Prognostic Subcellular Notch2, Notch3 and Jagged1 Localization Patterns in Early Triple-negative Breast Cancer.

BACKGROUND: The Notch pathway has been implicated in triple-negative breast cancer (TNBC). Herein, we studied the subcellular localization of the less investigated Notch2 and Notch3 and that of the Jagged1 (Jag1) ligand in patients with operable TNBC. PATIENTS AND METHODS: We applied immunohistochemistry for Notch2, Notch3 and Jag1 in 333 tumors from TNBC patients treated with adjuvant anthracycline-based chemotherapy. We evaluated cytoplasmic (c), membranous (m) and nuclear (n) protein localization. RESULTS: c-Notch2 (35% positive tumors), c-Notch3 (63%), c-Jag1 (43%), m-Notch3 (23%) and n-Jag1 (17%) were analyzed individually and by using hierarchical clustering for prognostic evaluation. Upon multivariate analysis, compared to high m-Notch3 in the absence of n-Jag1 (cluster 4), all other marker combinations (clusters 1, 2, 3) conferred significantly higher risk for relapse (p<0.05). CONCLUSION: Specific Notch3 and Jag1 subcellular localization patterns may provide clues for the behavior of the tumors and potentially for Jag1 targeting in TNBC patients.

Prognostic Evaluation of Epidermal Growth Factor Receptor (EGFR) Genotype and Phenotype Parameters in Triple-negative Breast Cancers.

BACKGROUND: Epidermal growth factor receptor (EGFR) aberrations have been implicated in the pathogenesis of triple-negative breast cancer (TNBC) but their impact on prognosis and, therefore, druggability, remain controversial. Herein, we studied EGFR aberrations at different molecular levels and assessed their prognostic impact in patients with operable TNBC treated with adjuvant anthracycline-based chemotherapy. MATERIALS AND METHODS: We evaluated the prognostic impact of EGFR gene status by fluorescent in situ hybridization (FISH), EGFR coding mutations by Sanger and next-generation sequencing, relative EGFR messenger RNA (mRNA) levels by qPCR (upper quartile) and EGFR and p53 protein expression by immunohistochemistry (IHC), in 352 centrally-assessed tumors from an equal number of TNBC patients. RESULTS: Approximately 53.5% of the tumors expressed EGFR, 59.3% p53 and 35.9% both EGFR and p53 proteins; 4.1% showed EGFR gene amplification and 4.4% carried EGFR mutations. The latter were located outside the druggable kinase domain region and presented at low frequencies. Amplification and mutations overlapped only in one case of glycogen-rich carcinoma. EGFR and CEN7 copies were higher in tumors from older patients (p=0.002 and p=0.003, respectively). Patients with amplified tumors (n=11) had excellent prognosis (0 relapses and deaths). Upon multivariate analysis, high EGFR copies conferred significantly favorable disease-free survival (HR=0.57, 95% CI 0.36-0.90, Wald's p=0.017) and high CEN7 copies favorable overall survival (HR=0.49, 95% CI=0.29-0.83, Wald's p=0.008). Patients with EGFR-/p53+ and EGFR+/p53- tumors had significantly higher risk for relapse than those with EGFR-/p53- and EGFR+/p53+ tumors (HR=1.73, 95% CI=1.12-2.67, Wald's p=0.013). CONCLUSION: EGFR gene amplification and mutations are rare in TNBC, the latter of no apparent clinical relevance. Surrogate markers of EGFR-related chromosomal aberrations and combined EGFR/p53 IHC phenotypes appear to be associated with favorable prognosis in patients with operable TNBC receiving conventional adjuvant chemotherapy.

The fate of BRCA1-related germline mutations in triple-negative breast tumors.

The preservation of pathogenic BRCA1/2 germline mutations in tumor tissues is usually not questioned, while it remains unknown whether these interact with somatic genotypes for patient outcome. Herein we compared germline and tumor genotypes in operable triple-negative breast cancer (TNBC) and evaluated their combined effects on prognosis. We analyzed baseline germline and primary tumor genotype data obtained by Sanger and Next Generation Sequencing in 194 TNBC patients. We also performed multiple tests interrogating the preservation of germline mutations in matched tumors and breast tissue from carriers with available material. Patients had been treated within clinical trials with adjuvant anthracyclines-taxanes based chemotherapy. We identified 50 (26%) germline mutation carriers (78% in BRCA1) and 136 (71%) tumors with somatic mutations (83% in TP53). Tumor mutation patterns differed between carriers and non-carriers (P<0.001); PIK3CA mutations were exclusively present in non-carriers (P=0.007). Germline BRCA1/2 mutations were not detected in matched tumors and breast tissues from 14 out of 33 (42%) evaluable carriers. Microsatellite markers revealed tumor loss of the germline mutant allele in one case only. Tumors that had lost the germline mutation demonstrated a higher incidence of somatic TP53 mutations as compared to tumors with preserved germline mutations (P=0.036). Germline mutation status significantly interacted with tumor TP53 mutations for patient disease-free survival (interaction P=0.026): In non-carriers, tumor TP53 mutations did not affect outcome; In carriers, those with mutated TP53 tumors experienced more relapses compared to those with wild-type TP53 tumors (36% vs. 9% relapse rate, respectively). In conclusion, we show that loss of germline BRCA1/2 mutations is not a rare event in TNBC. This finding, the observed differences in tumor genotypes with respect to germline status and the prognostic interaction between germline BRCA1-related and tumor TP53 mutation status prompt for combined germline and tumor genotyping for the classification of TNBC, particularly in the context of clinical trials evaluating synthetic lethality drugs.

Tumor Infiltrating Lymphocytes Affect the Outcome of Patients with Operable Triple-Negative Breast Cancer in Combination with Mutated Amino Acid Classes.

BACKGROUND: Stromal tumor infiltrating lymphocytes (TILs) density is an outcome predictor in triple-negative breast cancer (TNBC). Herein we asked whether TILs are related to coding mutation load and to the chemical class of the resulting mutated amino acids, i.e., charged, polar, and hydrophobic mutations. METHODS: We examined paraffin tumors from TNBC patients who had been treated with adjuvant chemotherapy mostly within clinical trials (training cohort, N = 133; validation, N = 190) for phenotype concordance; TILs density; mutation load and types. RESULTS: Concordance of TNBC phenotypes was 42.1% upon local / central, and 72% upon central / central pathology assessment. TILs were not associated with mutation load, type and class of mutated amino acids. Polar and charged mutation patterns differed between TP53 and PIK3CA (p<0.001). Hydrophobic mutations predicted for early relapse in patients with high nodal burden and <50% TILs tumors (training: HR 3.03, 95%CI 1.11-8.29, p = 0.031; validation: HR 2.90, 95%CI 0.97-8.70, p = 0.057), especially if compared to patients with >50% TILs tumors (training p = 0.003; validation p = 0.015). CONCLUSIONS: TILs density is unrelated to mutation load in TNBC, which may be regarded as an unstable phenotype. If further validated, hydrophobic mutations along with TILs density may help identifying TNBC patients in higher risk for relapse.

Effects of TP53 and PIK3CA mutations in early breast cancer: a matter of co-mutation and tumor-infiltrating lymphocytes.

The purpose of this study is to investigate whether the outcome of breast cancer (BC) patients treated with adjuvant chemotherapy is affected by co-mutated TP53 and PIK3CA according to stromal tumor-infiltrating lymphocytes (TILs). Paraffin tumors of all clinical subtypes from 1661 patients with operable breast cancer who were treated within 4 adjuvant trials with anthracycline-taxanes chemotherapy were informative for TP53 and PIK3CA mutation status (semiconductor sequencing genotyping) and for stromal TILs density. Disease-free survival (DFS) was examined. TP53 mutations were associated with higher (p < 0.001) and PIK3CA with lower (p = 0.004) TILs in an ER /PgR-specific manner (p < 0.001). Mutations did not affect the favorable DFS of patients with lymphocyte-predominant (LP) BC. Within non-LPBC, PIK3CA-only mutations conferred best, while TP53-PIK3CA co-mutations (6 % of all tumors) conferred worst DFS (HR 0.59; 95 % CI 0.44-0.79; p = 0.001 for PIK3CA-only). TP53-only mutations were unfavorable in patients with lower TILs, while patients with lower TILs performed worse if their tumors carried TP53-only mutations (interaction p = 0.046). Multivariate analysis revealed favorable PIK3CA-only mutations in non-LPBC (HR 0.64; 95 % CI 0.47-0.88; p = 0.007), and unfavorable TP53 mutations in ER/PgRpos/HER2neg (HR 1.55; 95 % CI 1.07-2.24; p = 0.021). Mutations did not interact with TILs in non-LP triple-negative and HER2-positive patients. TP53 and PIK3CA mutations appear to have diverse effects on the outcome of early BC patients, according to whether these genes are co-mutated or not, and for TP53 according to TILs density and ER/PgR-status. These findings need to be considered when evaluating the effect of these two most frequently mutated genes in the context of large clinical trials.

Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens.

BACKGROUND: MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements. METHODS: Tumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content. RESULTS: Individual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and 0.20 Cqs accompanied by >94 % concordant single marker assignments for all four markers. In platform 2, the inter-site SD estimates were between 0.40 and 0.66 Cqs while the concordance for single marker assignments was >94 % for all four markers. The agreement reached between the two qPCR systems located in one site was 100 % for ERBB2, 96.9 % for ESR1, 97.2 % for PGR and 98.6 % for MKI67. RT-qPCR for individual markers was stable up to a 64-fold dilution for a typical clinical sample. There was no change in assay performance detected at the level of individual markers or subtypes after using different RNA isolation methods. The presence of up to 80 % of surrounding non-tumor tissue including in situ carcinoma did not affect the assay output. Sixteen out of 20 RNXtract eluates yielded more than 50 ng/µl of RNA (average RNA output: 233 ng/µl), whereas DNA contamination per sample was restricted to less than 15 ng/µl. Median recovery rate of RNA extraction was 91.0 %. CONCLUSIONS: In this study the performance characteristics of MammaTyper were successfully validated. The various sources of analytical perturbations resulted in negligible variations in individual marker assessments. Therefore, MammaTyper may serve as a technical improvement to current standards for decentralized FFPE-based routine assessment of the commonly used breast cancer biomarkers and for molecular subtyping of breast cancer specimens.

Biological subtyping of early breast cancer: a study comparing RT-qPCR with immunohistochemistry.

The biological subtype of breast cancer influences the selection of systemic therapy. Distinction between luminal A and B cancers depends on consistent assessment of Ki-67, but substantial intra-observer and inter-observer variability exists when immunohistochemistry (IHC) is used. We compared RT-qPCR with IHC in the assessment of Ki-67 and other standard factors used in breast cancer subtyping. RNA was extracted from archival breast tumour tissue of 769 women randomly assigned to the FinHer trial. Cancer ESR1, PGR, ERBB2 and MKI67 mRNA content was quantitated with an RT-qPCR assay. Local pathologists assessed ER, PgR and Ki-67 expression using IHC. HER2 amplification was identified with chromogenic in situ hybridization (CISH) centrally. The results were correlated with distant disease-free survival (DDFS) and overall survival (OS). qPCR-based and IHC-based assessments of ER and PgR showed good concordance. Both low tumour MKI67 mRNA (RT-qPCR) and Ki-67 protein (IHC) levels were prognostic for favourable DDFS [hazard ratio (HR) 0.42, 95 % CI 0.25-0.71, P = 0.001; and HR 0.56, 0.37-0.84, P = 0.005, respectively] and OS. In multivariable analyses, cancer MKI67 mRNA content had independent influence on DDFS (adjusted HR 0.51, 95 % CI 0.29-0.89, P = 0.019) while Ki-67 protein expression had not any influence (P = 0.266) whereas both assessments influenced independently OS. Luminal B patients treated with docetaxel-FEC had more favourable DDFS and OS than those treated with vinorelbine-FEC when the subtype was defined by RT-qPCR (for DDFS, HR 0.52, 95 % CI 0.29-0.94, P = 0.031), but not when defined using IHC. Breast cancer subtypes approximated with RT-qPCR and IHC show good concordance, but cancer MKI67 mRNA content correlated slightly better with DDFS than Ki-67 expression. The findings based on MKI67 mRNA content suggest that patients with luminal B cancer benefit more from docetaxel-FEC than from vinorelbine-FEC.

TP53 mutations and protein immunopositivity may predict for poor outcome but also for trastuzumab benefit in patients with early breast cancer treated in the adjuvant setting.

BACKGROUND: We investigated the impact of PIK3CA and TP53 mutations and p53 protein status on the outcome of patients who had been treated with adjuvant anthracycline-taxane chemotherapy within clinical trials in the pre- and post-trastuzumab era. RESULTS: TP53 and PIK3CA mutations were found in 380 (21.5%) and 458 (25.9%) cases, respectively, including 104 (5.9%) co-mutated tumors; p53 immunopositivity was observed in 848 tumors (53.5%). TP53 mutations (p < 0.001) and p53 protein positivity (p = 0.001) were more frequent in HER2-positive and triple negative (TNBC) tumors, while PIK3CA mutations were more frequent in Luminal A/B tumors (p < 0.001). TP53 mutation status and p53 protein expression but not PIK3CA mutation status interacted with trastuzumab treatment for disease-free survival; patients with tumors bearing TP53 mutations or immunopositive for p53 protein fared better when treated with trastuzumab, while among patients treated with trastuzumab those with the above characteristics fared best (interaction p = 0.017 for mutations; p = 0.015 for IHC). Upon multivariate analysis the above interactions remained significant in HER2-positive patients; in the entire cohort, TP53 mutations were unfavorable in patients with Luminal A/B (p = 0.003) and TNBC (p = 0.025); p53 immunopositivity was strongly favorable in patients treated with trastuzumab (p = 0.009). MATERIALS AND METHODS: TP53 and PIK3CA mutation status was examined in 1766 paraffin tumor DNA samples with informative semiconductor sequencing results. Among these, 1585 cases were also informative for p53 protein status assessed by immunohistochemistry (IHC; 10% positivity cut-off). CONCLUSIONS: TP53 mutations confer unfavorable prognosis in patients with Luminal A/B and TNBC tumors, while p53 immunopositivity may predict for trastuzumab benefit in the adjuvant setting.

Genes associated with histopathologic features of triple negative breast tumors predict molecular subtypes.

Distinct subtypes of triple negative (TN) breast cancer have been identified by tumor expression profiling. However, little is known about the relationship between histopathologic features of TN tumors, which reflect aspects of both tumor behavior and tumor microenvironment, and molecular TN subtypes. The histopathologic features of TN tumors were assessed by central review and 593 TN tumors were subjected to whole genome expression profiling using the Illumina Whole Genome DASL array. TN molecular subtypes were defined based on gene expression data associated with histopathologic features of TN tumors. Gene expression analysis yielded signatures for four TN subtypes (basal-like, androgen receptor positive, immune, and stromal) consistent with previous studies. Expression analysis also identified genes significantly associated with the 12 histological features of TN tumors. Development of signatures using these markers of histopathological features resulted in six distinct TN subtype signatures, including an additional basal-like and stromal signature. The additional basal-like subtype was distinguished by elevated expression of cell motility and glucose metabolism genes and reduced expression of immune signaling genes, whereas the additional stromal subtype was distinguished by elevated expression of immunomodulatory pathway genes. Histopathologic features that reflect heterogeneity in tumor architecture, cell structure, and tumor microenvironment are related to TN subtype. Accounting for histopathologic features in the development of gene expression signatures, six major subtypes of TN breast cancer were identified.

Tumors with high-density tumor infiltrating lymphocytes constitute a favorable entity in breast cancer: a pooled analysis of four prospective adjuvant trials.

BACKGROUND: Tumor infiltrating lymphocytes (TILs) are considered in the prognosis of breast cancer (BC) patients. Here, we investigated the prognostic/predictive effect of TILs in patients treated in the frame of four prospective trials with adjuvant anthracycline-based chemotherapy in the pre- and post-trastuzumab era. METHODS: TILs density was histologically assessed as percentage of stromal area on whole routine sections of 2613 BC (1563 Luminal A/B; 477 Luminal HER2; 246 HER2-enriched; 327 triple negative [TNBC]) and were evaluated as high/low at three cut-offs (c/o; 50% [lymphocytic predominance, LP], 35% and 25%), in separate training and validation sets. RESULTS: High TILs were present in 3.5%, 6.5% and 11.5% of all tumors, using the 50%, 35% and 25% c/o, respectively. TILs status did not interact with BC subtypes or trastuzumab treatment. LPBC patient outcome was not affected by nodal status, while high TILs were favorable in TNBC with unfavorable nodal status. When adjusted for standard clinicopathological parameters and treatment, high TILs independently predicted for favorable outcome, e.g., disease-free survival with the 35% c/o in the entire cohort (HR = 0.44, 95% CI 0.28-0.69, p < 0.001) and in specific subtypes. CONCLUSIONS: High TILs tumors, especially LPBC seem worthy validating as a separate entity of favorable prognosis in breast cancer.

Evaluation of two highly-multiplexed custom panels for massively parallel semiconductor sequencing on paraffin DNA.

AIM: Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA. METHODS: Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible. RESULTS: Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman's rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8-99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1-95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3-76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality. CONCLUSIONS: Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.

Efficiency of different decalcification protocols for nasal osseous structures in a rat experimental model of allergic rhinitis, and their effects on epithelial histology: an attempt at standardization.

INTRODUCTION: Decalcification of osseous specimens is required for histological analysis; this however may cause tissue damage. In rodent models of allergic rhinitis (AR), epithelial histologic assessment necessitates prior decalcification of the nasal osseous structures. However, respiratory epithelium is highly susceptible to damage, and rat nasal architecture is elaborate and its sectioning is challenging. Nevertheless, decalcification is not standardized in experimental AR. We therefore undertook this task, in order to reduce experimental bias. METHODS: Six-to-eight week-old Wistar rats underwent an AR protocol. Subsequently, nasal structures were decalcified in the following mediums: (i) formic acid 10% for 5 and 20 days; (ii) formic acid 15% for 5 and 15 days; (iii) Morse Solution for 5 and 20 days and (iv) EDTA for 20 and 40 days. Decalcification efficiency/speed was evaluated via radiographic analysis. Furthermore, specimens were stained with hematoxylin and eosin and assessed for preservation of epithelial features. RESULTS: Specimens were appropriately decalcified in 5 days in the formic acid-based mediums and in 20 days in EDTA with minimal epithelial damage. EDTA for 40 days had no unacceptable adverse effects; conversely, 15 and/or 20 days in acid-based agents provided no extra benefit for decalcification and were detrimental to the epithelium. CONCLUSIONS: EDTA treatment for 20 days is appropriate for decalcification of nasal structures in rat models of allergic rhinitis; further incubation preserves epithelial integrity but is not required. When urgency is a factor, formic-acid-based decalcification for 5 days yields acceptable results.

The androgen receptor as a surrogate marker for molecular apocrine breast cancer subtyping.

The Androgen Receptor (AR) is a potential prognostic marker and therapeutic target in breast cancer. We evaluated AR protein expression in high-risk breast cancer treated in the adjuvant setting. Tumors were subtyped into luminal (ER+/PgR±/AR±), molecular apocrine (MAC, [ER-/PgR-/AR+]) and hormone receptor negative carcinomas (HR-negative, [ER-/PgR-/AR-]). Subtyping was evaluated with respect to prognosis and to taxane therapy. High histologic grade (p < 0.001) and increased proliferation (p = 0.001) more often appeared in MAC and HR-negative than in luminal tumors. Patients with MAC had outcome comparable to the luminal group, while patients with HR-negative disease had increased risk for relapse and death. MAC outcome was favorable upon taxane-containing treatment; this remained significant upon multivariate analysis for overall survival (HR 0.31, 95%CI 0.13-0.74, interaction p = 0.035) and as a trend for time to relapse (p = 0.15). In conclusion, AR-related subtyping of breast cancer may be prognostic and serve for selecting optimal treatment combinations.

Genome-wide association study identifies 25 known breast cancer susceptibility loci as risk factors for triple-negative breast cancer.

Triple-negative (TN) breast cancer is an aggressive subtype of breast cancer associated with a unique set of epidemiologic and genetic risk factors. We conducted a two-stage genome-wide association study of TN breast cancer (stage 1: 1529 TN cases, 3399 controls; stage 2: 2148 cases, 1309 controls) to identify loci that influence TN breast cancer risk. Variants in the 19p13.1 and PTHLH loci showed genome-wide significant associations (P < 5 × 10(-) (8)) in stage 1 and 2 combined. Results also suggested a substantial enrichment of significantly associated variants among the single nucleotide polymorphisms (SNPs) analyzed in stage 2. Variants from 25 of 74 known breast cancer susceptibility loci were also associated with risk of TN breast cancer (P < 0.05). Associations with TN breast cancer were confirmed for 10 loci (LGR6, MDM4, CASP8, 2q35, 2p24.1, TERT-rs10069690, ESR1, TOX3, 19p13.1, RALY), and we identified associations with TN breast cancer for 15 additional breast cancer loci (P < 0.05: PEX14, 2q24.1, 2q31.1, ADAM29, EBF1, TCF7L2, 11q13.1, 11q24.3, 12p13.1, PTHLH, NTN4, 12q24, BRCA2, RAD51L1-rs2588809, MKL1). Further, two SNPs independent of previously reported signals in ESR1 [rs12525163 odds ratio (OR) = 1.15, P = 4.9 × 10(-) (4)] and 19p13.1 (rs1864112 OR = 0.84, P = 1.8 × 10(-) (9)) were associated with TN breast cancer. A polygenic risk score (PRS) for TN breast cancer based on known breast cancer risk variants showed a 4-fold difference in risk between the highest and lowest PRS quintiles (OR = 4.03, 95% confidence interval 3.46-4.70, P = 4.8 × 10(-) (69)). This translates to an absolute risk for TN breast cancer ranging from 0.8% to 3.4%, suggesting that genetic variation may be used for TN breast cancer risk prediction.

The prognostic significance of WNT pathway in surgically-treated colorectal cancer: ß-catenin expression predicts for disease-free survival.

BACKGROUND: The wingless-type MMTV integration site family of proteins (WNT) pathway is highly involved in colorectal cancer development. The aim of this study was to explore the prognostic significance and clinicopatological correlations of this pathway in a cohort of surgically-treated patients with non-metastatic colorectal cancer in relation to the site of expression of pathway proteins. MATERIALS AND METHODS: Immunohistochemical expression of nuclear cyclin D1, membranous E-cadherin and P-cadherin, membranous and nuclear ß-catenin in the invasive front (IF), the tumor center (TC), as well as their mean, were assessed in 106 paraffin-embedded tissue samples. Adenomatous Polyposis Coli (APC), Axin-2 (AXIN2), cyclin-D1 (CCND1), Matrix Metalloproteinase-7 (MMP7), Secreted Frizzled Related Protein (SFRP) 1, 2 and 4 and WNT5A were evaluated by RT PCR. RESULTS: Membranous ß-catenin expression was statistically reduced in the IF. Cyclin-D1 was reduced in tumors arising closer to the rectum. Reduced nuclear expression of cyclin-D1 in the IF was associated with lymphatic, venous and perineural invasion. Loss of membranous ß-catenin in the TC was more common among N2 tumors. Higher SFRP4 mRNA was associated with advanced T stage. In univariate analysis, membranous expression of ß-catenin in TC and IF, and their mean, was associated with longer disease-free survival (DFS). In multivariate analysis, tumor stage and mean ß-catenin expression were prognostic for longer DFS (hazard ratio=0.33; p=0.01). ß-Catenin expression in the IF remained significant when the mean expression was not included in the multivariate analysis (hazard ratio=0.41; p=0.028). CONCLUSION: Mean membranous expression of ß-catenin, as well as that in the IF, is prognostic for longer DFS in patients with non metastatic colorectal cancer.

Papillary cystadenoma of epididymis: Is there a need for further investigation in unilateral cases?

INTRODUCTION: The presence of a mass in the epididymis is not a common entity. The papillary cystadenoma of epididymis is a benign tumor which may occur sporadically or as a characteristic of von Hippel-Lindau disease. PRESENTATION OF CASE: We present a case of a 27-year-old man with a right scrotal mass who was treated with surgical excision. Histopathological examination revealed a clear cell epididymal papillary cystadenoma. A computed tomography scan that was performed later showed no other abnormality or any signs of von Hippel-Lindau disease. DISCUSSION: In this report, a case of a young man suffering from this rare tumor is discussed, focusing on the need of further evaluation in order to determinate if it occurs as a feature of VHL disease or as a sporadic form. CONCLUSION: In unilateral cases of papillary cystadenoma of epididymis such as our patient's, literature advocates that no further examinations and expensive genetic testing is required.

AMACR is associated with advanced pathologic risk factors in sporadic colorectal adenomas.

AIM: To analyze alpha-methylacyl CoA racemase (AMACR) expression in relation to various dysplasia phenotypes and clinicopathological parameters of sporadic colorectal adenomas. METHODS: Fifty-five cases of sporadic colorectal adenomas were categorized according to the Vienna classification for Gastrointestinal Neoplasia. These corresponded to a total of 98 different intra-lesion microscopic fields that were further independently assigned a histological grade based on the old nomenclature (mild, moderate, severe dyplasia and carcinoma in situ). AMACR expression was evaluated by immunohistochemistry and statistical analysis was performed to investigate possible associations with various clinicopathologic parameters of adenomas i.e. gender, age, localization, grade of dysplasia, size and configuration. RESULTS: Patient age ranged from 41 to 84 years (mean 65 +/- 13.2 years); 37 patients were males and 18 were females. Adenomas ranged in size between 0.5 and 30 cm (mean 2 +/- 1.3 cm), including 18 tubular, 16 villous, 20 mixed or tubulovillous, and 1 giant sessile villous adenoma. AMACR expression was observed in 3 out of 16 (18.8%) of low-grade vs 23 out of 35 (62.8%) of high-grade lesions (P = 0.002). Most adenomas exhibiting high grade dysplasia with in situ carcinoma-like areas stained positive for AMACR (15/17 or 88.2%) as compared to adenomas with high grade dysplasia which contained severe dysplasia-like foci (6/15 or 40%), (P = 0.005). In AMACR positive adenomas featuring severe dysplasia-like or in situ carcinoma-like areas, AMACR staining was not necessarily observed in the in situ component. Positivity in intra-lesion of mild, moderate or severe dysplasia-like foci was more often encountered in adenomas harboring in situ, intramucosal or infiltrative carcinoma [21/33 (63.6%) vs 9/40 (22.5%), P < 0.001]. Strong AMACR expression was found in 11 out of 17 villous adenomas, but in only 1 out of 18 tubular lesions (P = 0.005). Larger lesions, i.e. > 1 cm stained more frequently for AMACR than smaller ones [27/45 (60%) vs 2/10 (20%), P = 0.02]. Overall, AMACR expression was associated with the grade of dysplasia, as well as with the size and configuration of adenomas, i.e. the consensus risk factors applied to colorectal adenoma patient surveillance. CONCLUSION: It may be worthy to further evaluate the possible use of AMACR as an additional risk factor for the assessment of colorectal adenoma patients.

Spontaneous perforation of solitary ulcer of transverse colon.

Spontaneous ruptures of the colon and rectum are extremely uncommon clinical entities and always require laparotomy. A 44-year-old female was admitted with a 12-hour history of severe abdominal pain periumbilically and at the right hypochondrium. The patient was immediately transferred to the department of surgery for close surgical observation. Computed tomography (CT) of the entire abdomen performed just before the operation demonstrated thickening of the wall of the ascending colon with pericolic fat stranding. Surgery revealed a perforation at the antimesenteric wall of the transverse colon and segmental colectomy of the transverse colon was performed. The histological evaluation demonstrated a perforated solitary ulcer of the transverse colon. There are only few known etiologic factors concerning spontaneous ruptures of the colon and rectum and usually none of these causative factors can easily be recognised. Their clinical appearance is most of the times acute abdomen and, despite the use of all appropriate diagnostic methods, the diagnosis is usually set postoperatively.

Benign spindle cell tumor not otherwise specified (NOS) in a male breast.

Breast spindle cell tumors (BSCTs), although uncommon, constitute a heterogeneous group of benign and malignant lesions, often necessitating different therapeutic approaches. This study describes the case of a 62-year-old man who displayed a gradually growing retroareolar tumor of the left breast. The mass was well circumscribed, unilateral, and grossly nodular. The patient eventually underwent wide local excision of the mass. The lesion was made up of spindle cells arranged in fascicular clusters, separated by bands of collagen. No mitotic figures were observed. Immunohistochemically, the mass expressed strong and diffuse cytoplasmic staining for vimentin, CD34, CD10, and bcl-2, whereas it was negative for cytokeratins, smooth muscle actin, desmin, S-100 protein, p53, Ki-67, estrogen and progesterone receptors. Diverse histological results and immunohistochemical features established the diagnosis of benign BSCT, not otherwise specified. The patient remains disease-free 12 months after lumpectomy. This case report adds to the spectrum of the benign BSCTs and delineates the nature of different types of these lesions, in order to carefully select optimal therapeutic regimes.