The Evolution of Clinically Aggressive Triple-Negative Breast Cancer Shows a Large Mutational Diversity and Early Metastasis to Lymph Nodes.

In triple-negative breast cancer (TNBC), only 30% of patients treated with neoadjuvant chemotherapy achieve a pathological complete response after treatment and more than 90% die due to metastasis formation. The diverse clinical responses and metastatic developments are attributed to extensive intrapatient genetic heterogeneity and tumor evolution acting on this neoplasm. In this work, we aimed to evaluate genomic alterations and tumor evolution in TNBC patients with aggressive disease. We sequenced the whole exome of 16 lesions from four patients who did not respond to therapy, and took several follow-up samples, including samples from tumors before and after treatment, as well as from the lymph nodes and skin metastases. We found substantial intrapatient genetic heterogeneity, with a variable tumor mutational composition. Early truncal events were MCL1 amplifications. Metastatic lesions had deletions in RB1 and PTEN, along with TERT, AKT2, and CCNE1 amplifications. Mutational signatures 06 and 12 were mainly detected in skin metastases and lymph nodes. According to phylogenetic analysis, the lymph node metastases occurred at an early stage of TNBC development. Finally, each patient had three to eight candidate driving mutations for targeted treatments. This study delves into the genomic complexity and the phylogenetic and evolutionary development of aggressive TNBC, supporting early metastatic development, and identifies specific genetic alterations associated with a response to targeted therapies.

Association of Lung Adenocarcinoma Subtypes According to the IASLC/ATS/ERS Classification and Programmed Cell Death Ligand 1 (PD-L1) Expression in Tumor Cells.

Background: Programmed cell death-ligand 1 (PD-L1) protein expression is one of the most extensively studied biomarkers in patients with non-small cell lung cancer (NSCLC). However, there is scarce information regarding its association with distinct adenocarcinoma subtypes. This study evaluated the frequency of PD-L1 expression according to the IASLC/ATS/ERS classification and other relevant histological and clinical features. Patients and Methods: PD-L1 expression was assessed by immunohistochemistry (IHC). According to its positivity in tumor cells membrane, we stratified patients in three different tumor proportions score (TPS) cut-off points: a) <1% (negative), b) between 1 and 49%, and c) ≥50%; afterward, we analyzed the association among PD-L1 expression and lung adenocarcinoma (LADC) predominant subtypes, as well as other clinical features. As an exploratory outcome we evaluated if a PD-L1 TPS score ≥15% was useful as a biomarker for determining survival. Results: A total of 240 patients were included to our final analysis. Median age at diagnosis was 65 years (range 23-94 years). A PD-L1 TPS ≥1% was observed in 52.5% of the entire cohort; regarding specific predominant histological patterns, a PD-L1 TPS ≥1 was documented in 31.2% of patients with predominant-lepidic pattern, 46.2% of patients with predominant-acinar pattern, 42.8% of patients with a predominant-papillary pattern, and 68.7% of patients with predominant-solid pattern (p = 0.002). On the other hand, proportion of tumors with PD-L1 TPS ≥50% was not significantly different among adenocarcinoma subtypes. At the univariate survival analysis, a PD-L1 TPS cut-off value of ≥15% was associated with a worse PFS and OS. Conclusion: According to IASLC/ATS/ERS lung adenocarcinoma classification, the predominant-solid pattern is associated with a higher proportion of PD-L1 positive samples, no subtype was identified to be associated with a high (≥50%) TPS PD-L1.

Comprehensive Genomic Profile of Heterogeneous Long Follow-Up Triple-Negative Breast Cancer and Its Clinical Characteristics Shows DNA Repair Deficiency Has Better Prognostic.

Triple-negative breast cancer (TNBC) presents a marked diversity at the molecular level, which promotes a clinical heterogeneity that further complicates treatment. We performed a detailed whole exome sequencing profile of 29 Mexican patients with long follow-up TNBC to identify genomic alterations associated with overall survival (OS), disease-free survival (DFS), and pathologic complete response (PCR), with the aim to define their role as molecular predictive factors of treatment response and prognosis. We detected 31 driver genes with pathogenic mutations in TP53 (53%), BRCA1/2 (27%), CDKN1B (9%), PIK3CA (9%), and PTEN (9%), and 16 operative mutational signatures. Moreover, tumors with mutations in BRCA1/2 showed a trend of sensitivity to platinum salts. We found an association between deficiency in DNA repair and surveillance genes and DFS. Across all analyzed tumors we consistently found a heterogeneous molecular complexity in terms of allelic composition and operative mutational processes, which hampered the definition of molecular traits with clinical utility. This work contributes to the elucidation of the global molecular alterations of TNBC by providing accurate genomic data that may help forthcoming studies to improve treatment and survival. This is the first study that integrates genomic alterations with a long follow-up of clinical variables in a Latin American population that is an underrepresented ethnicity in most of the genomic studies.

Dysregulated expression of hypoxia-inducible factors augments myofibroblasts differentiation in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an age-related, progressive and lethal disease, whose pathogenesis is associated with fibroblasts/myofibroblasts foci that produce excessive extracellular matrix accumulation in lung parenchyma. Hypoxia has been described as a determinant factor in its development and progression. However, the role of distinct members of this pathway is not completely described. METHODS: By western blot, quantitative PCR, Immunohistochemistry and Immunocitochemistry were evaluated, the expression HIF alpha subunit isoforms 1, 2 & 3 as well, as their role in myofibroblast differentiation in lung tissue and fibroblast cell lines derived from IPF patients. RESULTS: Hypoxia signaling pathway was found very active in lungs and fibroblasts from IPF patients, as demonstrated by the abundance of alpha subunits 1 and 2, which further correlated with the increased expression of myofibroblast marker αSMA. In contrast, HIF-3α showed reduced expression associated with its promoter hypermethylation. CONCLUSIONS: This study lends further support to the involvement of hypoxia in the pathogenesis of IPF, and poses HIF-3α expression as a potential negative regulator of these phenomena.

Stage I-III colon cancer patients with tumor deposits behave similarly to stage IV patients. Cross-section analysis of 392 patients.

BACKGROUND AND OBJECTIVES: Tumor deposits (TDs) are associated with adverse prognostic factors and decreased survival in colorectal cancer. However, controversy exists regarding their definition, evaluation, and staging categories. This study aimed to determine the survival and recurrence impact of the TD in colon adenocarcinomas; and to determine if TD patients behave similarly to stage IV patients. METHODS: Cross-section study from 392 patients with colon adenocarcinoma from 2005 to 2012. We performed survival analysis and further stratified patients considering TD patients as a "stage IV-TD" to demonstrate if they behave similarly than stage IV patients. RESULTS: From 392 patients, 204 (52%) were men, the mean age was 57.4 ± 13.9 years and 11.5% of cases had TD. In a multivariate analysis, TD failed to predict mortality and recurrence. Considering cases with TD as stage IV-TD, their mean survival was similar to stage IV patients (69.3 and 64.6 months, respectively) and different to those in stage III (110.5 months), II (135.7 months), and I (114.9 months) (P < 0.001). CONCLUSIONS: TD failed to predict mortality and recurrence. Patients with TD in stage I-III shows similar mortality than stage IV patients; then, we suggest putting them into a substage IV category instead of the N1c category.

CD117 immunoexpression in oral and sinonasal mucosal melanoma does not correlate with somatic driver mutations in the MAPK pathway.

BACKGROUND: Mutations on KIT and downstream genes of MAPK pathway that overstimulate cellular proliferation have been associated with primary oral and sinonasal melanomas (POSNM), but there is limited information that allows the use of personalized therapy. Thus, the aim of the present study was to determine a possible association between the C-KIT immunohistochemical expression with the presence of somatic driver mutations in NRAS, BRAF, KIT, MITF and PTEN on POSNM. METHODS: A retrospective study included 62 tumour samples of an oncological reference centre in Mexico City (17-year period). Immunohistochemistry stain of C-KIT was carried out. Genomic DNA was obtained and used to assess hotspot mutations of KIT, NRAS, BRAF, MITF and PTEN through qPCR. Chi-square, Fisher's exact and the Mann-Whitney U tests were applied when necessary. The significance was set at P < 0.05. RESULTS: Sixty-two cases were included, 74% were positive for C-KIT immunoexpression, all exhibited moderate/strong intensity. Ten (16.1%) samples harboured at least one mutation, 6.4% and 6.6% for NRASQ 61R and BRAFV 600E , respectively, followed by KITK624E (3.2%). No KITL 576P , MITF or PTEN mutations were identified. No significant correlation was observed between mutations and immunostaining (rs = -0.057, P = 0.765). CONCLUSIONS: Regardless of the high immunoexpression of C-KIT, there was no association with the MAPK mutations among POSNM samples. Thus, C-KIT immunohistochemistry is not a reliable tool to detect POSNM candidates for biological therapy.

A pharmacoeconomic analysis of the collection and preservation of samples in the biobank of the "Instituto Nacional de Cancerología" in Mexico City.

In this work we estimated the budgetary impact of the samples produced by the biobank of the "Instituto Nacional de Cancerología" (BT-INCan) to set a recuperation fee from the perspective of the Health Ministry of Mexico. The study is an observational retrospective review of the direct medical costs (DMCs) of the processes involved in cryopreservation of the samples collected, on a per sample basis, including materials, laboratory tests, personnel, and administrative costs. Materials and labor costs were determined by information collected from the BT-INCan. DMCs were provided depending on the type of sample: plasma, tissue and biopsy; they were calculated according to the process required to preserve them. Sensitivity analysis was performed using bootstrap. Recuperation costs ranged from 130 to 155 USD. Costs were considered on a 5-year time frame for the maintenance per sample, which is the average time that a sample is kept in the BT-INCan. The cost analysis is perceived as an approximation to the most adequate recuperation fee per sample needed to guarantee the correct development of the BT-INCan. This work provides a basis and valuable information about costs, to enable several health institutions to strategically plan and manage a biobank or even motivate to establish their own biobank.

CTCF-KDM4A complex correlates with histone modifications that negatively regulate CHD5 gene expression in cancer cell lines.

Histone demethylase KDM4A is involved in H3K9me3 and H3K36me3 demethylation, which are epigenetic modifications associated with gene silencing and RNA Polymerase II elongation, respectively. KDM4A is abnormally expressed in cancer, affecting the expression of multiple targets, such as the CHD5 gene. This enzyme localizes at the first intron of CHD5, and the dissociation of KDM4A increases gene expression. In vitro assays showed that KDM4A-mediated demethylation is enhanced in the presence of CTCF, suggesting that CTCF could increase its enzymatic activity in vivo, however the specific mechanism by which CTCF and KDM4A might be involved in the CHD5 gene repression is poorly understood. Here, we show that CTCF and KDM4A form a protein complex, which is recruited into the first intron of CHD5. This is related to a decrease in H3K36me3/2 histone marks and is associated with its transcriptional downregulation. Depletion of CTCF or KDM4A by siRNA, triggered the reactivation of CHD5 expression, suggesting that both proteins are involved in the negative regulation of this gene. Furthermore, the knockout of KDM4A restored the CHD5 expression and H3K36me3 and H3K36me2 histone marks. Such mechanism acts independently of CHD5 promoter DNA methylation. Our findings support a novel mechanism of epigenetic repression at the gene body that does not involve promoter silencing.

Reproducibility of the EGFR immunohistochemistry scores for tumor samples from patients with advanced non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) is overexpressed in >60% of non-small cell lung cancer (NSCLC) cases. In combination with radiotherapy or chemotherapy, first-line treatments with antibodies against EGFR, including cetuximab and necitumumab, have demonstrated benefits by increasing overall survival (OS), particularly in patients who overexpress EGFR. The present study evaluated the interobserver agreement among three senior pathologists, who were blinded to the clinical outcomes and assessed tumor samples from 85 patients with NSCLC using the H-score method. EGFR immunohistochemistry was performed using a qualitative immunohistochemical kit. The reported (mean ± standard deviation) H-scores from each pathologist were 111±102, 127±103 and 128.53±104.03. The patients with average H-scores ≥1, ≥100, ≥200 and between 250-300 were 85.9, 54.1, 28.2 and 12.9, respectively. Patients who had an average H-score >100 had a shorter OS time compared with those with lower scores. Furthermore, patients with EGFR mutations who were treated with EGFR-tyrosine kinase inhibitors (TKIs) and had an average H-score >100 had a longer OS time compared with those with an average H-score <100. The interobserver concordance for the total H-scores were 0.982, 0.980 and 0.988, and for a positive H-score ≥200, the interobserver concordance was 0.773, 0.710 and 0.675, respectively. The determination of EGFR expression by the H-score method is highly reproducible among pathologists and is a prognostic factor associated with a poor OS in all patients. Additionally, the results of the present study suggest that patients with EGFR mutations that are treated with EGFR-TKIs and present with a high H-score have a longer OS time.

Thyroid Transcription Factor-1 Expression in Adenocarcinomas of the Bile Duct.

BACKGROUND: The immunoreactivity of thyroid transcription factor-1 (TTF-1) is a very specific marker for lung and thyroid neoplasms; the expression of TTF-1 has also been demonstrated in extrapulmonary carcinomas. We examined the expression of TTF-1 in 15 intestinal-type adenocarcinomas of the extrahepatic bile duct. We then compared the expression to TTF-1 staining with other immunohistochemical markers including cytokeratin (CK) 7, CK20, caudal-type homeobox transcription factor 2 (CDX2), Napsin A, and MUC2. We additionally compared the clinicopathological prognostic factors with the TTF-1 expression status. RESULTS: Nuclear TTF-1 staining was detected in 2 cases (13.3%), and Napsin A was positive in the same 2 cases (13.3%). All cases were positive for CK20, CDX2, and MUC2; 5 cases were positive for CK7. There was no correlation between TTF-1 expression and the clinicopathological characteristics. CONCLUSIONS: To avoid potential pitfalls, TTF-1 should be interpreted in conjunction with the clinical setting, histology, and the results of markers such as CK7, CK20, Napsin A, and CDX2. This report is the first of TTF-1 positivity in adenocarcinomas from the extrahepatic biliary tract.

Effects of 2-methoxyestradiol on apoptosis and HIF-1α and HIF-2α expression in lung cancer cells under normoxia and hypoxia.

Hypoxic tumor cells are known to be more resistant to conventional chemotherapy and radiation than normoxic cells. However, the effects of 2-methoxyestradiol (2-ME), an anti-angiogenic, antiproliferative and pro-apoptotic drug, on hypoxic lung cancer cells are unknown. The aim of the present study was to compare the effects of 2-ME on cell growth, apoptosis, hypoxia-inducible factor 1α (HIF-1α) and HIF-2α gene and protein expression in A549 cells under normoxic and hypoxic conditions. To establish the optimal 2-ME concentration with which to carry out the apoptosis assay and to examine mRNA and protein expression of HIFs, cell growth analysis was carried out through N-hexa-methylpararosaniline staining assays in A549 cell cultures treated with one of five different 2-ME concentrations at different times under normoxic or hypoxic growth conditions. The 2-ME concentration of 10 mM at 72 h was selected to perform all further experiments. Apoptotic cells were analyzed by flow cytometry. Western blotting was used to determine HIF-1α and HIF-2α protein expression in total cell extracts. Cellular localization of HIF-1α and HIF-2α was assessed by immunocytochemistry. HIF-1α and HIF-2α gene expression was determined by real-time PCR. A significant increase in the percentage of apoptosis was observed when cells were treated with 2-ME under a normoxic but not under hypoxic conditions (p=0.006). HIF-1α and HIF-2α protein expression levels were significantly decreased in cells cultured under hypoxic conditions and treated with 2-ME (p<0.001). Furthermore, 2-ME decreased the HIF-1α and HIF-2α nuclear staining in cells cultured under hypoxia. The HIF-1α and HIF-2α mRNA levels were significantly lower when cells were exposed to 2-ME under normoxia and hypoxia. Our results suggest that 2-ME could have beneficial results when used with conventional chemotherapy in an attempt to lower the invasive and metastatic processes during cancer development due to its effects on the gene expression and protein synthesis of HIFs.

A study of the impact of the 21-gene breast cancer assay on the use of adjuvant chemotherapy in women with breast cancer in a Mexican public hospital.

BACKGROUND: The majority of breast cancer patients in Mexico are treated through the public health system and >80% receive adjuvant chemotherapy. The aim of this prospective study was to characterize the impact of the Oncotype DX assay on adjuvant therapy decision making and the confidence in those decisions amongst public sector physicians in Mexico. METHODS: Ninety-eight consecutive patients with ER+, HER2-, stage I-IIIa, N0/N1-3 node-positive breast cancer from the Instituto Nacional de Cancerología were eligible for the study. The primary endpoint was the overall change in treatment recommendations after receiving the assay results. RESULTS: Of 96 patients, 48% received a chemohormonal therapy recommendation prior to testing. Following receipt of results, treatment decisions changed for 31/96 (32%) patients, including 17/62 (27%) node-negative patients and 14/34 (41%) node-positive patients. The proportion of patients with a chemotherapy-based recommendation decreased from 48% pre- to 34% post-assay (P=0.024). 92% of physicians agreed that they were more confident in their treatment recommendation after ordering the assay. CONCLUSIONS: These results suggest that use of the 21-gene assay in the Mexican public health system has a meaningful impact on adjuvant treatment recommendations that may reduce the overall use of chemotherapy.

Idiopathic granulomatous mastitis. Experience at a cancer center.

BACKGROUND: Idiopathic granulomatous mastitis (IGM) is a benign breast disease that has been described as a rare granulomatous inflammation (GI). It can mimic inflammatory breast cancer. MATERIAL AND METHODS: We included women with a diagnosis of IGM referred to an oncologic hospital between January 01, 2007 and to March 31, 2011, with diagnosis of breast cancer, in whom biopsy reported GI, without other cause related. The aim of this study was to review the clinical, radiologic and pathologic characteristics of a cohort of women with IGM. RESULTS: We analyzed 58 patients; mean age was 38 ± 12 years. Mammography showed diffuse asymmetry (n = 19) and focal asymmetry (n = 13); breast ultrasound showed heterogeneous and hypoechoic areas (n = 28) and lumps (n = 21) as the most frequent lesions. All biopsies showed lobulocentric GI. Treatment included antibiotics (n = 20), steroids (n = 8), both treatments (n = 20), surgical excision (n = 3) and observation (n = 7). Forty-three patients (74%) had complete remission; mean time to remission was 9.5 ± 5.8 months. Fifteen (26%) had partial remission. Any patient had progression or relapse. CONCLUSIONS: IGM is a benign breast condition that may mimic breast inflammatory cancer. Ultrasonography and mammography findings reveal characteristic data that can be useful for establishing the diagnosis; however, biopsy is the gold standard for its diagnosis and should be taken in any patient even with a mild suspicion of cancer.

Thalidomide combined with neoadjuvant chemotherapy in angiosarcoma of the breast with complete pathologic response: case report and review of literature.

BACKGROUND: Primary angiosarcoma of the breast is a rare malignancy. CASE REPORT: We report on a 41-year-old female patient who initially presented with locally advanced disease. Core biopsy showed angiosarcoma of the breast, grade 1, CD31-positive. The patient was treated with neoadjuvant systemic chemotherapy based on cisplatin, doxorubicin, and paclitaxel, given concurrently with thalidomide. After treatment completion, the patient underwent radical mastectomy. Pathologic complete response in the breast and axillary lymph nodes was achieved. The patient has no evidence of disease recurrence 6 months after her initial diagnosis. CONCLUSION: Anti-angiogenic therapy may be considered as part of the management of primary angiosarcoma of the breast.

Loss of expression of DNA mismatch repair proteins in aberrant crypt foci identified in vivo by magnifying colonoscopy in subjects with hereditary nonpolyposic and sporadic colon rectal cancer.

PURPOSE: The aims of this study are to characterize the frequency, density, and distribution of aberrant crypt foci (ACF) and its histological features and to determine the frequency of loss of expression of DNA mismatch repair (MMR) proteins of subjects with hereditary nonpolyposic colorectal cancer (HNPCC) and sporadic colon rectal cancer (CRC). METHODS: Patients with HNPCC, first-degree relatives of subjects with HNPCC, sporadic CRC, and average risk subjects of sporadic CRC were included prospectively. Total colonoscopy with chromoendoscopy using methylene blue 0.5% and magnification in the right colon (cecum and 20 cm of the ascending colon) and in the left colon (rectum) was performed; loss of expression of MLH1 and MSH2 was evaluated by immunohistochemistry in confirmed ACF. RESULTS: Fifty-two subjects were included. Thirty-eight of the 119 ACF detected by endoscopy were biopsied. In 14 of the 38 specimens (36.8%), ACF were confirmed by histology (Cohen's kappa, 0.44). In subjects with HNPCC, ACF were identified more frequently in the right segment of the colon than in the left (73.1% vs. 26%); in contrast, ACF predominated in the left segment of the colon (89.3% vs. 10.6%) in subjects with sporadic CRC. There was a loss of MLH1 expression in ACF in subjects with HNPCC. CONCLUSIONS: In HNPCC, we found a greater density of ACF in the right colon, and in sporadic CRC, greater density in the left. ACF present loss in the expression of DNA MMR protein and can be used as an early marker in patients with a risk of HNPCC in whom carcinogenesis appears to be accelerated.

Frequency of high-grade prostatic intraepithelial neoplasia in Mexican population.

BACKGROUND: High-grade intraepithelial neoplasia (HGPIN) is the only lesion regarded as precursor of prostatic carcinoma, though its frequency is unknown in many countries. Here we studied the frequency of HGPIN in a population with high grade frequency of prostatic carcinoma. MATERIAL AND METHODS: A total of 486 cases of sextant prostatic biopsies performed from January 2001 to January 2006 were reviewed. These included 280 biopsies from patients belonging to an urban population, with medium or high socioeconomic status, from two hospitals in Mexico City. For comparison, 206 cases from the Regional Hospital of Tabasco located in the tropical zone of the country were included. This hospital receives patients from a rural population with low income and socioeconomic status. RESULTS: Of the total 486 cases, 162 (33.33%) cases were diagnosed as prostatic carcinoma and 319 (65.64%) as benign conditions. Only in five (1.03%) biopsies was HGPIN found. Three of these patients were from Mexico City, and two from the Regional Hospital of Tabasco. CONCLUSIONS: Even when our results were obtained only in three hospitals, they suggest that a low frequency of HGPIN on needle prostate biopsies does not necessarily mean a low frequency of prostatic carcinoma in the same population. The reason for such a disparity could be related to a reduced extension of HGPIN areas in the prostate gland. In populations with low frequency of HGPIN and high incidence of prostatic carcinoma, perhaps more biopsy cores should be obtained in order to minimize false negative results for premalignant lesions or early adenocarcinoma.