Jean-Christophe Sabourin

Clinical value of chip-based digital-PCR platform for the detection of circulating DNA in metastatic colorectal cancer.

BACKGROUNDnThe detection of circulating DNA is considered a promising strategy in cancer patients. Digital PCR has emerged as a sensitive method able to quantify both circulating free and tumour DNA.nnnAIMnThe aim of this study was to prospectively evaluate the clinical value of a chip-based digital PCR for the detection of circulating DNA.nnnMETHODSnDigital PCR was used in 34 metastatic colorectal cancer patients to detect and quantify circulating free and tumour DNA based on K-ras mutational status. Clinical outcomes were analyzed according to circulating DNA measurements.nnnRESULTSnDigital PCR yielded a detection rate of 69% for circulating tumour DNA. The median concentrations of circulating free and tumour DNA were 20 and 6.8 ng/mL, respectively, with significant correlation between both biomarkers (p<0.001). Median overall survival was 4.8 months in patients with high circulating free DNA (>75% quartile) versus not reached in patients with a low level (<25% quartile) (p=0.029). Moreover, median overall survival was significantly decreased in patients with detectable circulating tumour DNA compared to those without (respectively 11.8 months versus not reached, p=0.04).nnnCONCLUSIONSnChip-based digital PCR is a simple and non-invasive method allowing the efficient detection of circulating DNA. Our results highlight that levels of these circulating markers may have a potential prognostic value.

Oligodendroglioma Arising in an Ovarian Mature Cystic Teratoma

We describe a case of oligodendroglioma arising in an ovarian mature cystic teratoma associated with a loss of heterozygosity on the long arm of chromosomes 19 and 10. To our knowledge it is the second case reported in the literature at this site and the first one described in association with a characterized genetic alteration. The patient was 29 years old and presented with a history of 4 months of pelvic pain. Ultrasound examination showed a cystic mass arising in the left adnexa suggesting a teratoma. At laparotomy a cystic ovoid mass was found arising from the left adnexa, completely replacing the ovary. An ovariectomy was performed. Macroscopically a multilocular cyst containing hair, sebum, and a relatively well-defined solid zone of grayish-pink color strongly suggestive of a cerebral tissue, was observed. Microscopic analyses confirmed the teratomatous nature of the cyst. The solid area was composed of mature glial tissue in which was observed a proliferation of monotonous cells with round and homogenous nuclei, surrounded by a clear halo of cytoplasm (“honeycomb appearance”) which immunohistochemically showed positivity for glial fibrilar acidic protein and for neurofilament protein. Ki-67 labeling index was about 3%. These findings were consistent with a low-grade oligodendroglioma arising in a mature ovarian cystic teratoma. Reverse transcription-polymerase chain reaction analysis showed a characterized loss of heterozygosity occurring in tumor DNA on chromosomes 10q and 19q13.

Tissue Microarray Cytometry Reveals Positive Impact of Homeodomain Interacting Protein Kinase 2 in Colon Cancer Survival Irrespective of p53 Function

The human p53 gene is a tumor suppressor mutated in half of colon cancers. Although p53 function appears important for proliferation arrest and apoptosis induced by cancer therapeutics, the prognostic significance of p53 mutations remains elusive. This suggests that p53 function is modulated at a posttranslational level and that dysfunctions affecting its modulators can have a prognostic impact. Among p53 modulators, homeodomain interacting protein kinase (HIPK) 2 emerges as a candidate switch governing p53 transition from a cytostatic to a proapoptotic function. Thus, we investigated the possible prognostic role of HIPK2 on a retrospective series of 80 colon cancer cases by setting up a multiplexed cytometric approach capable of exploring correlative protein expression at the single tumor cell level on TMA. Crossing the data with quantitative PCR and p53 gene sequencing and p53 functional assays, we observed the following: despite a strong impact on p21 transcription, the presence of disabling p53 mutations has no prognostic value, and the increased expression of the HIPK2 protein in tumor cells compared with paired normal tissue cells has a strong impact on survival. Unexpectedly, HIPK2 effect does not appear to be mediated by p53 function because it is also observed in p53-disabling mutated backgrounds. Thus, our results point to a prominent and p53-independent role of HIPK2 in colon cancer survival.

Copy Number Variations in DCC/18q and ERBB2/17q are Associated with Disease-Free Survival in Microsatellite Stable Colon Cancer.

We conducted a prospective study to assess the prognostic impact of selected copy number variations (CNVs) in Stage II–III microsatellite stable (MSS) colon cancer. A total of 401 patients were included from 01/2004 to 01/2009. The CNVs in 8 selected target genes, DCC/18q, EGFR/7p, TP53/17p, BLK/8p, MYC/8q, APC/5q, ERBB2/17q and STK6/20q, were detected using a quantitative multiplex polymerase chain reaction of short fluorescent fragment (QMPSF) method. The primary end‐point was the impact of the CNVs on the 4‐year disease‐free survival (DFS). The recurrence rate at 4 years was 20.9%, corresponding to 14% Stage II patients versus 31% Stage III patients (pu2009<u20090.0001). The 4‐year DFS was significantly decreased in patients with a loss at DCC/18q (pu2009=u20090.012) and a gain at ERBB2/17q (pu2009=u20090.041). The multivariate analysis demonstrated that Stage III, a loss at DCC/18q and a gain at ERBB2/17q were independent factors associated with DFS. A combination of DCC/18q and ERBB2/17q was also associated with relapse, with the hazard ratio increasing from 1 to 2.4 (95% confidence interval (CI), 1.5–4.1) and 3.1 (95% CI, 1.2–8.4) in the presence of 0, 1 or 2 alterations, respectively (pu2009=u20090.0013). CNVs in DCC/18q and ERBB2/17q are significantly associated with DFS in Stage II–III MSS colon cancer.

Pathologie moléculaire tumorale : à propos du génotypage de KRAS dans les carcinomes colorectaux

Resume La pathologie moleculaire (PM) est une branche emergeante de l’anatomie et cytologie pathologiques. La mise en place des techniques de PM dans les laboratoires de pathologie a ete initialement facilitee par la proximite d’un laboratoire de genetique ou d’une unite de recherche. En effet, la reussite de ces transferts technologiques a ete en partie liee aux fortes interactions existantes entre les pathologistes et les geneticiens/biologistes moleculaires. La complementarite des pathologistes (maitrisant la conservation et l’identification des tissus) et des geneticiens (maitrisant les outils de BM) a permis ainsi de garantir la qualite des examens effectues a partir des tissus. Cependant, c’est avec le genotypage de KRAS effectue « a grande echelle » pour tous les carcinomes colorectaux metastatiques que la PM est apparu comme un outil indispensable a la pratique de la pathologie et egalement comme une evolution irremediable de notre discipline.

Les biomarqueurs circulants du cancer : avantages et perspectives

Resume Les principaux biomarqueurs circulants du cancer sont les cellules tumorales circulantes (CTC) et l’ADN tumoral circulant (ADNtc). Les CTC, cellules issues de la tumeur, presentes dans la circulation sanguine, peuvent etre detectees apres diverses techniques d’enrichissement. Les CTC isolees par technique Veridex® ont ete validees par la Food and Drug Administration (FDA) comme un marqueur pronostique et de suivi dans les cancers mammaires, prostatiques et colorectaux. Les CTC sont egalement prometteuses comme marqueur diagnostique et predictif de reponse aux therapies ciblees. Les techniques microfluidiques, nanostructures permettant la manipulation de microlitres de fluides, permettent d’augmenter la precision et la sensibilite de l’enrichissement. L’ADNtc, issu de la secretion active des cellules vivantes ou passive de cellules en necrose, est decele par la recherche de mutations dans l’ADN libre plasmatique par differentes techniques. Des mutations de sensibilite ou de resistance a des therapies ciblees peuvent etre analysees. L’ADNtc montre aussi un interet comme marqueur pronostique et de suivi tumoral. Dans les tumeurs difficilement accessibles, l’ADNtc peut etre une aide diagnostique. De nouvelles plateformes automatisees proposent la recherche d’ADNtc en moins de 150 minutes. Dans l’avenir, des techniques plus sensibles et plus rapides pour ces deux biomarqueurs circulants, pourraient permettre un veritable monitoring et ainsi une therapie personnalisee adaptee a chaque etape de la maladie tumorale.

Anatomie pathologique : quels renseignements pour quelle décision thérapeutique ?

Le genotypage de KRAS dans les cancers colorectaux metastatiques est devenu aujourd’hui un standard dans la prise en charge des patients : l’absence de mutations dans les codons 12 et 13 de l’exon 2 de ce gene est un bon marqueur predictif de reponse aux therapies ciblees utilisant des anticorps monoclonaux diriges contre le recepteur membranaire de l’ Epidermal growth factor. Ce test moleculaire est effectue a partir d’un bloc tissulaire tumoral (fixe en formol et inclus en paraffine). Aujourd’hui, realise au niveau des plateformes regionales d’oncologie moleculaire mise en place par l’INCa, le genotypage KRAS necessite une collaboration etroite entre les pathologistes (responsables du diagnostic et de la phase preanalytique) et les biologistes/pathologistes moleculaires (responsables du test moleculaire). Cette fructueuse cooperation a permis des 2009, la realisation de pres de 19 000 tests, ce qui correspond a l’ensemble des patients francais susceptibles d’etre traite par un anti-EGFR. Avec ce nouveau marqueur predictif, l’oncologie digestive est reellement entree dans l’ere de la medecine personnalisee.