PLA2G12A as a Novel Biomarker for Colorectal Cancer with Prognostic Relevance.

Metastasis is the leading cause of colorectal cancer (CRC)-related deaths. Therefore, the identification of accurate biomarkers predictive of metastasis is needed to better stratify high-risk patients to provide preferred management and reduce mortality. In this study, we identified 13 new genes that modified circulating tumor cell numbers using a genome-wide genetic screen in a whole animal CRC model. Candidate genes were subsequently evaluated at the gene expression level in both an internal human CRC cohort of 153 patients and an independent cohort from the TCGA including 592 patients. Interestingly, the expression of one candidate, PLA2G12A, significantly correlated with both the time to recurrence and overall survival in our CRC cohort, with its low expression being an indicator of a poor clinical outcome. By examining the TCGA cohort, we also found that low expression of PLA2G12A was significantly enriched in epithelial-mesenchymal transition signatures. Finally, the candidate functionality was validated in vitro using three different colon cancer cell lines, revealing that PLA2G12A deficiency increases cell proliferation, migration, and invasion. Overall, our study identifies PLA2G12A as a prognostic biomarker of early-stage CRC, providing evidence that its deficiency promotes tumor growth and dissemination.

Epithelial cell adhesion molecule (EpCAM) from pleural fluid cell lysates is a highly accurate diagnostic biomarker of adenocarcinomatous effusions.

BACKGROUND AND OBJECTIVE: The discovery of highly accurate pleural fluid (PF) biomarkers of malignancy remains elusive. We assessed the operating characteristics of the PF epithelial cell adhesion molecule (EpCAM), claudin 4 (CL4) and human epididymis protein 4 (HE4) as potential markers of epithelial malignancies. METHODS: The three markers were quantified by immunoassays in the supernatants (s) and cell lysates (cl) of 175 PF samples. The cut-off values with 100% specificity were selected for malignant-benign discrimination. An immunocytochemical staining index score for each marker was also evaluated on PF cell blocks. The resulting best biomarker was further validated in two independent populations of 73 and 48 patients with pleural effusions (PE). RESULTS: An EpCAM(cl) >98 pg/g total lysate protein yielded 75% sensitivity, 100% specificity, negative likelihood ratio of 0.25 and area under the curve of 0.94 for labelling adenocarcinomatous effusions. Sensitivity reached 88% if EpCAM(cl) was combined with EpCAM immunostaining. One-third or more of the malignant effusions exhibiting a false-negative cytological fluid examination were correctly classified by EpCAM(cl) concentrations. Immunoassays for CL4 and HE4 were diagnostically useless. CONCLUSION: EpCAM(cl) is a new biomarker of adenocarcinomatous PE with meaningful discriminating properties.

Prognostic Factors Involved in the Epithelial-Mesenchymal Transition Process in Colorectal Cancer Have a Preponderant Role in Oxidative Stress: A Systematic Review and Meta-Analysis.

Epithelial-to-mesenchymal transition (EMT) is one of the most accepted mechanisms leading to metastasis, which is responsible for most of the cancer-related deaths. In order to identify EMT-related biomarkers able to predict clinical outcomes in colorectal cancer (CRC), a systematic review and meta-analysis of prognostic factors associated to overall survival (OS) and progression free survival (PFS) was conducted. The systematic literature search included studies from June 2014 to June 2019 available at PubMed and Scopus databases. Meta-analysis was performed for those markers appearing in minimum three works with a total number of 8656 participants. The rest were enlisted and subjected to functional enrichment. We identified nine clinical biomarkers and 73 EMT-related molecular biomarkers associated to OS and/or PFS in CRC. The significant enrichment of biomarkers found involved in cellular oxidoreductase activity suggests that ROS generation plays an active role in the EMT process. Clinical practice needs new biomarkers with a reliable prognostic value able to predict clinical outcomes in CRC. Our integrative work supports the role of oxidative stress in tumorigenesis and EMT progress highlighting the importance of deciphering this specific mechanism to get a better understanding of metastasis.

Novel biomarkers in primary breast core biopsies to predict poor response to neoadjuvant chemotherapy and appearance of metastases.

Drug resistance has been one of the major obstacles limiting the success of cancer chemotherapy. In two thirds of breast cancer patients, large (>1cm) residual tumors are present after neoadjuvant chemotherapy (NCT). The residual tumor and involved nodes have been indicators of relapse and survival very important in breast cancer. The goal of this preliminary study was to assess the predictive significance of a panel of molecular biomarkers, related with the response to treatment or drug resistance to NCT, as determined on the diagnostic tumor. The expression of 22 proteins was examined using immunohistochemistry in tissue microarrays (TMA) from 115 patients of stage II-III breast cancer, treated with NCT. Among studied proteins, there are some that are anti-apoptotic, pro-proliferative, cancer stem cell markers and the Vitamin D Receptor. Other proteins are involved in the identification of molecular subtype, cell cycle regulation or DNA repair. Next, a predictive signature of poor response was generated from independent markers of predictive value. Tumors that expressed four or five conditions (biomarkers of chemoresistance with a determinated cutoff) were associated with a 9-fold increase in the chances of these patients of having a poor response to NCT. Additionally, we also found a worse prognostic signature, generated from independent markers of prognostic value. Tumors which expressed two or three conditions of worst prognostic, were associated with a 6-fold reduction in Distant Disease Free Survival. In conclusion, finding biomarkers of chemoresitance (ypTNM II-III) and metastases can become a stepping stone for future studies that will need to be assessed in a bigger scale.

Prognostic value of c-FLIPL/s, HIF-1α, and NF-κß in stage II and III rectal cancer.

Locally advanced rectal cancer (LARC) is associated with a 25 % rate of metastases. The prognostic and predictive relevances of the expression of five proteins (c-FLIPL/s, HIF-1α, ß-catenin, p65, and p105/p50 NF-κß) were assessed. This is a retrospective study. From 1998 to 2009, 152 patients with stage II/III rectal cancer were treated with radio-chemotherapy. TMAs constructed with tumor and normal tissue from the diagnostic endoscopic biopsy and the surgical specimen after chemoradiotherapy were subjected to immunohistochemical (IHC) analysis. Results were correlated with clinical and pathological data, including progression-free survival (PFS). Four different IHC conditions were independent prognostic parameters for PFS: (1) cytoplasmic c-FLIPL/s (p = 0.007), (2) nuclear HIF-1α (p = 0.020), (3) a change in the cytoplasmic p65 between the diagnostic biopsy and the post-treatment specimen (p = 0.004), and (4) a change in the cytoplasmic c-FLIPL/s between the diagnostic biopsy and the post-treatment specimen (p = 0.021). Three different protein expression profiles, combining biomarkers, showed prognostic significance. IHC evaluation of these biomarkers in our three protein expression profiles may help to identify patients with worse prognosis and design more effective therapeutic strategies to personalize the treatment of rectal cancer.

Gene expression microarray-based assay to determine tumor site of origin in a series of metastatic tumors to the ovary and peritoneal carcinomatosis of suspected gynecologic origin.

The origin of the primary tumor is sometimes difficult to determine in peritoneal and ovarian metastases. A series of 25 metastatic tumors to the ovary and 7 cases of peritoneal carcinomatosis of suspected gynecologic origin were collected. Total RNA was extracted from frozen tumor tissue and studied by the Tissue of Origin-Frozen test, a microarray-based gene expression test from Pathwork Diagnostics (Redwood City, CA). Independently, formalin-fixed, paraffin-embedded tumor tissue was subjected to pathologic analysis. Immunohistochemical stains included keratins 7 and 20, estrogen and progesterone receptors, CDX2, villin, CEA, WT-1, TTF-1, mammoglobin, GCDF-15, and CD31. Clinical data were considered as gold standard, and after clinicopathologic evaluation, the tissue of origin was found in 29 cases. The Tissue of Origin-Frozen test correctly identified the ovary as site of origin in 7 of 7 peritoneal carcinomatosis cases, whereas immunohistochemical stains only allowed appropriate recognition in 5. In addition, the Tissue of Origin-Frozen test identified correctly the site of origin in 18 of the 22 metastatic tumors to the ovary with known origin. In the remaining 4 tumors, the correct origin was the second option in 2 cases and was not determined in the other 2. Immunohistochemistry correctly identified the site of origin in 17 of these 22 ovarian metastases. A combination of Tissue of Origin-Frozen and immunohistochemistry correctly identified the site of origin in 19 of 22 ovarian metastases of known origin. Although conventional pathologic examination and immunohistochemistry are commonly used for assessing the tumor site of origin, Tissue of Origin testing can be useful in difficult cases.

BRCA1 loss activates cathepsin L-mediated degradation of 53BP1 in breast cancer cells.

Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)-mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and poly(adenosine diphosphate-ribose) polymerase inhibitors, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor, and 53BP1 emerged as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identify here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy.

Importance of assessing CK19 immunostaining in core biopsies in patients subjected to sentinel node study by OSNA.

Analysis of sentinel lymph node (SLN) by means of One-Step Nucleic Acid Amplification (OSNA) is being used increasingly as a very sensitive and quick method for intraoperative axillary staging in patients with breast cancer. This molecular diagnostic assay detects the expression level of cytokeratin 19 (CK19), a luminal epithelial cell marker broadly expressed in most breast carcinomas and not normally found in lymph nodes. Almost all breast cancers express this cytoskeleton protein, but some breast tumors have been found to lose the expression of CK19. CK19 immunostaining in core biopsies has been recommended in selecting patients eligible for OSNA analysis because SLNs with metastatic involvement by CK19-negative breast cancers may result in a false negative result by OSNA. However, the real frequency of CK19-negative breast cancer has to be elucidated. In this study, we have assessed the frequency and molecular profile of CK19-negative breast carcinomas in three series of cases. The first is a prospective series of 197 breast carcinomas, 111 of which were subjected to SLN evaluation by OSNA. The second is a retrospective series of 41 triple-negative (TN) breast carcinomas, and the third is a retrospective series of 68 breast cancer patients (matched core biopsies and metastatic lymph nodes) that had been evaluated by conventional procedures before the OSNA methodology was adopted in our institution. Our results not only demonstrate that lack of expression of CK19 is infrequent in breast cancers but also that performing CK19 immunohistochemical staining is important on diagnostic core biopsies in taking the decision of using OSNA methodology in the evaluation of sentinel nodes in breast cancer patients.