Marie-Pierre Gaub

Frequent factor II G20210A mutation in idiopathic portal vein thrombosis

BACKGROUND & AIMS Despite extensive investigations of portal vein thrombosis, no underlying cause is identifiable in up to 30% of patients. A recently described mutation of the prothrombin gene at nucleotide position 20210 is associated with history of venous thrombosis and was assessed in this study. METHODS We compared the frequency of factor II G20210A and factor V G1691A (factor V Leiden) mutations in 10 patients with idiopathic portal vein thrombosis, 10 patients with nonidiopathic portal vein thrombosis, 60 patients with deep vein thrombosis of the legs, and 42 control subjects. RESULTS The frequency of factor II G20210A mutation was increased in patients with idiopathic portal vein thrombosis (40.0%; confidence interval, 3.1%-76.9%) compared with controls (4.8%; confidence interval, 0%-11.5%) or patients with nonidiopathic portal vein thrombosis or deep vein thrombosis (P = 0.0001). In contrast, the frequency of the factor V G1691A mutation was similar in subjects with portal vein thrombosis and in controls but was increased in patients with deep vein thrombosis (P = 0.0001). CONCLUSIONS The factor II G20210A mutation is frequent in patients with idiopathic portal vein thrombosis and should therefore be assessed under this circumstance.

Marked Activity of Irinotecan and Rapamycin Combination toward Colon Cancer Cells In vivo and In vitro Is Mediated through Cooperative Modulation of the Mammalian Target of Rapamycin/Hypoxia-Inducible Factor-1α Axis

Purpose: Despite recent progress, colon cancer is often resistant to combination chemotherapy, highlighting the need for development of novel therapeutic approaches. An attractive target is hypoxia-inducible factor-1α (HIF-1α), a key transcription factor with a pivotal role in tumor cell metabolism. One potential class of therapeutic agents targeting HIF-1α are mammalian target of rapamycin inhibitors such as rapamycin. A second class are topoisomerase I inhibitors, such as irinotecan, which are able to inhibit the accumulation of HIF-1α. We here investigated whether combination of rapamycin and irinotecan was active in human colon cancer models. Experimental Design: Human metastatic tumors were xenografted in nude mice and treated with low doses of irinotecan alone, rapamycin alone, or combination of both drugs. The cellular effects of irinotecan and rapamycin were further characterized for HT-29 and HCT-116 colon cancer cells in vitro. Results: In contrast to single-agent therapy, xenografted tumors treated with combination of irinotecan and rapamycin showed potent inhibition of the mammalian target of rapamycin/HIF-1α axis, which was accompanied by a dramatic reduction in tumor volume. In vitro experiments showed that exposure to low concentrations of the two drugs resulted in massive HT-29 cell death under hypoxic, but not normoxic, conditions, in full agreement with a cytotoxic effect mediated through HIF-1α rather than through induction of genotoxic lesions. HCT-116 cells were less sensitive to the combined treatment due to constitutive activation of phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways. Conclusion: These results identify HIF-1α as a promising target and provide a rationale for clinical trials of low-dose irinotecan and rapamycin combination toward metastatic colon cancer.

Retrospective Comparison of Chemoradiotherapy Followed by Adjuvant Chemotherapy, With or Without Prior Gliadel Implantation (Carmustine) After Initial Surgery in Patients With Newly Diagnosed High-Grade Gliomas

PURPOSE Retrospective study of patients treated for high-grade glioma, with or without biodegradable carmustine wafers and according to the Stupp protocol. METHODS AND MATERIALS Between May 2007 and June 2008, 65 patients underwent surgery for high-grade glioma, 28 had implantation of Gliadel and 37 patients did not. Patients received radiotherapy with concomitant temozolomide followed by 5 consecutive days of temozolomide every month for 6 months. RESULTS Overall median follow-up was 17.1 months; the median relapse-free survival (RFS) was 14 months with a RFS of 54% at 12 months, and 38% at 24 months. For patient with and without Gliadel, median and 1-year RFS were 12.9 months and 52% vs. 14 months and 42%, respectively (p = 0.89). According to pathology, Gliadel did not influence RFS of patients with Grade III or glioblastoma. However, for all patients, in multivariate analysis, non-methylated methylguanine methyltransferase (MGMT) was the only unfavorable prognostic factor of RFS (p = 0.017; HR 2.8; CI [1.2-7]). Median overall survival (OS) was 20.8 months; the OS rate at 12 months was 78.5%, and at 24 months 35.4%. For patients treated with and without Gliadel, median and 1-year OS were 20.6 months and 78.6% vs. 20.8 months and 78.4%, respectively. According to pathology, Gliadel did not influence OS of patients with Grade III or glioblastoma. For all patients, in multivariate analysis, unfavorable prognosticators for OS were non-methylated MGMT (p = 0.001; HR: 6.5; CI [2-20]) and irradiation dose <60 Gy (p = 0.02; HR: 6.3; CI [2-20]). With carmustine wafers, before irradiation, median gross tumor volume plus edema was 84 mL (27-229), whereas it was 68 mL (10-362) without carmustine (p = nonsignificant). Four cases of Grade 3 thrombopenia occurred, all in the carmustine wafer group. CONCLUSION In patients with high-grade gliomas, adding Gliadel before performing a Stupp protocol did not improve survival.

IR spectral imaging for histopathological characterization of xenografted human colon carcinomas.

This study aims to develop IR imaging of tumor tissues for generating an automated IR-based histology. Formalin-fixed paraffin-embedded xenografts of human colon carcinomas were analyzed. Chemometric and statistical multivariate treatments of spectral data permitted to probe the intrinsic chemical composition of tissues, directly from paraffinized sections without previous dewaxing. Reconstructed color-coded spectral images revealed a marked tumor heterogeneity. We identified three spectral clusters associated to tumoral tissues, whereas HE staining revealed only a single structure. Nine other clusters were assigned to either necrotic or host tissues. This spectral histology proved to be consistent over multiple passages of the same xenografted tumor confirming that intratumoral heterogeneity was maintained over time. In addition, developing an innovative image analysis, based on the quantification of neighboring pixels, permitted the identification of two main sequences of spectral clusters related to the tissue spatial organization. Molecular attribution of the spectral differences between the tumor clusters revealed differences of transcriptional activity within these tumor tissue subtypes. In conclusion, IR spectral imaging proves to be highly effective both for reproducible tissue subtype recognition and for tumor heterogeneity characterization. This may represent an attractive tool for routine high throughput diagnostic challenges, independent from visual morphology.

UGT1A1 genotype and irinotecan therapy: general review and implementation in routine practice

Irinotecan is a major drug in the treatment of advanced colorectal cancer. Its active form is the SN38 metabolite, which is cleared by the biliary route after glucuronidation by uridine diphosphate–glucuronosyltransferase 1A1 (UGT1A1). UGT1A1 activity exhibits a wide intersubject variability, in part related to UGT1A1 gene polymorphisms. The present review on the impact of the deficient UGT1A1*28 variant on irinotecan efficacy and toxicity was produced by a French joint workgroup comprising the Group of Clinical Onco‐pharmacology (GPCO‐Unicancer) and the National Pharmacogenetics Network (RNPGx). It clearly emerges that for irinotecan doses at least equal to 180 mg/m2, patients homozygous for the UGT1A1*28 allele are at increased risk of developing hematological and/or digestive toxicities. Irinotecan dose reduction is thus recommended in homozygous *28/*28 patients. In addition, this personalized medicine strategy aims to secure high‐dose irinotecan administration (≥240 mg/m2) that have proven to be safe in homozygous *1/*1 patients only. The clinical relevance of this test is discussed in terms of treatment efficacy improvement, as increasing the irinotecan dose appears to be safe in patients not bearing a deficient allele. Best execution practices, cost‐effectiveness, and result interpretation are discussed with the aim of facilitating the implementation of this analysis in clinical practice. The existence of networks of laboratories performing this test in routine hospital treatment, as in France, offers the prospect of widespread screening, thus guaranteeing equal access to safe treatment and optimized therapy for patients receiving irinotecan‐based therapy in advanced colorectal cancer.

Post‐transplant Lymphoproliferative Disorders: Determination of Donor/Recipient Origin in a Large Cohort of Kidney Recipients

Although in previous studies most post‐transplant lymphoproliferative disorders (PTLD) were reported to derive from recipient cells, some cases derived from donor lymphocytes have been reported. To provide a better description of the features and outcome of PTLD according to the origin of the lymphoma, we performed histologic and molecular studies of PTLD in kidney recipients. Forty‐three specimens were analyzed by histochemistry, fluorescent hybridization of the Y chromosome and analysis of multiple short tandem repeat microsatellite loci. Sixteen tumors were shown to be of donor origin and 27 of recipient origin. Time to PTLD was shorter in donor‐derived PTLDs (20±27 vs. 69±67 months, p = 0.013). Ten‐year patient survival was similar among patients with recipient‐ and donor‐derived PTLD, but when PTLD‐related mortality was analyzed, there was a trend to better survival in patients with donor lymphomas. Among the 21 PTLDs localized in the allograft, 14 lymphomas were of donor origin and seven of recipient origin. No difference was found between the two groups. Our analysis of the origin of PTLDs in the largest cohort studied to date with a description of the clinical and histological characteristics of donor and recipient PTLDs should lead to a better understanding of lymphomagenesis.

Prognostic Significance of Allelic Imbalance at the c-kit Gene Locus and c-kit Overexpression by Immunohistochemistry in Pediatric Osteosarcomas

PURPOSE Since the recent development of biologic agents targeting oncogenes, increasing attention has been focused on determining the role of tyrosine kinase receptors in the pathogenesis of tumors. Our study was designed to investigate the status of region 4q12, which contains the candidate gene c-kit, and the expression of c-kit by immunohistochemistry (IHC). PATIENTS AND METHODS Paired blood and biopsy specimens of 68 children treated for high-grade primary osteosarcomas were collected. Microsatellite analysis at two genomic sites containing c-kit gene was performed on paired DNA using a sensible fluorescent polymerase chain reaction technology. To confirm the DNA data, we studied c-kit protein expression by IHC in 56 available paraffin-embedded tumor tissues. RESULTS The frequency of allelic imbalance (AI) at locus 4q12 was 39% in the overall population. In agreement with previous studies, we did not detect microsatellite instability, allowing us to hypothesize that this pathway is not implicated. Furthermore, the normal status at locus 4q12 was associated with a significantly better survival in the whole osteosarcoma population (P = .05). IHC overexpression of c-kit was concordant in all cases presenting an AI. However, normal status at locus 4q12 was correlated to an absence of c-kit protein expression in 19 (65.5%) of 29 informative cases. CONCLUSION Allelotyping of locus 4q12, which contains the c-kit gene, could help pediatric osteosarcoma prognostic screening and showed a strong correlation with overexpression of c-kit protein. These results allowed us to hypothesize that, in some cases, a mutation of c-kit gene could lead to a protein overexpression.

Analysis of Allelic Imbalance in Patients With Colorectal Cancer According to Stage and Presence of Synchronous Liver Metastases

ObjectiveTo investigate the relationship between number and location of allelic imbalances (AI) and local tumor progression according to Astler-Coller classification. Summary Background DataSpontaneous errors in DNA replication (i.e., allelic imbalance or microsatellite instability) have been suggested to play an important role in carcinomatous transformation as reflecting alterations of gene function. MethodsOne hundred two consecutive patients with colorectal carcinoma undergoing surgical resection were included in this study. Patients were distributed according to the Astler-Coller classification as stages A (n = 7), B1 (n = 15), B2 (n = 24), C (n = 31), and D (n = 25). Fluorescent polymerase chain reaction was performed on frozen tumor, normal colon mucosa, and blood DNA at 35 microsatellite markers. Allelic imbalance frequency was compared with tumor staging. ResultsThe percentage of AI was significantly higher in stage D than in A/B1 and B2. In addition, the percentage of AI was significantly higher in 10 synchronous colorectal liver metastases than in stage A/B1 and B2 tumors. However, the allelotyping revealed a subgroup of A/B1 tumors with a high AI frequency. Statistical analysis showed that the presence of AI at microsatellites D1S305, D2S138, D3S1282, D17S790, and D22S928 presented a significantly positive correlation with stages. ConclusionThe frequency of AI significantly correlates with tumor progression of colorectal cancer. Primary tumors with synchronous colorectal liver metastases showed a higher percentage of AI, suggesting that a frequency of AI greater than 35% with this selection of markers indicates a high risk of local progression and of development of metastases.

In Vivo Topoisomerase I Inhibition Attenuates the Expression of Hypoxia-Inducible Factor 1α Target Genes and Decreases Tumor Angiogenesis

Topoisomerase I is a privileged target for widely used anticancer agents such as irinotecan. Although these drugs are classically considered to be DNA-damaging agents, increasing evidence suggests that they might also influence the tumor environment. This study evaluates in vivo cellular and molecular modifications induced by irinotecan, a topoisomerase I-directed agent, in patient-derived colon tumors subcutaneously implanted in athymic nude mice. Irinotecan was given intraperitoneally at 40 mg/kg five times every 5 d, and expression profiles were evaluated at d 25 in tumors from treated and untreated animals. Unexpectedly, the in vivo antitumor activity of irinotecan was closely linked to a downregulation of hypoxia-inducible factor-1α (HIF1A) target genes along with an inhibition of HIF1A protein accumulation. The consequence was a decrease in tumor angiogenesis leading to tumor size stabilization. These results highlight the molecular basis for the antitumor activity of a widely used anticancer agent, and the method used opens the way for mechanistic studies of the in vivo activity of other anticancer therapies.

KIT gene in pediatric osteosarcomas: could it be a new therapeutic target?

In our previous study, a frequent rearrangement at 4q12 has been identified by allelotyping in our large and homogeneous population of pediatric osteosarcomas and it was significantly linked to c‐kit protein overexpression. To confirm and understand the involvement of KIT in this tumor, the next step of the study was designed to detect the potential mutations of KIT gene by sequencing the frequently mutated exons 6, 8, 10, 11, 13, 17 and 21 and, in case of unmutated samples, to confirm the genomic amplifications of the wild‐type receptor by real‐time quantitative PCR (QPCR). A new microsatellite and QPCR targeting PDGFRA was also added to check the accuracy of the 4q11‐12 locus. These techniques were performed in 74 pediatric high‐grade osteosarcomas treated with the OS94 protocol. Surprisingly, no mutations were found, but, only DNA amplification of KIT gene in the entire population. PDGFRA gene QPCR revealed an unexpected result of predominant deletions in the rearranged tumors. All these results confirm the major role of the 4q11‐12 locus and specifically the involvement of c‐kit wild‐type receptor overexpression in pediatric osteosarcomas and leads us to believe that inhibitors targeting this receptor could have a therapeutic effect in a selected group of patients.