Hereditary breast cancer: beyond BRCA genetic analysis; PALB2 emerges

Despite the initial enthusiasm following the discovery of the association of BRCA germline mutations with hereditary breast and/or ovarian cancer, in many families affected by the syndrome no pathogenic mutations were detected in the two genes, although exhaustively searched. Many other genes have also been implicated due to their role in the same pathway of DNA repair where the BRCA1/2 genes are involved: homologous recombination (HR). Among them, PALB2 clearly emerges as the third breast cancer susceptibility gene. Its mutations have been detected in most populations investigated so far, albeit rarely: in 1%-4% of families negative for BRCA mutations, with either partial or complete penetrance. In some populations, PALB2 recurrent mutations have been identified and the estimated hazard risks are comparable to those of BRCA mutations. Since new effective targeted therapeutic options are becoming available (”synthetic lethality” with novel PARP inhibitors, etc.) that are applicable to all those patients with a defect in HR pathway, it is imperative to detect all these candidate patients. Data obtained from laboratory tests in the tumor (simple immunohistochemistry, gene expression analysis, etc.) can assist in the recognition of a specific pattern (BRCA1ness, HRless) so that even patients that look “sporadic” could benefit from these targeted therapies. Therefore, a genetic analysis algorithm is proposed, although with the advent of Next Generation Sequencing it is predicted that in the future most germline genetic alterations and also somatic or epigenetic events in the tumor of these genes will be detected

Human papilloma virus (HPV) molecular diagnostics

Human Papilloma Virus (HPV) is becoming a menace worldwide, especially to the developing world, due to its involvement in a variety of malignancies, with cervical cancer being the most important and prevalent. There are many HPV types; HPV 16/18 are the most carcinogenic but few others are also characterized as high-risk (HR). They can cause a variety of low- or high-grade cellular abnormalities, most frequently detected in a routine Pap test. Most infections clear within 2 years, however, a minority persists and potentially could progress to cervical cancer. Molecular tests detecting HPV DNA, RNA or proteins are now being available either commercially or in-house developed. DNA detection is nowadays an established tool for diagnosis and monitoring of HPV-related disease, however, there is lack of a reference method and standardization with reference materials. The various available test formats create confusion on which molecular test to choose and what are its limitations. Therefore, the need for lab accreditation and participation in proficiency testing has to be stressed. Novel HPV biomarkers (RNA, protein etc.) are now intensively examined for their inclusion as adjunct tools. Recently, developed prophylactic vaccines for HPV 16/18 have already proven safe and efficient and raise high expectations for the complete eradication of these types in the future

Molecular study of BRCA1 and BRCA2 genes in greek patients with hereditary breast and ovarian cancer

85 Greek patients with hereditary breast and ovarian cancer were tested with the protein truncation test (PTT) for mutations in peripheral blood DNA in exon 11 of the BRCA1 gene and exons 10 and 11 of the BRCA2 gene (60% of the coding region of both genes). Totally eight deleterious mutations were detected and identified with automated DNA Sequencing: six in BRCA1 (3741insA, 1623del5, 3099delT, 3277insG, R1203X και 3896delT) and two in BRCA2 (2024del5 in exon 10 και 6631del5 in exon 11). Two of them are novel mutations reported for the first time in the literature (3099delT, 3277insG). Then, in tumor samples of 20 patients from the above group, prognostic factors of breast cancer were evaluated like ER, PgR, p53, Ki-67 and c-erbB-2 with immunohistochemistry and ploidy, DNA Index and proliferative phase with image cytometry. The tumors of five patients that tested positive for BRCA1 mutation (in peripheral blood) are negative for ER content and c-erbB-2 overexpression while in parallel they possessed intense proliferation (high Ki-67 and proliferative phase, giant cells in two). Two of the BRCA1 patients were recorded as medullary breast carcinomas in their histology report. The above reported phenotypic biological characteristics could assist in the detection of more genotypic changes in the highly penetrant BRCA1 gene. Therefore in a population-based study of patients fulfilling the aforementioned criteria, the recurrent 5382insC BRCA1 mutation was detected in one patient with PSM-PCR-RFLP methodology. Also, an immunohistochemical technique for the detection of BRCA1 protein was developed (MS110 mAb) and then applied: a) in a group of 30 sporadic breast cancer tumors where the range and intensity of positivity were recorded and b) in the 5 BRCA1 positive tumors (3 were assigned negative for BRCA1 protein). Finally, methods for the measurement of BRCA1 mRNA levels were developed: a) a semi quantitative hybridization method for PCR products and chemiluminescent detection in an ELISA microtiter plate and b) a sensitive quantitative method for BRCA1-mRNA with real time RT-PCR with the use of a Taqman DNA probe, conjugated with the fluorescent fluorescein dye and continuous monitoring in the Light Cycler instrument. This second method was applied: a) in peripheral blood samples of patients with breast cancer for the investigation of detection of circulating cancer cells where after checking healthy blood donors, positive BRCA1 signal was detected in 13/41 (32%) samples of metastatic breast cancer and b) in cultures of the MCF-7 breast cancer cell line after treatment with chemotherapeutic agents or radiation.Σε 85 Έλληνες ασθενείς με κληρονομούμενο καρκίνο μαστού και ωοθηκών εφαρμόστηκε σε DNA περιφερικού αίματος, δοκιμασία πρόωρου τερματισμού της πρωτεϊνοσύνθεσης (PTT) στο εξόνιο 11 του γονιδίου BRCA1 και στα εξόνια 10 και 11 του γονιδίου BRCA2 (60% της κωδικοποιούσης περιοχής των δύο γονιδίων). Ανιχνεύθηκαν συνολικά οκτώ παθογνωμικές μεταλλάξεις και ταυτοποιήθηκαν με την αυτοματοποιημένη μέθοδο του προσδιορισμού της αλληλουχίας DNA: έξι στο BRCA1 (3741insA, 1623del5, 3099delT, 3277insG, R1203X και 3896delT) και δύο στο BRCA2 (2024del5 στο εξόνιο 10 και 6631del5 στο εξόνιο 11). Οι δύο από τις οκτώ αναφέρονται για πρώτη φορά στη βιβλιογραφία (3099delT, 3277insG). Στη συνέχεια αξιολογήθηκαν σε δείγματα όγκου από 20 από τους παραπάνω ασθενείς, προγνωστικοί δείκτες καρκίνου μαστού όπως οι ER, PgR, p53, Ki-67 και c-erbB-2 με ανοσοϊστοχημική μέθοδο και πλοειδισμός, δείκτης DNA και αναπαραγωγική φάση με κυτταρομετρητή εικόνας. Οι όγκοι πέντε ασθενών θετικών για μετάλλαξη στο BRCA1 (στο περιφερικό αίμα), είναι αρνητικοί ως προς ER και υπερέκφραση c-erbB-2 ενώ παράλληλα διαθέτουν έντονο πολλαπλασιασμό (υψηλά Ki-67 και αναπαραγωγική φάση, γιγαντοκύτταρα σε δύο). Δύο από τους ασθενείς με μεταλλάξεις στο γονίδιο BRCA1 διέθεταν τον ιστολογικό υπότυπο του μυελοειδούς καρκινώματος μαστού. Τα παραπάνω ευρεθέντα φαινοτυπικά βιολογικά χαρακτηριστικά μπορούν να χρησιμοποιηθούν στην ανεύρεση περισσότερων γονοτυπικών αλλαγών στο υψηλής διεισδυτικότητας BRCA1 γονίδιο. Έτσι σε πληθυσμιακή μελέτη ασθενών που πληρούσαν τα ανωτέρω κριτήρια, ανιχνεύθηκε σε έναν ασθενή η συχνή μετάλλαξη 5382insC του BRCA1 με PSM-PCR-RFLP μεθοδολογία. Αναπτύχθηκε επίσης ανοσοϊστοχημική μέθοδος για την ανίχνευση της πρωτεΐνης BRCA1 (MS110 mAb) και στη συνέχεια εφαρμόστηκε: α) σε δείγμα 30 σποραδικών καρκίνων μαστού όπου και καταγράφηκε το εύρος και η ένταση της θετικότητας και β) στους 5 θετικούς για μετάλλαξη BRCA1 όγκους (οι 3 ευρέθησαν αρνητικοί ως προς την παρουσία πρωτεΐνης BRCA1). Τέλος, για την μέτρηση των επιπέδων του BRCA1 mRNA αναπτύχθηκαν: α) ημιποσοτική μέθοδος υβριδισμού προϊόντων PCR αντίδρασης και χημειοφωταυγούς ανίχνευσης σε μικροπλακίδιο τύπου ELISA και β) ευαίσθητη ποσοτική μέθοδος προσδιορισμού του BRCA1-mRNA με μεθοδολογία RT-PCR σε πραγματικό χρόνο (real time), με χρήση ειδικού DNA ανιχνευτή υβριδισμού τεχνολογίας Taqman, επισημασμένου με τη φθορίζουσα χρωστική φλουορεσκεΐνη και παρακολούθηση της αντίδρασης στο όργανο Light Cycler. Η δεύτερη μέθοδος εφαρμόστηκε: α) σε δείγματα περιφερικού αίματος ασθενών με καρκίνο μαστού για την διερεύνηση της ανίχνευσης κυκλοφορούντων καρκινικών κυττάρων όπου αφού ελέγχθηκαν εθελοντές υγιείς αιμοδότες, θετικό BRCA1 σήμα ελήφθη σε 13/41 (32%) δείγματα με μεταστατικό καρκίνο μαστού και β) σε καλλιέργειες της καρκινικής σειράς MCF-7 έπειτα από επίδραση χημειοθεραπευτικών ουσιών ή ακτινοβολιών

Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

AIM: To investigate three isoforms of survivin in colorectal adenocarcinomas

Development of novel real-time PCR methodology for quantification of COL11A1 mRNA variants and evaluation in breast cancer tissue specimens

Background: Collagen XI is a key structural component of the extracellular matrix and consists of three alpha chains. One of these chains, the alpha 1 (XI), is encoded by the COL11A1 gene and is transcribed to four different variants at least (A, B, C and E) that differ in the propensity to N-terminal domain proteolysis and potentially in the way the extracellular matrix is arranged. This could affect the ability of tumor cells to invade the remodeled stroma and metastasize. No study in the literature has so far investigated the expression of these four variants in breast cancer nor does a method for their accurate quantitative detection exist. Methods: We developed a conventional PCR for the general detection of the general COL11A1 transcript and real-time qPCR methodologies with dual hybridization probes in the LightCycler platform for the quantitative determination of the variants. Data from 90 breast cancer tissues with known histopathological features were collected. Results: The general COL11A1 transcript was detected in all samples. The developed methodologies for each variant were rapid as well as reproducible, sensitive and specific. Variant A was detected in 30 samples (33 %) and variant E in 62 samples (69 %). Variants B and C were not detected at all. A statistically significant correlation was observed between the presence of variant E and lymph nodes involvement (p = 0.037) and metastasis (p = 0.041). Conclusions: With the newly developed tools, the possibility of inclusion of COL11A1 variants as prognostic biomarkers in emerging multiparameter technologies examining tissue RNA expression should be further explored