze alle terapie sono ancora limitati. Parallelamente alle importanti scoperte sull'obesit\ue0 come fattore di rischio per diverse malattie e tipi di tumore, i ricercatori hanno iniziato a utilizzare la patologia digitale per caratterizzare il microambiente tumorale. Studi pionieristici hanno scoperto l'esistenza di un tipo particolare di adipociti chiamati adipociti associati al tumore presenti nel microambiente di tumori che crescono a stretto contatto con il tessuto adiposo. Nel momento in cui \ue8 stata scritta questa tesi, solo pochi studi con piccole coorti di pazienti e spesso, un numero limitato di adipociti analizzati, per lo pi\uf9 distanti dal tumore, hanno mostrato la rilevanza del tessuto adiposo e degli adipociti come componente del microambiente tumorale. Tuttavia, allo stato attuale manca ancora una conoscenza approfondita del ruolo degli adipociti nel carcinoma della mammella. Lo studio presentato in questa tesi descrive le potenzialit\ue0 della patologia digitale e fornisce una rappresentazione dello stato dell'arte sui software con algoritmi dedicati per la misurazione degli adipociti, con una attenzione per gli adipociti presenti nel microambiente di carcinomi mammari. Oltre a stabilire criteri istopatologici per l'analisi degli adipociti, i risultati presentati dimostrano che \ue8 possibile effettuare l'analisi degli adipociti associati al tumore come di quelli distanti, utilizzando un software di patologia digitale. Inoltre, abbiamo mostrato che gli adipociti associati al tumore sono pi\uf9 piccoli degli adipociti distanti, riflettendo il processo di delipidazione subito dagli stessi. Infine, l'indice di massa corporea del paziente \ue8 associato alle dimensioni degli adipociti distanti e associati al tumore, fornendo nuove prospettive per la valutazione dell'adiposit\ue0 mammaria di un paziente. I dati qui presentati, sono preliminari per un cambiamento diretto nella pratica clinica, ma forniscono i criteri per eseguire l'analisi degli adipociti su coorti pi\uf9 ampie di pazienti ed evidenziano l\u2019importanza della valutazione del tessuto adiposo nella gestione del carcinoma della mammella.Increased adiposity is a significant risk factor for many cancers, including breast cancer. Patients with breast cancer who are overweight or obese have an increased risk of recurrence, and breast cancer-related mortality. While several hypotheses have been proposed to explain the biological mechanisms that drive the obesity-breast cancer interconnections, progress in our understanding of the adipose tissue tumor microenvironment and its relevance to breast cancer initiation and progression or the emergence of resistance to therapy has been limited. In parallel to major breakthroughs on the increased adiposity as risk factor for several diseases and cancers, researchers have begun to utilize digital pathology to characterize the tumor microenvironment in diverse types of adipose tissue-rich neoplasia. Pioneering studies have uncovered the existence of a peculiar type of adipocytes named cancer-associated adipocytes in the microenvironment of tumors that grow in close contact with adipose tissue. At the time this thesis was started, only a few studies with small cohorts of patients and often, a limited number of adipocytes analyzed, mostly distant from the tumor, have shown the importance of the adipose tissue tumor microenvironment. However, an in-depth knowledge was currently lacking for breast cancer. The work presented here harnesses the strength of digital pathology and state of the art of adipocyte measurement tools to analyze adipocytes in the adipose tissue microenvironment of breast cancer. Beyond providing histopathological criteria for adipocyte analysis, these results suggest that the analysis of mammary cancer-associated adipocytes is feasible using image analysis software. Moreover, our analyses reveal that cancer-associated adipocytes are smaller than distant adipocytes, reflecting the delipidation process undergone by cancer-associated adipocytes. The body mass index of the patient is associated with the size of distant and cancer-associated adipocytes giving new insights in the evaluation of mammary adiposity of a patient. The data presented here are too preliminary to derive practice-changing evidence but provide the basis for performing adipocyte analysis on larger number of patients and support the concept that the evaluation of adipose tissue should be taken into account in breast cancer management

Her2 assessment using quantitative reverse transcriptase polymerase chain reaction reliably identifies Her2 overexpression without amplification in breast cancer cases

Background: Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination. Patients and methods: We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting. Results: The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results. Significance: qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination

Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting

Squalene epoxidase as a promising metabolic target in cancer treatment

Oncogenic alteration of the cholesterol synthesis pathway is a recognized mechanism of metabolic adaptation. In the present review, we focus on squalene epoxidase (SE), one of the two rate-limiting enzymes in cholesterol synthesis, retracing its history since its discovery as an antimycotic target to its description as an emerging metabolic oncogene by amplification with clinical relevance in cancer. We review the published literature assessing the association between SE over-expression and poor prognosis in this disease. We assess the works demonstrating how SE promotes tumor cell proliferation and migration, and displaying evidence of cancer cell demise in presence of human SE inhibitors in in vitro and in vivo models. Taken together, robust scientific evidence has by now accumulated pointing out SE as a promising novel therapeutic target in cancer treatment

Schlafen-11 expression is associated with immune signatures and basal-like phenotype in breast cancer

PURPOSE: Breast cancer (BC) is a heterogeneous disorder, with variable response to systemic chemotherapy. Likewise, BC shows highly complex immune activation patterns, only in part reflecting classical histopathological subtyping. Schlafen-11 (SLFN11) is a nuclear protein we independently described as causal factor of sensitivity to DNA damaging agents (DDA) in cancer cell line models. SLFN11 has been reported as a predictive biomarker for DDA and PARP inhibitors in human neoplasms. SLFN11 has been implicated in several immune processes such as thymocyte maturation and antiviral response through the activation of interferon signaling pathway, suggesting its potential relevance as a link between immunity and cancer. In the present work, we investigated the transcriptional landscape of SLFN11, its potential prognostic value, and the clinico-pathological associations with its variability in BC. METHODS: We assessed SLFN11 determinants in a gene expression meta-set of 5061 breast cancer patients annotated with clinical data and multigene signatures. RESULTS: We found that 537 transcripts are highly correlated with SLFN11, identifying "immune response", "lymphocyte activation", and "T cell activation" as top Gene Ontology processes. We established a strong association of SLFN11 with stromal signatures of basal-like phenotype and response to chemotherapy in estrogen receptor negative (ER-) BC. We identified a distinct subgroup of patients, characterized by high SLFN11 levels, ER- status, basal-like phenotype, immune activation, and younger age. Finally, we observed an independent positive predictive role for SLFN11 in BC. CONCLUSIONS: Our findings are suggestive of a relevant role for SLFN11 in BC and its immune and molecular variability.status: publishe

Clinico-pathological associations and concomitant mutations of the RAS/RAF pathway in metastatic colorectal cancer

BACKGROUND: Over the past few years, next-generation sequencing (NGS) has become reliable and cost-effective, and its use in clinical practice has become a reality. A relevant role for NGS is the prediction of response to anti-EGFR agents in metastatic colorectal cancer (mCRC), where multiple exons from KRAS, NRAS, and BRAF must be sequenced simultaneously. METHODS: We optimized a 14-amplicon NGS panel to assess, in a consecutive cohort of 219 patients affected by mCRC, the presence and clinico-pathological associations of mutations in the KRAS, NRAS, BRAF, and PIK3CA genes from formalin-fixed, paraffin-embedded specimens collected for diagnostics and research at the time of diagnosis. RESULTS: We observed a statistically significant association of RAS mutations with sex, young age, and tumor site. We demonstrated that concomitant mutations in the RAS/RAF pathway are not infrequent in mCRC, and as anticipated by whole-genome studies, RAS and PIK3CA tend to be concurrently mutated. We corroborated the association of BRAF mutations in right mCRC tumors with microsatellite instability. We established tumor side as prognostic parameter independently of mutational status. CONCLUSIONS: To our knowledge, this is the first monocentric, consecutively accrued clinical mCRC cancer cohort tested by NGS in a real-world context for KRAS, NRAS, BRAF, and PIK3CA. Our study has highlighted in clinical practice findings such as the concomitance of mutations in the RAS/RAF pathway, the presence of multiple mutations in single gene, the co-occurrence of RAS and PIK3CA mutations, the prognostic value of tumor side and possible associations of sex with specific mutations.status: publishe

Digital analysis of distant and cancer-associated mammary adipocytes.

Adipocytes and cancer-associated adipocytes (CAAs) are poorly investigated cells in the tumor microenvironment. Different image analysis software exist for identifying and measuring these cells using scanned hematoxylin and eosin (H&E)-stained slides. It is however unclear which one is the most appropriate for breast cancer (BC) samples. Here, we compared three software (AdipoCount, Adiposoft, and HALO®). HALO® outperformed the other methods with regard to adipocyte identification, (> 96% sensitivity and specificity). All software performed equally good with regard to area and diameter measurement (concordance correlation coefficients > 0.97 and > 0.96, respectively). We then analyzed a series of 10 BCE samples (n = 51 H&E slides) with HALO®. Distant adipocytes were defined >2 mm away from cancer cells or fibrotic region, whereas CAAs as the first three lines of adipocytes close to the invasive front. Intra-mammary heterogeneity was limited, implying that measuring a single region of ∼500 adipocytes provides a reliable estimation of the distribution of their size features. CAAs had smaller areas (median fold-change: 2.62) and diameters (median fold-change: 1.64) as compared to distant adipocytes in the same breast (both p = 0.002). The size of CAAs and distant adipocytes was associated with the body mass index (BMI) of the patient (area: rho = 0.89, p = 0.001; rho = 0.71, p = 0.027, diameter: rho = 0.87 p = 0.002; rho = 0.65 p = 0.049, respectively). To conclude, we demonstrate that quantifying adipocytes in BC sections is feasible by digital pathology using H&E sections, setting the basis for a standardized analysis of mammary adiposity in larger series of patients.info:eu-repo/semantics/publishe

Circulating tumor DNA using tagged targeted deep sequencing to assess minimal residual disease in breast cancer patients undergoing neoadjuvant chemotherapy

In breast cancer patients undergoing neoadjuvant chemotherapy before surgery, there is an unmet need for noninvasive predictive biomarkers of response. The analysis of circulating tumor DNA (ctDNA) in particular has been the object of several reports, but few of them have studied the applicability of tagged targeted deep sequencing (tTDS) to clinical practice and its performance compared with droplet digital PCR (ddPCR). Here, we present the first results from an ongoing study involving a prospectively accrued, monocentric cohort of patients affected by invasive breast cancer, undergoing neoadjuvant chemotherapy followed by surgery with curative intent as per clinical practice. A pretreatment tumor biopsy and plasma samples were collected before and during treatment, after surgery, and every six months henceforth or until relapse, whichever came first. Pretreatment biopsies were sequenced with a 409-gene massive parallel sequencing (MPS) panel, allowing the identification of target mutations and their research in plasma by tTDS and ddPCR as a complementary approach. Using tTDS, we demonstrated the presence of at least one deleterious mutation in all the relapsed cases we studied (n = 4), with an average lead time of six months before clinical relapse. The association with ddPCR was suboptimal, and only one relapsed patient could be identified with such method. tTDS shows potential as an early noninvasive method for the detection of MRD in BC patients

The association between adiposity and anti-proliferative response to neoadjuvant endocrine therapy with letrozole in post-menopausal patients with estrogen receptor positive breast cancer.

The impact of adiposity on the efficacy of endocrine treatment in patients with estrogen receptor positive breast cancer is poorly investigated. Here, we retrospectively investigated in a cohort of 56 patients whether body mass index and/or mammary adiposity are associated with anti-proliferative response in the neoadjuvant setting. Anti-proliferative response was defined as high Ki67 at baseline (Ki67bl) and low Ki67 at surgery (Ki67srg), using the 14% cut-off. Mammary adipocyte size was assessed on hematoxylin and eosin slides from the surgical samples using digital pathology. A higher proportion of tumors with an anti-proliferative response was observed in patients with obesity (54.5%) as compared to patients with normal weight (9.0%) and patients with overweight (40.0%) (p = 0.031), confirmed by multivariable regression analysis adjusted for baseline Ki67 (OR, obese vs normal weight: 13.76, 95%CI: 1.49-207.63, p = 0.020). Larger adipocyte diameter was identified as predictor of anti-proliferative response (OR per increase in diameter of 5 μm for adipocytes distant from the tumor: 2.24, 95%CI: 1.01-14.32, p = 0.046). This study suggests that anti-proliferative response to neoadjuvant letrozole might be more frequent in patients with increased systemic or mammary adiposity.info:eu-repo/semantics/publishe

Circulating Tumor DNA Using Tagged Targeted Deep Sequencing to Assess Minimal Residual Disease in Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy

In breast cancer patients undergoing neoadjuvant chemotherapy before surgery, there is an unmet need for noninvasive predictive biomarkers of response. The analysis of circulating tumor DNA (ctDNA) in particular has been the object of several reports, but few of them have studied the applicability of tagged targeted deep sequencing (tTDS) to clinical practice and its performance compared with droplet digital PCR (ddPCR). Here, we present the first results from an ongoing study involving a prospectively accrued, monocentric cohort of patients affected by invasive breast cancer, undergoing neoadjuvant chemotherapy followed by surgery with curative intent as per clinical practice. A pretreatment tumor biopsy and plasma samples were collected before and during treatment, after surgery, and every six months henceforth or until relapse, whichever came first. Pretreatment biopsies were sequenced with a 409-gene massive parallel sequencing (MPS) panel, allowing the identification of target mutations and their research in plasma by tTDS and ddPCR as a complementary approach. Using tTDS, we demonstrated the presence of at least one deleterious mutation in all the relapsed cases we studied (n = 4), with an average lead time of six months before clinical relapse. The association with ddPCR was suboptimal, and only one relapsed patient could be identified with such method. tTDS shows potential as an early noninvasive method for the detection of MRD in BC patients.status: publishe