Francisco Beca

Cancer stem cells markers CD44, CD24 and ALDH1 in breast cancer special histological types.

Aims CD44, CD24 and ALDH1 are the most consistently used biomarkers to identify and characterise the breast cancer stem cell (CSC) phenotype. However, most studies performed until now analysed samples of invasive ductal carcinomas of no special type (IDC-NST). Therefore, prevalence and clinical significance of these CSC markers in breast carcinomas of special histological types (SHT) is largely unknown. For that reason, this study aims to determine the distribution of the breast CD44, CD24 and ALDH1 CSC markers among a series of invasive breast carcinomas of SHT, in comparison with a series of IDC-NST. Methods 117 invasive SHT breast carcinomas were analysed for the expression of CD44, CD24 and ALDH1, by immuhohistochemistry. The distribution of these CSC markers was evaluated among the distinct histological special types, and the results were compared with a series of 466 IDC-NST. Results The expression prevalence of the breast CSC markers differed between special types and IDC-NST. Medullary, papillary and tubular carcinomas were enriched in the CSC phenotype CD44+/CD24−/low (80.0%, 100.0% and 100.0%, respectively, vs 45.3% in IDC-NST). Considering the ALDH1 cytoplasmic tumour expression, only medullary and metaplastic carcinomas displayed significant increase in CD44+/CD24-/low/ALDH1+ CSC phenotype frequency (36.4% and 28.6%, respectively, vs 4.8% in IDC-NST). Conclusions The expression distribution of breast CSC markers is largely dependent on histological type. Interestingly, within the distinct SHT, medullary and metaplastic carcinomas are the two types highly associated with high-grade carcinomas, basal-like and claudin-low molecular subtypes, and to the CSC phenotype CD44+/CD24−/low/ALDH1+.

Diagnostic assessment of deep learning algorithms for detection of lymph node metastases in women with breast cancer

Importance Application of deep learning algorithms to whole-slide pathology images can potentially improve diagnostic accuracy and efficiency. Objective Assess the performance of automated deep learning algorithms at detecting metastases in hematoxylin and eosin–stained tissue sections of lymph nodes of women with breast cancer and compare it with pathologists’ diagnoses in a diagnostic setting. Design, Setting, and Participants Researcher challenge competition (CAMELYON16) to develop automated solutions for detecting lymph node metastases (November 2015-November 2016). A training data set of whole-slide images from 2 centers in the Netherlands with (n = 110) and without (n = 160) nodal metastases verified by immunohistochemical staining were provided to challenge participants to build algorithms. Algorithm performance was evaluated in an independent test set of 129 whole-slide images (49 with and 80 without metastases). The same test set of corresponding glass slides was also evaluated by a panel of 11 pathologists with time constraint (WTC) from the Netherlands to ascertain likelihood of nodal metastases for each slide in a flexible 2-hour session, simulating routine pathology workflow, and by 1 pathologist without time constraint (WOTC). Exposures Deep learning algorithms submitted as part of a challenge competition or pathologist interpretation. Main Outcomes and Measures The presence of specific metastatic foci and the absence vs presence of lymph node metastasis in a slide or image using receiver operating characteristic curve analysis. The 11 pathologists participating in the simulation exercise rated their diagnostic confidence as definitely normal, probably normal, equivocal, probably tumor, or definitely tumor. Results The area under the receiver operating characteristic curve (AUC) for the algorithms ranged from 0.556 to 0.994. The top-performing algorithm achieved a lesion-level, true-positive fraction comparable with that of the pathologist WOTC (72.4% [95% CI, 64.3%-80.4%]) at a mean of 0.0125 false-positives per normal whole-slide image. For the whole-slide image classification task, the best algorithm (AUC, 0.994 [95% CI, 0.983-0.999]) performed significantly better than the pathologists WTC in a diagnostic simulation (mean AUC, 0.810 [range, 0.738-0.884]; P < .001). The top 5 algorithms had a mean AUC that was comparable with the pathologist interpreting the slides in the absence of time constraints (mean AUC, 0.960 [range, 0.923-0.994] for the top 5 algorithms vs 0.966 [95% CI, 0.927-0.998] for the pathologist WOTC). Conclusions and Relevance In the setting of a challenge competition, some deep learning algorithms achieved better diagnostic performance than a panel of 11 pathologists participating in a simulation exercise designed to mimic routine pathology workflow; algorithm performance was comparable with an expert pathologist interpreting whole-slide images without time constraints. Whether this approach has clinical utility will require evaluation in a clinical setting.

Intratumor Heterogeneity in Breast Cancer

Intratumor heterogeneity is the main obstacle to effective cancer treatment and personalized medicine. Both genetic and epigenetic sources of intratumor heterogeneity are well recognized and several technologies have been developed for their characterization. With the technological advances in recent years, investigators are now elucidating intratumor heterogeneity at the single cell level and in situ. However, translating the accumulated knowledge about intratumor heterogeneity to clinical practice has been slow. We are certain that better understanding of the composition and evolution of tumors during disease progression and treatment will improve cancer diagnosis and the design of therapies. Here we review some of the most important considerations related to intratumor heterogeneity. We discuss both genetic and epigenetic sources of intratumor heterogeneity and review experimental approaches that are commonly used to quantify it. We also discuss the impact of intratumor heterogeneity on cancer diagnosis and treatment and share our perspectives on the future of this field.

Prostate cancer–associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4

The bromodomain and extraterminal (BET) family of proteins comprises four members—BRD2, BRD3, BRD4 and the testis-specific isoform BRDT—that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer–associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

Loss of caveolin-1 and gain of MCT4 expression in the tumor stroma : key events in the progression from an in situ to an invasive breast carcinoma

The progression from in situ to invasive breast carcinoma is still an event poorly understood. However, it has been suggested that interactions between the neoplastic cells and the tumor microenvironment may play an important role in this process. Thus, the determination of differential tumor-stromal metabolic interactions could be an important step in invasiveness. The expression of stromal Caveolin-1 (Cav-1) has already been implicated in the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Additionally, stromal Cav-1 expression has been associated with the expression of stromal monocarboxylate transporter 4 (MCT4) in invasive breast cancer. However, the role of stromal MCT4 in invasiveness has never been explored, neither the association between Cav-1 and MCT4 in the transition from breast DCIS to IDC. Therefore, our aim was to investigate in a series of breast cancer samples including matched in situ and invasive components, if there was a relationship between stromal Cav-1 and MCT4 in the progression from in situ to invasive carcinoma. We found loss of stromal Cav-1 in the progression to IDC in 75% of the cases. In contrast, MCT4 stromal expression was acquired in 87% of the IDCs. Interestingly, a concomitant loss of Cav-1 and gain of MCT4 was observed in the stroma of 75% of the cases, when matched in situ and invasive carcinomas were compared. These results suggest that alterations in Cav-1 and MCT4 may thus mark a critical point in the progression from in situ to invasive breast cancer.

Immune Escape in Breast Cancer During In Situ to Invasive Carcinoma Transition

To investigate immune escape during breast tumor progression, we analyzed the composition of leukocytes in normal breast tissues, ductal carcinoma in situ (DCIS), and invasive ductal carcinomas (IDC). We found significant tissue and tumor subtype-specific differences in multiple cell types including T cells and neutrophils. Gene expression profiling of CD45+CD3+ T cells demonstrated a decrease in CD8+ signatures in IDCs. Immunofluorescence analysis showed fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including in matched DCIS and recurrent IDC. T-cell receptor clonotype diversity was significantly higher in DCIS than in IDCs. Immune checkpoint protein TIGIT-expressing T cells were more frequent in DCIS, whereas high PD-L1 expression and amplification of CD274 (encoding PD-L1) was only detected in triple-negative IDCs. Coamplification of a 17q12 chemokine cluster with ERBB2 subdivided HER2+ breast tumors into immunologically and clinically distinct subtypes. Our results show coevolution of cancer cells and the immune microenvironment during tumor progression.Significance: The design of effective cancer immunotherapies requires the understanding of mechanisms underlying immune escape during tumor progression. Here we demonstrate a switch to a less active tumor immune environment during the in situ to invasive breast carcinoma transition, and identify immune regulators and genomic alterations that shape tumor evolution. Cancer Discov; 7(10); 1098-115. ©2017 AACR.See related commentary by Speiser and Verdeil, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Aspirin Suppresses Growth in PI3K-Mutant Breast Cancer by Activating AMPK and Inhibiting mTORC1 Signaling

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiologic studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA Here, we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on COX-2 and NF-κB. We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mTORC1 inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, whereas combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports the evaluation of aspirin and PI3K pathway inhibitors as a combination therapy for targeting breast cancer. Cancer Res; 77(3); 790-801. ©2016 AACR.

Genotypes and prevalence of HPV single and multiple concurrent infections in women with HSIL

The contribution of human papillomavirus (HPV) types to the carcinogenesis of cervical cancer has been established for a long time. However, the role of phylogenetically related and rare variants remains uncertain, as well as the influence of concurrent multiple HPV genotypes infection. We aimed at studying the prevalence of several HPV genotypes infecting women with single versus concurrent multiple HPV genotypes infection with a HSIL diagnosis in a cervical cytology. We conducted a cross‐sectional study using Thin‐Prep® liquid‐based cervical cytology specimens with the diagnosis of high‐grade squamous intraepithelial lesion (HSIL), in which HPV genotype was sequentially tested. Genotypes were determined with a PapilloCheck® system, a DNA‐Chip for the type‐specific identification of 18 high‐risk and six low‐risk types of HPV. Of the total study population, 176 cases had a diagnosis of HSIL and positive HPV genotyping result, being HPV16 the most prevalent genotype (48.86%; 95%CI: 41.58–56.19) followed by HPV31 (14.20%; 95%CI: 9.75–20.18). Concurrent multiple HPV genotypes were detected in 36.93% (95%CI: 30.15–44.27) of the patients. The prevalence of the 10 most common HPV genotypes detected varied significantly according to the presence of single vs. concurrent multiple HPV genotypes (P = 0.022). Moreover, women with concurrent multiple HPV genotypes were on average 3.53 (95%CI: 0.43–6.64) years younger than women with single genotype infection. Our results suggest that women with multiple genotype HPV infection differ in terms of age and distribution of the most prevalent HPV genotypes. Additionally, we provide further evidence of the predominance of HPV16 in HSIL lesions of the uterine cervix. Diagn. Cytopathol. 2014;42:919–923.

Growing indication for FNA to study and analyze tumor heterogeneity at metastatic sites.

In routine practice, suspected metastases in patients with cancer are only occasionally biopsied, primarily because of the cost and invasiveness of the procedure. However, biopsies of metastatic lesions can be valuable, not only in confirming the presence of metastatic disease, but also in revealing unsuspected benign disease or secondary malignancies. In addition, such biopsies also allow the assessment of biomarkers that might differ from those on primary tumor cells, and can thereby facilitate selection of the optimal treatment. Because of the increasing recognition of clonal and phenotypic heterogeneity of tumors, we anticipate that in the near future, biopsying of metastatic lesions will constitute a standard‐of‐care practice, allowing assessment of molecular differences between the primary tumor and metastatic lesions. In our opinion, fine‐needle aspiration is currently the best method for making repeated biopsies to monitor the tumor: it is minimally invasive, safe, and cost effective and can be coupled with modern ancillary techniques. Here we provide an up‐to‐date review of the clinical implications of tumor heterogeneity in metastatic disease and the ancillary molecular techniques used in cytology; we also discuss the role of modern cytology in contemporary diagnosis and management of metastatic cancer. Cancer (Cancer Cytopathol) 2014;122:504–511.

Oral squamous cell carcinoma in a Crohn's disease patient taking azathioprine: Case report and review of the literature

Thiopurines are widely used for remission maintenance and post-operative recurrence prevention in Crohns disease. The increased risk of cancer in transplant recipients on azathioprine is well recognized and there are concerns that this may also be true for inflammatory bowel disease patients. We report a case of a 33-year-old Caucasian woman with Crohns disease treated with azathioprine for 9 years who developed an ulcerated lesion at the right superior retromolar trigone. Biopsy specimen revealed a squamous cell carcinoma.