[Show abstract][Hide abstract] ABSTRACT: Background:
The PI3K-AKT pathway is frequently activated in breast cancer. PIK3CA mutations are most frequently found in the helical (exon 9) and kinase (exon 20) domains of this protein. The aim of the present study was to examine the role of different types of PIK3CA mutations in combination with molecular biomarkers related to PI3K-AKT signaling in patients with early breast cancer.
Tumor tissue samples from 1008 early breast cancer patients treated with adjuvant chemotherapy in two similar randomized trials of HeCOG were examined. Tumors were subtyped with immunohistochemistry (IHC) and FISH for ER, PgR, Ki67, HER2 and androgen receptor (AR). PIK3CA mutations were analyzed by Sanger sequencing (exon 20) and qPCR (exon 9) (Sanger/qPCR mutations). In 610 cases, next generation sequencing (NGS) PIK3CA mutation data were also available. PIK3CA mutations and PTEN protein expression (IHC) were analyzed in luminal tumors (ER and/or PgR positive), molecular apocrine carcinomas (MAC; ER/PgR negative / AR positive) and hormone receptor (ER/PgR/AR) negative tumors.
PIK3CA mutations were detected in 235/1008 tumors (23%) with Sanger/qPCR and in 149/610 tumors (24%) with NGS. Concordance between the two methods was good with a Kappa coefficient of 0.76 (95% CI 0.69-0.82). Lobular histology, low tumor grade and luminal A tumors were associated with helical domain mutations (PIK3CAhel), while luminal B with kinase domain mutations (PIK3CAkin). The overall incidence of PIK3CA mutations was higher in luminal as compared to MAC and hormone receptor negative tumors (p = 0.004). Disease-free and overall survival did not significantly differ with respect to PIK3CA mutation presence and type. However, a statistically significant interaction between PIK3CA mutation status and PTEN low protein expression with regard to prognosis was identified.
The present study did not show any prognostic significance of specific PIK3CA mutations in a large group of predominantly lymph-node positive breast cancer women treated with adjuvant chemotherapy. Further analyses in larger cohorts are warranted to investigate possible differential effect of distinct PIK3CA mutations in small subgroups of patients.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND—
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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Anaplastic lymphoma kinase (ALK) break-apart fluorescent in situ hybridization (FISH) is currently used in diagnostics for the selection of non-small cell lung cancer (NSCLC) patients to receive crizotinib. We evaluated ALK status in NSCLC with a novel ALK mRNA test based on the break-apart FISH concept, which we called break-apart transcript (BAT) test. ALK5' and ALK3' transcript patterns were established with qPCR for ALK-expressing controls including fusion-negative neuroblastomas, as well as fusion-positive anaplastic large cell lymphomas and NSCLC. The BAT test was evaluated on 271 RNA samples from routinely processed paraffin NSCLC tissues. Test results were compared with ALK FISH (n=121), immunohistochemical (IHC) analysis (n=86), and automated quantitative analysis (AQUA, n=83). On the basis of the nonoverlapping ALK BAT patterns in ALK-expressing controls (P<0.0001), 8/174 adenocarcinomas (4.6%) among 259 informative NSCLC were predicted as fusion positive. Overall concordance for paired method results was high (94.1% to 98.8%) but mainly concerned negative prediction because of the limited availability of positive-matched cases. Tumors with 100% cytoplasmic IHC staining of any intensity (n=3) were positive for AQUA, FISH, and BAT test; tumors with lower IHC positivity and different staining patterns were AQUA-negative. Upon multiple reevaluations, ALK gene status was considered as originally misinterpreted by FISH in 3/121 cases (2.5%). Tumors with >4 ALK gene copies were associated with longer overall survival upon first-line chemotherapy. In conclusion, application of the ALK BAT test on routinely processed NSCLC tissues yields the same fusion partner independent information as ALK break-apart FISH but is more robust and cost-effective. The BAT concept may be considered for the development of further drug-predictive translocation tests.
No preview · Article · Aug 2014 · Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry
[Show abstract][Hide abstract] ABSTRACT: Background
HER2 and TOP2A gene status are assessed for diagnostic and research purposes in breast cancer with fluorescence in situ hybridization (FISH). However, FISH probes do not target only the annotated gene, while chromosome 17 (chr17) is among the most unstable chromosomes in breast cancer. Here we asked whether the status of specifically targeted genes on chr17 might help in refining prognosis of early high-risk breast cancer patients.
Copy numbers (CN) for 14 genes on chr17, 4 of which were within and 10 outside the core HER2 amplicon (HER2- and non-HER2-genes, respectively) were assessed with qPCR in 485 paraffin-embedded tumor tissue samples from breast cancer patients treated with adjuvant chemotherapy in the frame of two randomized phase III trials.
HER2-genes CN strongly correlated to each other (Spearman’s rho >0.6) and were concordant with FISH HER2 status (Kappa 0.6697 for ERBB2 CN). TOP2A CN were not concordant with TOP2A FISH status (Kappa 0.1154). CN hierarchical clustering revealed distinct patterns of gains, losses and complex alterations in HER2- and non-HER2-genes associated with IHC4 breast cancer subtypes. Upon multivariate analysis, non-HER2-gene gains independently predicted for shorter disease-free survival (DFS) and overall survival (OS) in patients with triple-negative cancer, as compared to luminal and HER2-positive tumors (interaction p = 0.007 for DFS and p = 0.011 for OS). Similarly, non-HER2-gene gains were associated with worse prognosis in patients who had undergone breast-conserving surgery as compared to modified radical mastectomy (p = 0.004 for both DFS and OS). Non-HER2-gene losses were unfavorable prognosticators in patients with 1–3 metastatic nodes, as compared to those with 4 or more nodes (p = 0.017 for DFS and p = 0.001 for OS).
TOP2A FISH and qPCR may not identify the same pathology on chr17q. Non-HER2 chr17 CN patterns may further predict outcome in breast cancer patients with known favorable and unfavorable prognosis.
[Show abstract][Hide abstract] ABSTRACT: Cancer of unknown primary origin (CUP) had a poor prognosis, determined by clinico-histological characteristics, partly due to the lack of insights on its biology. We screened tumour DNA from 87 patients with CUP for CTNNB1 (coding exons 2,3,4,5), MET (coding exon 18), PIK3CA (coding exons 9,20), KRAS (coding exons 1,2), BRAF (coding exon 15) gene mutations by using dd-sequencing and evaluated their impact on prognosis. Mutated gene incidences in the 87 CUP cases were: KRAS 11 (12.6 %), BRAF 5 (5.7 %), PIK3CA 8 (9 %), MET 6 (6.7 %) and CTNNB1 18 (20.7 %). Several mutations in the KRAS gene were not the commonly encountered mutations in other solid tumours. Activating mutations were observed in 10.2 % in KRAS, 4.5 % in BRAF, 6.6 % in PIK3CA, 4.5 % in MET, and 19.5 % in CTNNB1. Activating mutations in PIK3CA coding exon 9 were inversely correlated with MET coding exon 18 activating mutations (p = 0.036). MET activating mutations were prognostic for poor Progression-Free Survival (median PFS 5 vs 9 months, p = 0.009) and Overall Survival (median OS 7 vs 20 months, p = 0.005). The complex profile of either CTNNB1 or MET mutations also had an adverse prognostic significance (median OS 11 vs 21 months, p = 0.015). No other gene mutation exhibited prognostic significance. In multivariate analysis, poor performance status, male gender, visceral disease and adenocarcinoma histology, but not gene mutations, were independently associated with poor patient outcome. CTNNB1 gene mutations are frequent, and along with MET mutations have an adverse prognostic effect in patients with CUP.
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Recurrent pregnancy loss (RPL) of unknown etiology is correlated with immunological alterations during pregnancy. Normally, changes in leukocyte subpopulations and HLA expression take place in pregnant uterus in order to tolerate the semi-allogenic embryo.
Our research tries to enlighten the immunological changes that take place in the uterus of women with recurrent abortions of unknown etiology during first trimester of pregnancy.
Materials and methods:
The miscarriage group was obtained from 25 women who miscarried between the ages of 35 to 42 years and controls consisted of 25 healthy women between the ages of 27 to 39 years, who had electively terminated their pregnancies during the first trimester of pregnancy. The abortion was processed and specimens taken were studied using immunohistochemical methods. Specimens were taken from decidua basalis and decidua parietalis. Monoclonal antibodies were used against HLAG (Human Leukocyte Antigen G), CD68( Cluster of Differentiation 68), CD56, CD16 and CD25. The results were statistically analysed with Mann-Whitney test.
HLA-G expression in decidua basalis from miscarriage group was found to be decreased. CD25+ cell expression was found to be invariable in deciduas from both groups. CD16+ cell and CD68 + cell expression was found to be increased in deciduas from the miscarriage group. CD56+ cell expression was found to be increased in decidua parietalis from miscarriage group. Conclusion : Several differences in the immunological profile of deciduas from RPL group were observed. Changes in feto-protective HLA-G expression and a possible implication of macrophages and NK cells were found.
No preview · Article · Feb 2014 · Histology and histopathology
[Show abstract][Hide abstract] ABSTRACT: 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.
Immunohistochemical expression of nuclear cyclin D1, membranous E-cadherin and P-cadherin, membranous and nuclear β-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.
Membranous β-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 β-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 β-catenin in TC and IF, and their mean, was associated with longer disease-free survival (DFS). In multivariate analysis, tumor stage and mean β-catenin expression were prognostic for longer DFS (hazard ratio=0.33; p=0.01). β-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).
Mean membranous expression of β-catenin, as well as that in the IF, is prognostic for longer DFS in patients with non metastatic colorectal cancer.
No preview · Article · Oct 2013 · Anticancer research
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to evaluate the association of vascular endothelial growth factor (VEGF) genotypes with treatment efficacy in a phase II trial. This study evaluated weekly docetaxel, as first-line treatment for metastatic breast cancer. Existing data from in vitro and animal model experiments suggest that docetaxel at low doses has anti-angiogenic activity. DNA was extracted from blood samples of 86 patients participating in the trial. Genotyping was performed for selected single-nucleotide polymorphisms (SNPs; VEGF-2578, -1498, -1154, and +936). Moreover, due to the highly polymorphic nature of the studied areas, we were able to analyze additional registered SNPs. All candidate genotypes were evaluated for associations with overall survival (OS), progression-free survival (PFS) and response rate. The VEGF-1154 GG genotype was more frequent in patients not responding to treatment compared with responders (42.9% vs 0.0%, P=0.048). Moreover, the VEGF-2578 AA genotype was associated with longer PFS compared with CC (hazard ratio (HR)=0.40; 95% confidence interval (CI) 0.17-0.98; pairwise P=0.0457). Patients with the VEGF-1190 GG genotype demonstrated shorter PFS compared with those with the alternative genotypes (GA and AA) combined (HR=3.85; 95% CI: 1.20-12.50; P=0.0224). In addition, the VEGF-2551/-2534 homozygous del18bp and VEGF-2430/-2425 homozygous ins1bp genotypes were associated with worse PFS compared with no deletion and no insertion, respectively (HR=2.49; 95% CI: 1.02-6.07; pairwise P=0.0442 and HR=2.57; 95% CI: 1.05-6.27; pairwise P=0.0385, respectively). Furthermore, patients with the VEGF-1498 CC genotype exhibited longer median OS compared with those with the alternatives genotypes (CT and TT) combined (HR=0.27; 95% CI: 0.08-0.89; P=0.0311). In multivariate analysis, the VEGF-2578 AA genotype retained its significance (P=0.0220) for PFS. Our results support the association of specific VEGF genotypes with clinical outcome in patients with metastatic breast cancer treated with a potentially anti-angiogenic regimen, such as weekly docetaxel. However, current results should be validated prospectively in larger cohorts.The Pharmacogenomics Journal advance online publication, 24 September 2013; doi:10.1038/tpj.2013.36.
No preview · Article · Sep 2013 · The Pharmacogenomics Journal
[Show abstract][Hide abstract] ABSTRACT: Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer.
Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes.
In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01). ESR1 clusters were observed in 9.5% (57 gain, 38 amplification) of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression) in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification) fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003). Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066) and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029).
ESR1 gene deletion and amplification do not constitute per se prognostic markers, instead they can be classified to distinct prognostic groups according to their protein-mediated functional status.
[Show abstract][Hide abstract] ABSTRACT: Background–aim: ALK1 (ALK) translocation is a rare event in NSCLC, which more often seem to harbor aberrant ALK gene copies. Herein, we investigated the still undefined impact of ALK gene copies and of ALK mRNA expression on NSCLC patient outcome.
Methods: The presence and relative quantity of two ALK exon spanning transcript targets located before (ALK-5’) and after (ALK-3’) the translocation breakpoints of this gene were assessed with qRT-PCR in 198 NSCLC RNA samples from paraffin tissues upon stringent intra- and inter-run assay performance validation. ALK
mRNA expression was compared with ALK gene status assessed with FISH. Patients
had been treated in the adjuvant and/or 1st line setting. None of them received crizotinib.
Results: Four patterns of ALK mRNA expression emerged: tumors negative for both transcripts (85/198, 42.9%) or positive for both (56/198, 28.3%), which were considered as close to normal (ALK-N); and, ALK-5’ positive only (34/198, 17.2%) or ALK-3’ positive only (23/198, 11.6%), which were termed as aberrant (ALK-A).
ALK translocation was observed in 9/124 cases (7.3%). ALK copies >2.2 were noticed
in 40 cases (32.3%) with >6 copies in 26 cases (21%) and overall complex gene gain patterns. ALK mRNA was unrelated to ALK translocation, but ALK-3’ was associated with increased ALK gene copies (p = 0.017). ALK-A was more common in stage IIIB-IV tumors, while ALK mRNA and gene copy gains were not associated with gender, smoking and histology. ALK gene status was not associated with patient
outcome. In comparison to patients with tumors expressing ALK-N, those with ALK-A had a significantly shorter overall survival (OS, median 29.3 vs. 13.1 months, CI95% 19.1-39.6 vs. 5.4-20.9, p = 0.0007). The same unfavorable impact of ALK-N vs. ALK-A was observed on stage IIIB-IV patient OS (median 17.5 vs. 11 months, CI95% 9.1-26.0 vs. 6.6-15.3, p = 0.0050).
Conclusions: ALK gene status and mRNA expression seem to suffer a complex pathology in NSCLC. When expressed, ALK mRNA may be fragmented, possibly due to currently unknown genomic alterations. Aberrant ALK mRNA expression appears to have an unfavorable prognostic impact on NSCLC patient outcome; its
role on NSCLC biology merits further evaluation.
[Show abstract][Hide abstract] ABSTRACT: To examine the potential differences in the expression of Progesterone Receptor A and Estrogen Receptor A in intermediate trophoblastic cells at the implantation site in elective abortions and miscarriages by immunohistochemistry.
Twenty two (22) samples of miscarriages and eighteen (18) samples of elective abortions were obtained during gestational weeks 6 to 12. Monoclonal antibodies against Cytokeratin 7 and prolactin were used to help discriminate between trophoblastic and decidual cells at the feto-maternal interface on formalin-fixed paraffin-embedded sections. Samples were then stained with ERA and PRA antibodies. Nuclear expression was considered positive. Staining intensity was measured according to a 4 grade scale. Statistical analysis of the results was performed using the Mann-Whitney test and the Wilcoxon signed rank test.
PRA expression in intermediate trophoblastic cells was significantly higher in elective abortions (control group) compared to miscarriages. ERA expression was uniformly negative in both groups.
PRA expression is significantly lower in intermediate trophoblastic cells of miscarriages compared to elective abortion pregnancies. Although this could be solely a result of a secondary event, it is still an important finding in the effort to unravel the complex molecular pathobiology of spontaneous abortions.
No preview · Article · May 2011 · Histology and histopathology