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Background: Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI, a marker for defective DNA mismatch repair) as a first screen for Lynch syndrome (LS). In this study, we investigated whether it may be possible to improve the detection of MSI in CRC. We examined whether the HT17 DNA repeat (critical for correct splicing of the chaperone HSP110) might constitute a superior marker for diagnosis of the MSI phenotype in patients with CRC compared with the standard panel of markers (pentaplex)., Methods: The HT17 polymorphism was analysed in germline DNA from 1037 multi-ethnic individuals. We assessed its sensitivity and specificity for detecting MSI in a multicentre, population-based cohort of 685 patients with CRC and an additional series of 70 patients with CRC considered to be at-risk of LS. All cases were screened earlier for MSI using pentaplex markers. Cases showing discordant HT17/pentaplex results were further examined for the expression of mismatch repair proteins., Results: HT17 status was analysed independently and blinded to previous results from pentaplex genotyping. HT17 showed no germline allelic variation outside a very narrow range. Compared with the pentaplex panel, HT17 showed better sensitivity (0.984 (95% CI 0.968 to 0.995) vs 0.951 (95% CI 0.925 to 0.972)) and similar specificity (0.997 (95% CI 0.989 to 1.000) for both) for the detection of MSI. Furthermore, HT17 alone correctly classified samples judged to be uncertain with the pentaplex panel and showed excellent ability to detect MSI in patients with LS., Conclusions: HT17 simplifies and improves the current standard molecular methods for detecting MSI in CRC., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Dihydropyrimidine dehydrogenase is a crucial enzyme for the degradation of 5-fluorouracil (5FU). DPYD, which encodes dihydropyrimidine dehydrogenase, is prone to acquire genomic rearrangements because of the presence of an intragenic fragile site FRA1E. We evaluated DPYD copy number variations (CNVs) in a prospective series of 242 stage I-III colorectal tumours (including 87 patients receiving 5FU-based treatment). CNVs in one or more exons of DPYD were detected in 27% of tumours (deletions or amplifications of one or more DPYD exons observed in 17% and 10% of cases, respectively). A significant relationship was observed between the DPYD intragenic rearrangement status and dihydropyrimidine dehydrogenase (DPD) mRNA levels (both at the tumour level). The presence of somatic DPYD aberrations was not associated with known prognostic or predictive biomarkers, except for LOH of chromosome 8p. No association was observed between DPYD aberrations and patient survival, suggesting that assessment of somatic DPYD intragenic rearrangement status is not a powerful biomarker to predict the outcome of 5FU-based chemotherapy in patients with colorectal cancer.

MET pathway is a promising target in non-small cell lung cancers (NSCLC) requiring companion tests. The aim of this study was to compare MET expression/gene copy number in a Caucasian population of NSCLC patients.We analysed 201 NSCLC, with 141 adenocarcinomas classified according to 2011 IASLC recommendations, for MET expression by immunohistochemistry (IHC) and gene copy number (GCN) by silver in situ hybridisation (SISH) on tissue microarrays. Mutations in EGFR, KRAS, BRAF, HER2, PIK3CA genes and ALK rearrangements were determined.MET overexpression was observed in 44% and a high MET GCN (≥5 copies) in 14%. MET CGN was correlated with MET expression, regardless of IHC scores (p < 0.001) but only 31% of MET overexpressed cases were SISH positive. MET overexpression/GCN number was more frequent in ADC than the other types (p < 0.001), the highest in high grade (74%/34%) and sarcomatoid ADC (86%/43%). Mutations of current genes or ALK rearrangements were identified in overexpressed or amplified MET cases. MET overexpression was an independent prognostic factor for overall survival in non-smoker NSCLC in univariate (p = 0.01) and multivariate (p = 0.01) analyses.MET overexpression is more frequent than MET high GCN, particularly in high grade ADC, regardless of EGFR, KRAS, BRAF, HER2, PIK3CA and ALK status in NSCLC.

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/m(2) , 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/m(2) ) 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., (© 2015 Société Française de Pharmacologie et de Thérapeutique.)

PIK3CA, which encodes the p110α catalytic subunit of PI3Kα, is one of the most frequently altered oncogenes in colon cancer (CC), but its prognostic value is still a matter of debate. Few reports have addressed the association between PIK3CA mutations and survival and their results are controversial. In the present study, we aimed to clarify the prognostic impact of PIK3CA mutations in stage I-III CC according to mismatch repair status. Fresh frozen tissue samples from two independent cohorts with a total of 826 patients who underwent curative surgical resection of CC were analyzed for microsatellite instability and screened for activating point mutations in exon 9 and 20 of PIK3CA by direct sequencing. Overall, 693 tumors (84%) exhibited microsatellite stability (MSS) and 113 samples (14%) harbored PIK3CA mutation. In the retrospective training cohort (n = 433), patients with PIK3CA-mutated MSS tumors (n = 47) experienced a significant increased 5-year relapse-free interval compared with PIK3CA wild-type MSS tumors (n = 319) in univariate analysis (94% vs. 68%, Log-rank P = 0. 0003) and in multivariate analysis (HR = 0.12; 95% confidence interval, 0.029-0.48; P = 0.0027). In the prospective validation cohort (n = 393), the favorable prognostic impact of PIK3CA mutations in MSS tumors (n = 327) was confirmed (83% vs. 67%, Log-rank P = 0.04). Our study showed that PIK3CA mutations are associated with a good prognosis in patients with MSS stage I-III CC., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

High grade gliomas (HGG) are usually associated with a very dismal prognosis, which was moderately improving in the last decade with the introduction of the alkylating agent temozolomide in their treatment. The methylation status of MGMT (O6 methylguanine DNA-methyltransferase) promoter is one of the strongest predictive and prognostic factors for the patient chemoresponse. For instance, the molecular method of assessment for MGMT promoter status is not standardized. In this background, we developed a fluorescent capillary gel electrophoresis-based methylation specific-PCR. This technique allowed a semi-quantitative estimate of the relative ratio between methylated and unmethylated alleles. The efficacy and accuracy of the technique was assessed in a retrospective cohort of 178 newly diagnosed adult HGGs, who were homogeneously treated. First, we analyzed the impact on survival of different cut-off points in the MGMT promoter methylation and, to go further, we correlated these different rates to other well-known prognostic molecular factors involved in adult HGGs. This strategy allowed to validate our technique as a very sensitive technique (detection of a low methylation percentage, < 5%), which was feasible in fresh-frozen as well as in FFPE samples and had the propensity to detect intra-tumor heterogeneity. This technique identified a new sub-group of anaplastic oligodendrogliomas or oligoastrocytomas defined by a minor methylation and a worse outcome and, therefore, will help to substratify accurately into more homogeneous subgroups of methylated tumors.

Irinotecan is a cytotoxic agent administered by IV infusion in the treatment of advanced colorectal cancer. Its anticancer activity results from its bioactivation into SN-38 metabolite, which is cleared through glucuronidation by the hepatic enzyme uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1). In the general population, there is wide inter-subject variability in UGT1A1 enzyme activity related to UGT1A1 gene polymorphisms. The French joint workgroup coming from the National Pharmacogenetic Network (RNPGx) and the Group of Clinical Oncologic Pharmacology (GPCO) herein presents an updated review dealing with efficacy and toxicity clinical studies related to UGT1A1 genetic variants. From a critical analysis of this review it clearly emerges that, for doses higher than 180 mg/m(2), hematologic and digestive irinotecan-induced toxicities could be prevented in daily clinical practice by generalizing the use of a simple pharmacogenetic test before starting treatment. The clinical relevance of this test is also discussed in terms of treatment efficacy improvement, with the possibility of increasing the irinotecan dose in patients not bearing the deleterious allele. This test involves using a blood sample to analyze the promoter region of the UGT1A1 gene (UGT1A1*28 allele). Best execution practices, laboratory costs, as well as results interpretation are described with the aim of facilitating the implementation of this analysis in clinical routine. The existence of a French laboratories network performing this test in clinical routine makes it possible to generalize UGT1A1 deficiency screening, so as to guarantee equal access to safe treatment and optimized irinorecan-based therapy for the many patients receiving irinotecan-based therapy in advanced colorectal cancer.

Background: To test the prognostic value of tumour protein and genetic markers in colorectal cancer (CRC) and examine whether deficient mismatch repair (dMMR) tumours had a distinct profile relative to proficient mismatch repair (pMMR) tumours., Methods: This prospective multicentric study involved 251 stage I-III CRC patients. Analysed biomarkers were EGFR (binding assay), VEGFA, thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) expressions, MMR status, mutations of KRAS (codons 12-13), BRAF (V600E), PIK3CA (exons 9 and 20), APC (exon 15) and P53 (exons 4-9), CpG island methylation phenotype status, ploidy, S-phase, LOH., Results: The only significant predictor of relapse-free survival (RFS) was tumour staging. Analyses restricted to stage III showed a trend towards a shorter RFS in KRAS-mutated (P=0.005), BRAF wt (P=0.009) and pMMR tumours (P=0.036). Deficient mismatch repair tumours significantly demonstrated higher TS (median 3.1 vs 1.4) and TP (median 5.8 vs 3.5) expression relative to pMMR (P<0.001) and show higher DPD expression (median 14.9 vs 7.9, P=0.027) and EGFR content (median 69 vs 38, P=0.037) relative to pMMR., Conclusions: Present data suggesting that both TS and DPD are overexpressed in dMMR tumours as compared with pMMR tumours provide a strong rationale that may explain the resistance of dMMR tumours to 5FU-based therapy.

Background: HIF-1α and CXCR4/CXCL12 have crucial roles in the metastatic process of colorectal cancer. Our aim was to study the significance of targeting HIF-1α and the CXCR4/CXCL12 axis in colorectal cancer to prevent the dissemination process in vitro., Methods: We investigated CXCR4 and CXCR7 mRNA and protein expression in human colon carcinomas and the modulation of their expression by hypoxia and HIF-1α in colon cancer cell lines. The migration of tumor cells in a Boyden chamber was studied after CXCR4 inhibition with siRNA or the CXCR4/CXCL12 neutraligand, chalcone 4., Results: Analysis of a cohort of colon polyps and chromosome-unstable carcinomas showed that the expression of CXCR4 and CXCR7 was similar to that of the normal mucosa in the polyps and early-stage carcinomas but significantly increased in late stage carcinomas. Our data demonstrate that hypoxia strongly induced the expression of CXCR4 transcript and protein at the cell membrane, both regulated by HIF-1α, whereas CXCR7 expression was independent of hypoxia. After transient hypoxia, CXCR4 levels remained stable at the cell membrane up to 48 hours. Furthermore, reducing CXCR4 expression impaired CXCL12-induced Akt phosphorylation, whereas Erk activation remained unchanged. In contrast, reducing CXCR7 expression did not affect Akt nor Erk activation. In the presence of CXCR4 or CXCR7 siRNAs, a significant reduction in cell migration occurred (37% and 17%, respectively). Although irinotecan inhibited cell migration by 20% (p <0.001), the irinotecan and chalcone 4 combination further increased inhibition to 40% (p <0.001)., Conclusion: We demonstrated, for the first time, that hypoxia upregulated CXCR4 but not CXCR7 expression in tumor cells and that the CXCR4 receptor protein level remains high at the cell membrane when the tumor cells return to normoxia for up to 48 hours. In addition we showed the interest to inhibit the CXCR4 signaling by inhibiting both the HIF-1α and CXCR4/CXCL12 pathway. CXCR4 seems to be a relevant target because it is continuously expressed and functional both in normoxic and hypoxic conditions in tumor cells.

Background & Aims: Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil–based chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T(17) intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin.We investigated whether HSP110 T(17) could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin., Methods: We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T(17) affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage II–III colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T(17)., Results: HSP110 and HSP110DE9 interacted in a1:1 ratio. Tumor cells with large deletions in T(17) had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T(17) were mostly biallelic in primary tumor samples with MSI. Patients with stage II–III cancer who received chemotherapy and had large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012–0.8; P = .03). We found a significant interaction between chemotherapy and T17 deletion (P =.009)., Conclusions: About 25% of patients with stages II–III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T(17) intron repeat of HSP110 in tumor DNA.

Currently, the treatment of pediatric high-grade osteosarcomas systematically includes one topoisomerase IIα inhibitor. This chemotherapy is usually adapted to the response to the neo-adjuvant therapy after surgery. The current and unique marker of chemoresponsiveness is the percentage of viable residual cells in the surgical resection. This late patient management marker has to be evaluated earlier in the therapeutic history of the patients on initial biopsy. Therefore, new biomarkers, especially those involved in the topoisomerase IIα inhibitor response might be good candidates. Therefore, our study was designed to target TOP1, TOP2A and TOP2B genes in 105 fresh-frozen diagnostic biopsies by allelotyping and real-time quantitative PCR. Our analyses in those pediatric osteosarcomas, homogeneously treated, highlighted the frequent involvement of topo-isomerase genes. The main and most important observation was the statistical link between the presence of TOP2A amplification and the good response to neo-adjuvant chemotherapy. Compared to adult cancers, the 17q21 amplicon, including TOP2A and ERBB2 genes, seems to be differentially implicated in the osteosarcoma chemoresponsiveness. Surprisingly, there is no ERBB2 gene co-amplification and the patients harboring TOP2A amplification tend to show a worse survival, so TOP2A analyses remain a preliminary, but a good molecular approach for the evaluation at diagnosis of pediatric osteosarcoma chemoresponsiveness.

Background: Colon cancer (CC) pathological staging fails to accurately predict recurrence, and to date, no gene expression signature has proven reliable for prognosis stratification in clinical practice, perhaps because CC is a heterogeneous disease. The aim of this study was to establish a comprehensive molecular classification of CC based on mRNA expression profile analyses., Methods and Findings: Fresh-frozen primary tumor samples from a large multicenter cohort of 750 patients with stage I to IV CC who underwent surgery between 1987 and 2007 in seven centers were characterized for common DNA alterations, including BRAF, KRAS, and TP53 mutations, CpG island methylator phenotype, mismatch repair status, and chromosomal instability status, and were screened with whole genome and transcriptome arrays. 566 samples fulfilled RNA quality requirements. Unsupervised consensus hierarchical clustering applied to gene expression data from a discovery subset of 443 CC samples identified six molecular subtypes. These subtypes were associated with distinct clinicopathological characteristics, molecular alterations, specific enrichments of supervised gene expression signatures (stem cell phenotype-like, normal-like, serrated CC phenotype-like), and deregulated signaling pathways. Based on their main biological characteristics, we distinguished a deficient mismatch repair subtype, a KRAS mutant subtype, a cancer stem cell subtype, and three chromosomal instability subtypes, including one associated with down-regulated immune pathways, one with up-regulation of the Wnt pathway, and one displaying a normal-like gene expression profile. The classification was validated in the remaining 123 samples plus an independent set of 1,058 CC samples, including eight public datasets. Furthermore, prognosis was analyzed in the subset of stage II-III CC samples. The subtypes C4 and C6, but not the subtypes C1, C2, C3, and C5, were independently associated with shorter relapse-free survival, even after adjusting for age, sex, stage, and the emerging prognostic classifier Oncotype DX Colon Cancer Assay recurrence score (hazard ratio 1.5, 95% CI 1.1-2.1, p = 0.0097). However, a limitation of this study is that information on tumor grade and number of nodes examined was not available., Conclusions: We describe the first, to our knowledge, robust transcriptome-based classification of CC that improves the current disease stratification based on clinicopathological variables and common DNA markers. The biological relevance of these subtypes is illustrated by significant differences in prognosis. This analysis provides possibilities for improving prognostic models and therapeutic strategies. In conclusion, we report a new classification of CC into six molecular subtypes that arise through distinct biological pathways.

The Ikaros transcription factor is crucial for many aspects of hematopoiesis. Loss of function mutations in IKZF1, the gene encoding Ikaros, have been implicated in adult and pediatric B cell acute lymphoblastic leukemia (B-ALL). These mutations result in haploinsufficiency of the Ikaros gene in approximately half of the cases. The remaining cases contain more severe or compound mutations that lead to the generation of dominant-negative proteins or complete loss of function. All IKZF1 mutations are associated with a poor prognosis. Here we review the current genetic, clinical and mechanistic evidence for the role of Ikaros as a tumor suppressor in B-ALL.

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.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mismatch repair-deficient colorectal cancers (CRC) display widespread instability at DNA microsatellite sequences (MSI). Although MSI has been reported to commonly occur at coding repeats, leading to alterations in the function of a number of genes encoding cancer-related proteins, nothing is known about the putative impact of this process on non-coding microRNAs. In miRbase V15, we identified very few human microRNA genes with mono- or di-nucleotide repeats (n = 27). A mutational analysis of these sequences in a large series of MSI CRC cell lines and primary tumors underscored instability in 15 of the 24 microRNA genes successfully studied at variable frequencies ranging from 2.5% to 100%. Following a maximum likelihood statistical method, microRNA genes were separated into two groups that differed significantly in their mutation frequencies and in their tendency to represent mutations that may or may not be under selective pressures during MSI tumoral progression. The first group included 21 genes that displayed no or few mutations in CRC. The second group contained three genes, i.e., hsa-mir-1273c, hsa-mir-1303 and hsa-mir-567, with frequent (≥ 80%) and sometimes bi-allelic mutations in MSI tumors. For the only one expressed in colonic tissues, hsa-mir-1303, no direct link was found between the presence or not of mono- or bi-allelic alterations and the levels of mature miR expression in MSI cell lines, as determined by sequencing and quantitative PCR respectively. Overall, our results provide evidence that DNA repeats contained in human miRNA genes are relatively rare and preserved from mutations due to MSI in MMR-deficient cancer cells. Functional studies are now required to conclude whether mutated miRNAs, and especially the miR-1303, might have a role in MSI tumorigenesis.

Heat shock proteins (HSPs) are necessary for cancer cell survival. We identified a mutant of HSP110 (HSP110ΔE9) in colorectal cancer showing microsatellite instability (MSI CRC), generated from an aberrantly spliced mRNA and lacking the HSP110 substrate-binding domain. This mutant was expressed at variable levels in almost all MSI CRC cell lines and primary tumors tested. HSP110ΔE9 impaired both the normal cellular localization of HSP110 and its interaction with other HSPs, thus abrogating the chaperone activity and antiapoptotic function of HSP110 in a dominant-negative manner. HSP110ΔE9 overexpression caused the sensitization of cells to anticancer agents such as oxaliplatin and 5-fluorouracil, which are routinely prescribed in the adjuvant treatment of people with CRC. The survival and response to chemotherapy of subjects with MSI CRCs was associated with the tumor expression level of HSP110ΔE9. HSP110 may thus constitute a major determinant for both prognosis and treatment response in CRC.

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., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Colon carcinogenesis encompasses the stepwise accumulation of genomic aberrations correlated with the transition of aberrant crypt-adenoma-carcinoma. Recent data have revealed that, in addition to the microsatellite-instable phenotype, the chromosome instability pathway, representing four fifth of the colon carcinoma, could be involved in heterogeneous molecular alterations. Our project was aimed at determining the existence of distinct molecular subtypes in 159 non-microsatellite-instable colon polyps and their correlation with histology and dysplasia, using allelotyping, MGMT promoter gene methylation status, and K-RAS mutation analyses. Allelic imbalance, MGMT methylation, and K-RAS mutations arise in 62%, 39%, and 32% of polyps, respectively. Only 14% of polyps had no alterations. A 2-way hierarchical clustering analysis of the allelic imbalances identified subgroups of polyps according to their allelic imbalance frequency and distribution. Not only tubulovillous adenoma but also high-grade adenomas were correlated with high global allelic imbalance frequency (P = .005 and P = .003), with allelic imbalance at microsatellites targeting chromosomes 1, 6, and 9. In conclusion, the data presented in this study show that a large heterogeneity exists in the molecular patterns of alterations in precancerous colon lesions, favoring different modes of tumor initiation. Therefore, molecular alterations correlated with tubulovillous-type and high-grade dysplasia could represent targets identifying predictive factors of progression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

TWIST and adenomatosis polyposis coli (APC) are critical signaling factors in normal bone development. In previous studies examining a homogeneously treated cohort of pediatric osteosarcoma patients, we reported the frequent and concurrent loss of both TWIST and APC genes. On these bases, we created a related animal model to further explore the oncogenic cooperation between these two genes. We performed intercrosses between twist-null/+ and Apc1638N/+ mice and studied their progeny. The Apc1638N/+;twistnull/+ mice developed bone abnormalities observed by macroscopic skeletal analyses and in vivo imaging. Complementary histologic, cellular, and molecular analyses were used to characterize the identified bone tumors, including cell culture and immunofluorescence of bone differentiation markers. Spontaneous localized malignant bone tumors were frequently identified in Apc1638N/+;twist-null/+ mice by in vivo imaging evaluation and histologic analyses. These tumors possessed several features similar to those observed in human localized osteosarcomas. In particular, the murine tumors presented with fibroblastic, chondroblastic, and osteoblastic osteosarcoma histologies, as well as mixtures of these subtypes. In addition, cellular analyses and bone differentiation markers detected by immunofluorescence on tumor sections reproduced most murine and human osteosarcoma characteristics. For example, the early bone differentiation marker Runx2, interacting physically with hypophosphorylated pRb, was undetectable in these murine osteosarcomas, whereas phosphorylated retinoblastoma was abundant in the osteoblastic and chondroblastic tumor subtypes. These characteristics, similar to those observed in human osteosarcomas, indicated that our animal model may be a powerful tool to further understand the development of localized osteosarcoma.

Background and Aims: O(6)-Methylguanine-DNA methyltransferase (MGMT) removes methyl adducts from O(6)-guanine. Known as methylation tolerance, selection for mismatch repair (MMR)-deficient cells that are unable to initiate lethal processing of O(6)-methylguanine-induced mismatches in DNA is observed in vitro as a consequence of MGMT deficiency. It was therefore hypothesised that an MGMT field defect may constitute a preneoplastic event for the development of MMR-deficient tumours displaying microsatellite instability (MSI)., Methods: MGMT expression was investigated by immunohistochemistry and the methylation status of the gene promoter by PCR in neoplastic, adjacent and distant mucosal tissues of patients with MSI or non-MSI (MSS) colorectal cancer (CRC). The cancers were familial (42 MSI, 13 MSS) or sporadic (40 MSI, 49 MSS) in origin, or arose in the context of inflammatory bowel disease (IBD; 13 MSI, 36 MSS). Colonic mucosa from patients with diverticulitis (n=20) or IBD (n=39 in 27 patients) without cancer served as controls., Results: Loss of MGMT expression was more frequent in MSI than MSS CRC (p=0.047). In comparison with MSS tumours, MSI CRC occurred more frequently adjacent to patches of mucosa that lacked MGMT expression (p=0.002). Overall, loss of MGMT expression was associated with MGMT gene promoter methylation (p=0.03)., Conclusion: MGMT field defects are more frequently associated with MSI than MSS CRC. These findings indicate that methylation tolerance may be a crucial initiating step prior to MMR deficiency in the development of MSI CRC in familial, sporadic and IBD settings.

Background: Numerous studies reported genomic alterations in colorectal human tumors but few focused on rectal tumors with the specification of preoperative-treated or untreated tumors. The goals of this study were to list chromosome allelic imbalances and correlate their frequency with tumor progression and to identify potential molecular markers of progression in rectal chromosomally unstable tumors without preoperative treatment., Methods: Genomic alterations of 57 rectal tumors assessed by allelotyping targeting 33 chromosomal loci, were clusterised and compared to those of 151 left colon tumors., Results: Clustering separated the rectal tumors without preoperative treatment into three subtypes according to the allelic imbalance frequency and genomic alteration associations. The tumors without preoperative treatment displayed a significantly higher allelic imbalance frequency (54%) than the tumors with preoperative treatment (33%), suggesting that treatment could target highly altered tumor clones. Interestingly, the survival analysis identified three potential prognostic molecular survival markers, D1S197, D5S430, and D14S65, for tumors without preoperative treatment., Conclusion: Based on the genomic status of 33 chromosomal loci, we observed that rectal tumors without preoperative treatment segregate according to the global allelic imbalance frequency but without correlation to the tumor progression. Moreover, the detailed associations of alterations in rectal tumors are different from those described in colon tumors suggesting that rectal and left tumors should be considered as separate entities. Finally, potential prognostic genomic molecular markers for survival are proposed which status could specify the clinical course of the tumors.

Introduction: The purpose of this study was to investigate the gene expression of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) in circulating mononuclear cells harvested from septic shock patients on drotrecogin-α activated (DAA) in order to determine whether this treatment has any effect on the inflammation phase., Methods: We conducted a prospective cohort study in two intensive care departments. Blood samples were collected at inclusion (T1) and 36 hours later (T2) to measure plasma cytokines and the changes in intracellular TNF-α, IL-10 and IFN-γ mRNA expressions using the real-time quantitative polymerase chain reaction (RT-qPCR). Thirty-two septic shock patients were included: 16 with DAA at 24 μg/kg/h for 96 hours (DAA+) and 16 control (DAA-) eligible but contraindicated for DAA because of low platelet count., Results: The basal characteristics were similar in both groups: mortality (50%), plasma cytokine concentrations, and baseline IFN-γ, TNF-α and IL-10 mRNA expressions (DAA+ vs. DAA-). At T2, there was a significant IFN-γ gene down-regulation in DAA+ but not in DAA- patients (-0.34 (-0.62; +1.54) vs. +1.41 (+0.35; +5.87), P = 0.008). In survivors, DAA administration was associated with a down-expression of both IFN-γ (-0.65 (-0.93; 0.48) vs. +0.7 (-0.04; +1.26), P = 0.01) and IL-10 (-0.78 (-0.92; -0.6) vs. -0.18 (-0.68; +0.46), P = 0.038). In the non-survivors, DAA infusion was associated with IL-10 over-expression when compared with survivors (+0.54 (-0.35; +11.52) vs. -0.78 (-0.92; -0.6), P < 0.001)., Conclusions: In this study, lack of IL-10 gene down-expression despite a 36-hour infusion of DAA is an ominous sign in septic shock patients suggesting that DAA is not able to reverse the outcome. Our results suggest that DAA can decrease the expression of anti-inflammatory cytokines in septic shock patients. IL-10 or IFN-γ gene down-expression could represent markers of DAA response.

Crohn's disease is considered to be caused either by an excess of T-cell effector functions and/or by a defective regulatory T-cell compartment. The aim of this study was to assess in Crohn's disease the frequency of circulating CD4(+)CD25(high) T cells that possess regulatory T-cell functions and CD4(+)CD25(low) T cells that contain activated T cells. Flow cytometry of peripheral blood was used to assess CD4(+)CD25(high) and CD4(+)CD25(low) T-cell frequencies in a cohort of 66 patients with Crohn's disease in comparison to 19 patients with ulcerative colitis and 31 healthy individuals enrolled as controls. The CD4(+)CD25(high) T-cell frequency was significantly lowered in naïve Crohn's disease (P = 0.013) and in ulcerative colitis (P = 0.001). CD4(+)CD25(low) T-cell frequency was increased in Crohn's disease (P = 0.0001) and in ulcerative colitis (P = 0.0002). Both CD4(+)CD25(high) and CD4(+)CD25(low) T-cell frequencies are altered in naïve Crohn's disease resulting in an imbalance between both populations and a relative contraction of the CD4(+)CD25(high) T-cell population.

The genomic aberration profile of chromosome 20q in distal CIN colon carcinomas was analysed using allelotyping and CGH arrays. Allelotyping revealed carcinomas with allelic imbalance along the full long arm, and carcinomas with fully non-aberrant 20q. Oligonucleotide-based CGH showed that among the carcinomas without allelic imbalance, 47% had in fact a gain. In this subgroup, quantitative PCR for the TOPI gene (20q12) confirmed this gain, and fluorescence in situ hybridization showed that the chromosome 20q gain resulted from tetra/polysomy instead of aneusomy. The 20q gain correlated with a high frequency of aberrations, with allelic imbalance at TP53 locus but not at APC locus, and carcinomas with a disomic 20q showed low frequency of genomic aberrations and were significantly associated to mucinous phenotype. The prognostic value of 20q amplification was not demonstrated in this study. These results indicate that on the basis of aberration frequency, chromosome 20q and TP53/APC locus status, distal CIN carcinomas harbor a high degree of genetic heterogeneity suggesting several pathways for carcinogenesis. This study also indicates that allelotyping needs to be carried out with a complementary technique, such as quantitative PCR.

Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P<0.005), among which five in samples with low tumour cellularity. In adenocarcinoma, K-Ras mutation frequency increased from 7 out of 55 (13%) by direct sequencing to 15 out of 55 (27%) by clamped-PCR (P<0.005). K-Ras mutations detected by these sensitive techniques lost its prognostic value. In conclusion, a rapid and sensitive PCR-clamping test avoiding macro or micro dissection could be proposed in routine analysis especially for NSCLC samples with low percentage of tumour cells such as bronchial biopsies or after neoadjuvant chemotherapy.

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-1alpha (HIF-1alpha), a key transcription factor with a pivotal role in tumor cell metabolism. One potential class of therapeutic agents targeting HIF-1alpha 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-1alpha. 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-1alpha 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-1alpha 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-1alpha as a promising target and provide a rationale for clinical trials of low-dose irinotecan and rapamycin combination toward metastatic colon cancer.

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib improves survival of lung cancer as second- or third-line therapy. However, after an initial response, most patients will recur, particularly within the central nervous system. The present study reports the case of a 27-yr-old nonsmoking male presenting with a metastatic lung adenocarcinoma with EGFR exon 19 deletion, associated with sensitivity to EGFR-TKI. Gefitinib, followed by chemotherapy and finally erlotinib resulted in prolonged disease control, until multiple liver metastases were detected. After stopping EGFR-TKI, brain metastases with carcinomatous meningitis were diagnosed. A secondary T790M mutation, associated with resistance to EGFR-TKI, was found on the liver biopsy but not in the cerebrospinal fluid. Erlotinib was reintroduced and allowed a quick neurological improvement, even though the extra-cranial disease remained resistant to erlotinib. The present report underscores the interest of molecular monitoring in lung cancer. Persistent cerebral tyrosine kinase inhibitor sensitivity should be considered in patients presenting with an early central nervous system relapse after stopping epidermal growth factor receptor tyrosine kinase inhibitor, even with a T790M-resistant mutation in noncerebral metastases. Questions remain concerning the selection of sub-clones during epidermal growth factor receptor tyrosine kinase inhibitor therapy, which could differ according to metastatic sites, especially in the central nervous system.

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.

Introduction: Recent clinical success of epidermal growth factor (EGFR)-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) have raised hopes that targeting other deregulated growth factor signaling, such as the hepatocyte growth factor/MET pathway, will lead to new therapeutic options for NSCLC. Furthermore, NSCLC present secondary EGFR-TKIs resistance related to exons 20 and 19 EGFR mutations or more recently to MET amplification. The aim of this study was to determine MET copy number related to EGFR copy number and K-Ras mutations in a targeted TKI naive NSCLC cohort., Methods: We investigated 106 frozen tumors from surgically resected NSCLC patients. Genes copy number of MET and EGFR were assessed by quantitative relative real-time polymerase chain reaction and K-Ras mutations by sequencing., Results: MET is amplified in 22 cases (21%) and deleted in nine cases (8.5%). EGFR is amplified in 31 cases (29%). K-Ras is mutated in 11 cases (10.5%). As observed for EGFR amplification, MET amplification is never associated with K-Ras mutation. MET amplification could be associated with EGFR amplification. MET amplification is not related to clinical and pathologic features. MET amplification and EGFR amplification showed a trend toward poor prognosis in adenocarcinomas., Conclusion: In EGFR-TKIs naive NSCLC patients, MET amplification is a frequent event, which could be associated with EGFR amplification, but not with K-Ras mutation. MET amplification may identify a subset of NSCLC for new targeted therapy. It will also be important to evaluate MET copy number to properly interpret future clinical trials.

Several publications have recently focused on the erbB2 receptor in pediatric medulloblastomas (MBs) and its prognostic consequence. We determined erbB2 expression in 23 MBs at diagnosis. After DNA extraction, quantitative PCR targeting the erbB2 gene was performed and correlated with FISH analysis and immunohistochemistry. The samples were representative of the spectrum of the disease apart from the absence of large cell MBs. Using the tools validated for breast cancers by the FDA, we did not observe any expression or amplification of erbB2 and hence we speculate that MBs are not a good target for treatment with anti-erbB2 antibodies., ((c) 2007 Wiley-Liss, Inc.)

Association of a t(9;22)(q34;q11), BCR/ABL-positive, with a dic(19;21)(p13;p13) has been described in acute lymphoblastic leukemia in relapse, raising the question of whether this association is recurrent. Described here are two cases, one of myeloproliferative disease and one of acute lymphoblastic leukemia, both presenting a masked t(9;22) and t(19;21). Chromosomal rearrangements were ascertained by fluorescence in situ hybridization (FISH) using locus-specific probes, multicolor FISH, and bacterial artificial chromosome array. These additional observations suggest a nonrandom association.

Dysregulated cell growth or differentiation due to misexpression of developmental critical factors seems to be a decisive event in oncogenesis. As osteosarcomas are histologically defined by malignant osteoblasts producing an osteoid component, we prospected in pediatric osteosarcomas treated with OS94 protocol the genomic status of several genes implied in ossification processes. In 91 osteosarcoma cases, we focused on the analysis of the fibroblast growth factor receptors (FGFRs) TWIST, APC, and MET by allelotyping, real-time quantitative polymerase chain reaction, gene sequencing, and protein polymorphism study. Our study supports the frequent role of TWIST, APC, and MET as osteosarcoma markers (50%, 62%, and 50%, respectively). TWIST and MET were mainly found to be deleted, and no additional APC mutation was identified. Surprisingly, FGFRs are abnormal in only < 30%. Most of these factors and their abnormalities seem to be linked more or less to one clinical subgroup, but the most significant correlation is the link of MET, TWIST, and APC abnormalities to a worse outcome and their combination within abnormal tumors. A wider cohort is mandatory to define more robust molecular conclusions, but these results are to be considered as the beginning of a more accurate basis for diagnosis, in search of targeted therapies, and to further characterize prognostic markers.

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.

The expression of the CDX2 gene, a crucial regulator of gut homeostasis, is altered in human colorectal cancers in parallel with de-differentiation. Here, we have investigated the chromosomal status of CDX2 in human sporadic colorectal cancers with the phenotype of chromosomal instability. Allelic imbalance determination showed frequent rearrangements at the CDX2 locus. The rearrangements correlated with CDX2 gene amplification, as assessed by quantitative PCR analysis. However, they were not predictive of the Cdx2 protein pattern. These data suggest that mechanisms other than structural alterations at the CDX2 locus account for the change of expression in colorectal cancers.

In colorectal cancer, the molecular alterations that lead to metastasis are not clearly established, probably because of their high genetic complexity. To identify combinations of genetic changes involved in tumor progression and metastasis, we focused on chromosome instable (CIN) colon cancers. We compared by allelotyping of 33 microsatellites, the genomic alterations of 38 primary colon tumors with the synchronously resected matched liver metastases (CLM). We observed that (i) the number of patients with alterations at certain loci did not differ significantly between the whole primary tumor and the paired CLM, (ii) a group of patients had fewer alterations in the metastasis when compared with the matched primary tumor. A 2-way hierarchical unsupervised clustering of the allelotyping data revealed 2 tumor subtypes that have different levels of CIN (CIN-High, CIN-Low). Both subtypes have a minimal common set of alterations at chromosomes 8p, 17p and 18q, but does not include alteration at 5q or mutation at K-Ras. These 2 subtypes were also observed using a collection of 104 independent primary CIN colon tumors. In addition, we found a third subtype, consisting of tumors with a very low number of alterations not associated with specific loci (CIN-Very Low). We found that colon carcinogenesis may require a minimal set of alterations and that, in contrast to the current hypothesis, the level of CIN does not correlate with tumor progression. Therefore, our results suggest that metastasis potential could be present at very early stages of tumor development.

Objective: To detect the presence of tumour DNA in urine of patients with renal cancer based on microsatellite analysis., Material and Method: Blinded, comparative, experimental study conducted between July 1996 and December 2003. Preoperative urine and blood samples were collected from 69 patients with renal cancer (pT1 to pT4). The control population comprised 35 patients with a benign urological disease. Loss of heterozygosity (LOH) and allele amplification were investigated by analysing allele imbalances between urinary DNA and blood lymphocyte DNA. Twenty-six loci were analysed, including 23 microsatellites. We studied the sensitivity and specificity of this analysis as a function of stage, grade and invasion of renal cavities by the tumour., Results: The sensitivity of the test was 61% for pT1a and the global sensitivity was 62.3%. A significant difference was observed in favour of low nuclear grades (p < 0.05). More than 80% of tumours detected were identified by 4 microsatellites. The specificity was 83%. Renal cavities were invaded by the tumour in 38% of cases. No correlation was observed between invasion of renal cavities and the result of the test., Conclusion: The results are encouraging, particularly for small tumours and justify development of clinical applications.

Evaluation of the role of clonal heterogeneity in colon tumour sensitivity/resistance to drugs and/or in conferring metastatic potential requires an adequate experimental model in which the tumour cells maintain the initial genetic alterations and intra-tumoral heterogeneity through maintenance of the genetic clones present in the initial tumour. Therefore, we xenografted subcutaneously into nude mice seven human colonic tumours (from stages B1 to D) that showed chromosome instability and transplanted them sequentially for up to 14 passages. Maintenance after xenografting of the genetic alterations present in the initial tumours was scored by allelotype studies targeting 45 loci localized on 18 chromosomes. We show that xenografting does not alter the genetic or the histological profiles of the tumours even after 14 passages. Screening of the entire genome of one tumour by comparative genome hybridization also showed overall stability of the alterations between the initial and the xenografted tumour. In addition, intra-tumoral heterogeneity was maintained over time, suggesting that no clonal selection occurred in the nude mice. The observation that some loci showed partial allelic imbalance in the initial tumour but loss of heterozygosity after the first passage in nude mice when all the normal cells were lost may allow identification of interesting genetic defects that could be involved in tumour expansion. Thus, sequential xenografts of colon tumours will provide a powerful model for further study of tumour clonality and for the identification of genetic profiles responsible for differential resistance to therapeutic treatments. Our data also suggest that tumour expansion can result from alterations in several distinct genetic pathways.

The identification of genes as markers for chromosome aberrations in specific tumors might facilitate oncogenesis mechanism comprehension, cancer detection, prediction of clinical outcomes, and response to therapy. Previous physiologic and oncologic data identified the TWIST gene as a marker for mesodermal derivative and bone tissue differentiation, but its contribution to bone malignancies has not been investigated. In the present study, search for genomic alterations in high-grade pediatric osteosarcomas was focused on the 7p21 region, and more specifically on the TWIST gene. In a cohort of 74 patients, we observed by allelotyping that 31 of 68 informative tumors were rearranged at the TWIST locus. Among them, analysis by quantitative PCR (QPCR) revealed that, surprisingly, mostly deletions (22/68), but also amplifications (9/68), of the TWIST gene were detected. Furthermore, deletions at TWIST were statistically correlated to other molecular abnormalities, like alterations at the APC or c-kit loci, as well as to clinical features such as a poor outcome. This work shows that the TWIST gene seemed to be involved in high-grade pediatric osteosarcomas and is a new marker with a possible initial predictive value., ((c) 2005 Wiley-Liss, Inc.)

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.

Background: Posttransplant lymphoproliferative disorders (PTLD) occur in 0.5% to 2.5% of cases in renal-transplant recipients. Epstein-Barr virus (EBV) is usually detected in the tumor cells, suggesting a role for this virus as an agent of B-cell proliferation. It is unusual for patients receiving allografts from the same donor to develop PTLD simultaneously., Methods: we describe two patients who received renal allografts from the same donor and developed PTLD simultaneously. The presence of EBV in both tumors was confirmed. In this report, the origin of tumor cells was determined by immunohistochemical human leukocyte antigen (HLA) typing and microsatellite analysis. Clonality was studied by immunoglobulin gene rearrangement analysis., Results: Our results suggest that the tumor originated from donor cells in both patients but, because immunoglobulin gene rearrangements were different, this could mean that lymphoid cells proliferate independently in each recipient., Conclusions: We propose the following pathogenesis: immortalization of passenger B lymphocytes by EBV, proliferation of these cells, and development of PTLD by means of immunosuppression, antigenic stimulation, and HLA mismatch.

Objective: To assess the levels of expression of the antiapoptotic gene Bcl-2 and the proapoptotic gene Bax in circulating mononuclear cells (CMNC) harvested during the course of severe sepsis (SS) in formerly non-immunocompromised patients undergoing hospital-acquired infection, in parallel to cytokine levels., Design: Prospective study., Setting: Intensive care unit., Participants: A total of 24 patients without immunodeficiency undergoing standard goal-directed therapy for nosocomial SS, 10 critically ill patients without sepsis, and 10 healthy controls., Interventions: Blood was collected before infection and within 12 h, 1, 3 and 7 days after fever onset, to determine plasma concentrations of IL-6, IL-10, TNF-alpha, C-reactive protein, whole blood cell counts, lymphocyte subsets, annexin V labelling for apoptosis, and Bax and Bcl-2 relative RNA expression by real-time polymerase chain reaction., Results: SS patients displayed increased cytokine concentrations, TNF-alpha being significantly increased at full-blown sepsis. Within 12 h after onset of infection, lymphocyte counts were lower in SS patients than in critically ill controls ( p=0.001), and this phenomenon was marked in CD4+ and CD8+ subsets ( p<0.001). This was associated with enhanced apoptosis in CMNC (15.7+/-8.7% vs 3.4+/-2.1%, p<0.001) and a significant down-expression of the Bcl-2 gene throughout the study ( p<0.05). In contrast, the expression of Bax did not change significantly. Within 12 h of fever onset, non-survivors expressed a 10-fold down-expression of Bcl-2 when compared to survivors ( p<0.001)., Conclusions: An early transient down-expression of the gene Bcl-2 occurred in CMNC harvested from SS patients who died despite intensive care. In contrast, the expression of the gene Bax did not change significantly.

The purpose of this paper is the construction of an adapted statistical test for urinary detection of bladder cancer based on the assessment of allelic imbalance using biallelic Short Insertion/Deletion Polymorphism DNA markers. This test is based on the comparison of several of these markers, analysed from urinary DNA, to the distribution of their analogues in blood, which is taken as reference. A reproductibility study is first performed in blood in order to make a selection among the 23 available markers and 18 are retained. A global score based on the chi2 distribution is then built to test for allelic imbalance among all informative markers for each patient. It avoids the count of DNA abnormalities marker by marker and allows to take into account the degree of abnormality of each marker. That method is preliminarily evaluated on a sample of 53 patients and 27 controls. The estimated specificity (96.3%) on that reduced sample is satisfactory, whereas sensitivity (60.4%) could be improved by an augmentation of the number of tested markers. Several issues regarding the validity of the method are discussed., (Copyright 2003 John Wiley & Sons, Ltd.)

Fibroblast growth factors (FGF), hepatocyte growth factor (HGF) and their receptors, FGFR and c-Met, are essential components of the regulatory networks between the epithelium and mesenchyme in embryonic lung, but their respective roles in tumour growth are not clear. We performed allelotyping at loci containing the candidate genes FGFR-1-2-3-4, FGF-1-2-7-10, c-Met and HGF in 36 non-small cell lung cancer (NSCLC) (20 squamous-cell carcinomas (SQC) and 16 adenocarcinomas (ADC)), by surrounding each locus with two microsatellites (MS), as close as possible to the genes of interest. Unexpectedly, SQC and ADC were frequently altered at all of these loci, and SQC showed more simultaneously altered loci. In ADC, alterations at the 15q13-22 locus (FGF7 candidate gene) were significantly more frequent. Thus, these loci showed different patterns of molecular alterations between SQC and ADC. Finally, alterations at loci containing FGFR and HGF candidate genes were inversely correlated to the lymph node status in SQC and ADC, respectively.