The importance of markers HLA6 and CD68 in placenta tissues of recurrent pregnancy loss

Introduction: Recurrent pregnancy loss of unknown etiology is correlated with immunological factors during pregnancy. Changes in leukocyte subpopulations and HLA (Human Leukocyte Antigen) expression take place in pregnant uterus on both decidua basalis and decidua parietalis in order to carry the semiallogenic embryo. These changes affect the pregnancy course.Objective: Our research is focused 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 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) and CD68 (Cluster of Differentiation 68). The results were statistically analysed with Mann-Whitney test.Results: HLA-G expression in decidua basalis from miscarriage group was found decreased.CD68 + cell expression was found increased in both deciduas from the miscarriage group.Conclusion: The immunological profile of women with recurrent miscarriage is quite different comparing with controls. A possible role of CD68+cells in RPL was observed .Changes in HLA-G expression was observed

Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer

Background: Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy. Methods: Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial. Expression of ESR1, PGR and MKI67 by RT-qPCR was performed on RNA extracted from the same formalin-fixed paraffin-embedded tissue. Concordance between the three methods (vIHC, qIHC and RT-qPCR) was assessed for all 3 markers. The potential of Ki-67 IHC and RT-qPCR to predict pathological complete response (pCR) was evaluated using ROC analysis and non-parametric Mann-Whitney Test. Results: Correlation between methods (qIHC versus RT-qPCR) was high for ER and PgR (spearman´s r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively) resulting in high levels of concordance using predefined cut-offs. When comparing qIHC of ER and PgR with RT-qPCR of ESR1 and PGR the overall agreement was 96.6 and 91.4%, respectively, while overall agreement of visual IHC with RT-qPCR was slightly lower for ER/ESR1 and PR/PGR (91.2 and 92.9%, respectively). In contrast, only a moderate correlation was observed between qIHC and RT-qPCR continuous data for Ki-67/MKI67 (Spearman’s r = 0.50, p = 0.0001). Up to now no predictive cut-off for Ki-67 assessment by IHC has been established to predict response to neoadjuvant chemotherapy. Setting the desired sensitivity at 100%, specificity for the prediction of pCR (ypT0ypN0) was significantly higher for mRNA than for protein (68.9% vs. 22.2%). Moreover, the proliferation levels in patients achieving a pCR versus not differed significantly using MKI67 RNA expression (Mann-Whitney p = 0.002), but not with qIHC of Ki-67 (Mann-Whitney p = 0.097) or vIHC of Ki-67 (p = 0.131). Conclusion: Digital image analysis can successfully be implemented for assessing ER, PR and Ki-67. IHC for ER and PR reveals high concordance with RT-qPCR. However, RT-qPCR displays a broader dynamic range and higher sensitivity than IHC. Moreover, correlation between Ki-67 qIHC and RT-qPCR is only moderate and RT-qPCR with MammaTyper® outperforms qIHC in predicting pCR. Both methods yield improvements to error-prone manual scoring of Ki-67. However, RT-qPCR was significantly more specific

Genotype and phenotype characteristics of epithelial ovarian cancer in primary tumors and matched dissemination sites

Epithelial ovarian cancer (OVCA) is associated with genomic alterations in BRCA1/2 and TP53 and with molecular intra-patient heterogeneity (IPH), owing to tumor – host interactions, including host anti-tumor immune response. Morphological features, i.e., tissue phenotypes, are surrogates of the underlying genomic contexture. Genomic IPH, and morphological IPH may affect the efficacy of standard or targeted therapy and the validity of common predictive markers, including pathogenic mutations in BRCA1/2. We examined matched routine paraffin tissue samples (total n: 302) from 72 treatment naïve OVCA patients who were treated with surgery and platinum-based chemotherapy. Up to 8 different tissue samples per patient were obtained from the ovaries (primary site), sites of intraperitoneal spread, and from fallopian tubes. We assessed tumor morphological subtypes as immunoreactive (IR), solid – proliferative (SD), papilloglandular (PG), and mesenchymal transition (MT); subtype load per section and per patient; and, maximal stromal tumor infiltrative lymphocyte (TIL) density values (max-TILs) among all samples per patient, ovaries and implants. NGS was applied at avg mean depth >2000X. Based on tissue mutation patterns, we distinguished tissue genotypes into no mutation (33/297 samples; 11.1%), stable (173; 58.2%) and unstable (91; 30.7%). We profiled genotypes per patient and assessed genomic heterogeneity in 69 patients with multiple informative samples. The most frequent morphological subtype was PG (n=150 samples, 51.0%), followed by MT (71, 24.1%), SD (48, 16.3%) and IR (15, 5.1%), while combinations were observed in 67/294 sections (22.8%) and IPH in 48/70 patients (68.6%). PG prevailed in ovaries (p2000Χ). Με βάση τα προφίλ των γενωμικών αλλαγών στους ιστούς, ορίσαμε τρεις κατηγορίες γονοτύπων: χωρίς μεταλλάξεις ή φυσιολογικοί (33/297 samples; 11.1%), σταθεροί (173; 58.2%) και ασταθείς (91; 30.7%). Αποτυπώσαμε τα προφίλ ανά ασθενή και αναλύσαμε την γενωμική ετερογένεια των ιστών σε 69 ασθενείς με πολλαπλά αξιολογήσιμα δείγματα. Συχνότερος μορφολογικός υπότυπος ήταν ο αδενοθηλώδης (150 δείγματα, 51.0%) και ακολουθούσαν οι μεσεγχυματικός (71, 24.1%), συμπαγής (48, 16.3%) and ανοσοαντιδραστικός (15, 5.1%). Συνδυασμοί υποτύπων παρατηρήθηκαν σε 67/294 τομές (22.8%) και μορφολογική ετερογένεια σε 48/70 ασθενείς (68.6%). Ο αδενοθηλώδης κυριαρχούσε στις ωοθήκες (p<0.001), ενώ ο συμπαγής και ο μεσεγχυματικός στις θέσεις διασποράς (p=0.023 and p<0.001, αντίστοιχα). Η πυκνότητα sTIL ήταν υψηλότερη σε θέσεις διασποράς (p<0.001). Σε 71 ασθενείς με αξιολογήσιμα δείγματα, 46 (64.8%) είχαν παθογόνες BRCA1 μεταλλάξεις και 15 (21.7%) είχαν λειτουργική απώλεια BRCA1/2 στο πλαίσιο LOH. Διαπιστώσαμε γενωμική ετερογένεια ιστών σε 29 ασθενείς (42%), όλες με παθογόνες μεταλλάξεις BRCA1 στα ιστικά δείγματα. Ωστόσο, μόνο το 64% αυτών είχαν γενωμικά ετερογενή νόσο (p<0.001). Οι ασθενείς με γενωμική ετερογένεια είχαν χειρότερη έκβαση νόσου (log-rank p=0.048 [ελεύθερο διάστημα μέχρι την υποτροπή, PFS]; p=0.037 [συνολική επιβίωση, OS]), συμπεριλαμβανομένων 12/15 ασθενών με λειτουργική απώλεια BRCA1/2 και 3 φορέων BRCA1 με υπολειπόμενο παθογόνο αλλήλιο στους ιστούς. Με καλύτερη έκβαση σχετίζονταν το υψηλότερο φορτίο συμπαγούς υπότυπου (PFS) και οι υψηλότερες τιμές max-TILs (PFS, OS). Η ετερογένεια των μορφολογικών υποτύπων και της ανοσολογικής απάντησης του οργανισμού αποτελούν τον κανόνα στον ΚτΩ, ενώ η γενωμική ετερογένεια επίσης αφορά σημαντικό ποσοστό των ασθενών με τη νόσο. Οι παθογόνες μεταλλάξεις BRCA1 στους ιστούς εμφανίζονται ως αναγκαίες αλλά όχι επαρκείς για την εγκατάσταση της γενωμικής ετερογένειας των ιστών. Η παράμετρος αυτή ίσως σχετίζεται με δυσμενέστερη πρόγνωση, αφορά δε και τις ασθενείς με λειτουργική απώλεια BRCA1/2 που συνήθως θεωρείται ως ευμενής προγνωστικός δείκτης στο κλινικό πλαίσιο που εξετάσθηκε. Σε φορείς BRCA1/2, η κατάσταση της γαμετικής μετάλλαξης στους ιστούς ίσως σχετίζεται με γενωμική και μορφολογική ετερογένεια, κι ίσως είναι χρήσιμο να περιλαμβάνεται στις εκθέσεις αποτελεσμάτων NGS. Ένα μόνο δείγμα προ-εγχειρητικής βιοψίας ή μία «αντιπροσωπευτική τομή», όπως τις εξετάζουμε σήμερα, ενδέχεται να μην επαρκούν για την εκτίμηση της συνολικής κατάστασης του όγκου και της ασθενούς στο πλαίσιο της Ογκολογίας Ακριβείας

Low expression of Progesterone Receptor A in intermediate trophoblast of miscarriages

Summary. Objective: 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. Study Design: 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. Results: 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. Conclusion: 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

Immunohistochemical study of immunological markers: HLAG, CD16, CD25, CD56 and CD68 in placenta tissues in recurrent pregnancy loss

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. Objective: 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. Results: 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

Concordance between Comprehensive Cancer Genome Profiling in Plasma and Tumor Specimens

Introduction: Detection of somatic genomic alterations in the plasma of patients with cancer (liquid biopsy) are increasingly being used in the clinic. However, the concordance of alterations identified in liquid biopsies with those detected in cancer specimens is not routinely being determined. Methods: We sought to systematically compare alterations found by a massively parallel sequencing liquid biopsy assay covering 39 genes (NEOliquid [NEO New Oncology GmbH, Koln, Germany]) with those identified through routine diagnostic testing in a certified central pathology laboratory in a cohort of patients with nonsquamous NSCLC. NEOliquid is based on enrichment of the genomic territory of interest by hybrid capture and is thus capable of detecting point mutations, small insertions and deletions, copy number alterations, and gene rearrangements/fusions in a single assay. Results: In a cohort of 82 patients with matched blood/tissue samples, the concordance between NEOliquid and tissue-based routine testing was 98%, the sensitivity of NEOliquid was higher than 70%, and the specificity was 100%. Discordant cases included those with insufficient amounts of circulaating tumor DNA in plasma and cases in which known driver mutations (e.g., isocitrate dehydrogenase (NADP(+)), 1 systolic gene [IDH1] R132H, kinesin family member 5B gene [KIFSb-ret proto-oncogene [RET], or MNNG HOS Transforming gene [MET] exon 14) were found in the plasma but were not interrogated by routine tissue analyses. Conclusions: In summary, NEOliquid offers accurate and reliable detection of clinically relevant driver alterations in plasma of patients with cancer. (C) 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved

Mismatch repair deficiency and aberrations in the Notch and Hedgehog pathways are of prognostic value in patients with endometrial cancer.

The aim of this study was to investigate the prognostic value of the Hedgehog (Gli, Patched-1, Shh, Smo) and Notch (Jag1, Notch2, Notch3) pathway members, in comparison to a panel of proteins (ER, PgR, HER2/neu, Ki67, p53, p16, PTEN and MMR) previously suggested to be involved in the pathogenesis of endometrial cancer, in association with clinical outcome and standard clinicopathological characteristics. A total of 204 patients with histological diagnosis of endometrial cancer treated from 2004 to 2013 were included. The evaluation of protein expression was assessed by immunohistochemistry. Univariate analysis showed that higher Ki67 labeling, expression of PTEN, p16, Notch2 and Notch3 proteins, as well as MMR proficiency were associated with increased relapse and mortality rate. Additionally, Patched-1 protein expression was associated with worse DFS, while p53 overexpression was associated with worse OS. In multivariate analyses, patients with MMR proficient tumors had more than double risk for death than patients with MMR deficient (MMRd) tumors (adjusted HR = 2.19, 95% CI 1.05-4.58, p = 0.036). Jag1 positivity conferred reduced mortality risk (HR = 0.48, 95% CI 0.23-0.97, p = 0.042). However, as shown by hierarchical clustering, patients fared better when their tumors expressed high Jag1 protein in the absence of Notch2 and Notch3, while they fared worse when all three proteins were highly expressed. Patched-1 positivity conferred higher risk for relapse (HR = 2.04, 95% CI 1.05-3.96, p = 0.036). Aberrant expression of key components of the Notch and Hedgehog signaling pathways, as well as MMRd may serve as independent prognostic factors for recurrence and survival in patients with endometrial cancer