Raphaël Saffroy

Microsatellite instability in European hepatocellular carcinoma.

Genetic instability has been detected in many types of cancers but poorly investigated in hepatocellular carcinoma (HCC). We have studied the incidence of microsatellite instability (MI) at eight highly polymorphic microsatellite markers and the poly A tract BAT26 and tested for mutations at two sites of repetitive sequence (poly-A nucleotides 709 – 718 and GT repeat-nucleotides 1931 – 1936) in the Transforming Growth Factor β (TGFβ) type II receptor (RII) gene, in a group of 46 European HCCs and the surrounding nontumour tissue. This analysis showed that 63% of HCCs exhibit MI in at least one chromosome locus and 41% in two or more loci. No mutations of the TGFβRII gene were found in the MI positive tumours. No correlation was found with clinicopathological characteristics of the tumours such as cirrhosis, etiology, number of nodules, Edmondsons grade and vascular invasion. However, in patients who had a rearranged D16S402 microsatellite in their tumour, the recurrent disease and the number of nodules were significantly higher than in the others (P<0.005 and P<0.02, respectively). We propose to consider D16S402 rearrangement in HCC as a prognostic factor to identify patients presenting a higher risk of recurrence.

Exploration of global gene expression in human liver steatosis by high-density oligonucleotide microarray.

Understanding the molecular mechanisms underlying fatty liver disease (FLD) in humans is of major importance. We used high-density oligonucleotide microarrays (22.3 K) to assess the mechanisms responsible for the development of human liver steatosis. We compared global gene expression in normal (n=9) and steatotic (n=9) livers without histological signs of inflammation or fibrosis. A total of 34 additional human samples including normal (n=11), steatosis (n=11), HCV-related steatosis (n=4) or steatohepatitis associated with alcohol consumption (n=4) or obesity (n=4) were used for immunohistochemistry or quantitative real-time PCR studies. With unsupervised classification (no gene selection), all steatotic liver samples clustered together. Using step-down maxT multiple testing procedure for controlling the Family-Wise Error-Rate at level 5%, 110 cDNAs (100 over- and 10 underexpressed) were found to be differentially expressed in steatotic and normal livers. Of them were genes involved in mitochondrial phosphorylative and oxidative metabolism. The mean ratio of mitochondrial DNA to nuclear DNA content was higher in liver steatosis compared to normal liver biopsies (1.12±0.14 vs 0.67±0.10; P=0.01). An increased expression of genes involved in inflammation (IL-1R family, TGFB) was also observed and confirmed by quantitative RT-PCR or immunochemistry. In steatohepatitis, an increase of the protein expression of mitochondrial antigens, IL-1R1, IGF2 and TGFB1 was also observed, interleukin 1 receptor being always strongly expressed in steatohepatitis linked to alcohol or obesity. In conclusion, mitochondrial alterations play a major role in the development of steatosis per se. Activation of inflammatory pathways is present at a very early stage of steatosis, even if no morphological sign of inflammation is observed.

New perspectives and strategy research biomarkers for hepatocellular carcinoma.

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Cirrhosis caused by hepatitis B virus, hepatitis C virus or chronic alcohol intake is associated with major risk. Systematic screening for HCC of asymptomatic patients with cirrhosis is needed for earlier detection of small tumors requiring treatment (liver transplantation, surgical resection, percutaneous techniques). The recommended screening strategy among cirrhotic patients is based on regular liver ultrasonography associated with serum α-fetoprotein (AFP) assay. As the performance of AFP is not satisfactory, additional tumoral markers are proposed (des-γ-carboxyprothrombin, glycosylated AFP-L3 fraction). Currently, diagnosis of HCC in cirrhotic patients includes non-invasive tests (imaging after contrast administration, AFP assay); diagnostic biopsy is performed when imaging is limited. After treatment, tumor recurrence is assessed by regular follow-up (AFP assay and imaging). Despite the lack of accurate markers, recent developments in genomic and proteomic approaches will allow the discovery of new biomarkers for primary tumors, as well as for recurrence. This review summarizes the current state of biomarkers for screening, diagnosis and follow-up of HCC, and highlights new perspectives in the field. Clin Chem Lab Med 2007;45:1169–79.

Expression of T-cadherin in tumor cells influences invasive potential of human hepatocellular carcinoma

Overexpression of T‐cadherin (T‐cad) transcripts occurs in ≈50% of human hepatocellular carcinomas (HCCs). To elucidate T‐cad functions in HCC, we examined T‐cad protein expression in normal and tumoral human livers and hepatoma cell lines and investigated its influence on invasive potential of HCC using RNA interference silencing of T‐cad expression in Mahlavu cells. Whereas T‐cad expression was restricted to endothelial cells (EC) from large blood vessels in normal livers, it was up‐regulated in sinusoidal EC from 8/15 invasive HCCs. Importantly, in three of them (38%) T‐cad was detected in tumor cells within regions in which E‐cadherin expression was absent. Among six hepatoma cell lines, only Mahlavu expressed T‐cad but not E‐cadherin. T‐cad exhibited a globally punctuate distribution in quiescent Mahlavu and additionally it concentrated at the leading edge of migrating cells. Matrigel invasion assay revealed that Mahlavu possess a high invasive potential that was significantly inhibited by T‐cad silencing. Wound healing and random motility assays demonstrated that inhibition of T‐cad expression in Mahlavu significantly reduced their motility. We propose that T‐cad expression in tumor cells might occur by cadherin‐switching during epithelial‐mesenchymal transition and may represent an additional mechanism contributing to HCC metastasis.—Riou, P., Saffroy, R., Chenailler, C., Franc, B., Gentile, C., Rubinstein, E., Resink, T., Debuire, B., Piatier‐Tonneau, D., Lemoine, A. Expression of T‐cadherin in tumor cells influences invasive potential of human hepatocellular carcinoma. FASEB J. 20, 2291–2301 (2006)

Acquired KRAS mutations during progression of colorectal cancer metastases: possible implications for therapy and prognosis

PurposeDocumentation of a wild-type (wt) KRAS gene in tumor has become mandatory for the prescription of anti-EGFR monoclonal antibodies in patients with colorectal cancer (CRC). Acquired KRAS mutations have seldom been reported in metastases from wtKRAS primary CRC. We report the first case of multiple KRAS mutations acquired during the metastatic phase of CRC, and retrospectively reviewed all patients with CRC, in whom KRAS was analyzed in at least two tumor samples from distinct lesions.MethodsGenomic DNA purified from paraffin-embedded tissues was used after histological quantification of tumor tissue. The seven KRAS mutations located within codons 12 and 13 were screened using the allelic discrimination assay.ResultsA 35-year-old woman with CRC liver metastasis, resistant to all conventional cytotoxic agents, experienced for the first time significant tumor shrinkage while cetuximab was added, allowing hepatic resection. Further liver relapse occurred on cetuximab, but a new hepatic resection was attempted. No mutation in KRAS was detected in the primary colon tumor or in synchronous liver metastases. In contrast, in metachronous liver metastasis samples, two distinct mutations at codon 13 and 12 were detected. No acquired mutations were found in all the other 12 CRC cases with at least two serially performed KRAS analyses.ConclusionsOur findings suggest that late switch in KRAS mutational status could occur more frequently than currently recognized and account for acquired resistance to anti-EGFR therapies. Prospective studies are warranted to better estimate the incidence of change in KRAS mutational status and assess their clinical relevance.

Prospective evaluation of blood concentration of mitochondrial DNA as a marker of toxicity in 157 consecutively recruited untreated or HAART-treated HIV-positive patients.

Highly active antiretroviral therapy (HAART) can cause mitochondrial toxicity. The concentration of mitochondrial DNA (mtDNA) in peripheral blood cells has been reported to be a marker of this toxicity. However, these observations are controversial and were drawn from small series. Thus, we analysed the value of blood mtDNA as a marker of mitochondrial toxicity in a large cohort of human immunodeficiency virus (HIV)-infected out-patients during routine clinical evaluations. Real-time quantitative PCR was used to determine the mtDNA to nuclear DNA (nDNA) ratio in peripheral blood mononuclear cells from 157 consecutive HIV-1-infected patients (13 naive, 144 receiving HAART) and 30 HIV-1-uninfected patients. The mtDNA to nDNA ratio was significantly lower in both groups of HIV-infected patients than in the control group. No significant difference was observed between treated and naive HIV-infected patients. Lactataemia was significantly lower in controls than in the group of HIV-treated patients. None of the treated patients had lactataemia >5 mmol/l or bicarbonates <20 mmol/l. Triglyceride levels were significantly higher in the HAART-treated patients than in the nontreated patients. Clinical symptoms of lipodystrophy were observed in 62 HAART-treated patients. These symptoms were not associated with an abnormal mtDNA to nDNA ratio or plasma triglyceride concentration. The mtDNA to nDNA ratio was lower in DDI/D4T-treated patients than in AZT/3TC-treated patients. In conclusion, there are no obvious links between the mtDNA to nDNA ratio in peripheral mononuclear cells and any clinical symptoms or lactate level. Thus, the mtDNA to nDNA ratio in leukocytes does not seem to be an accurate marker of mild and/or long-term mitochondrial toxicity.

Clinical significance of circulating anti-p53 antibodies in European patients with hepatocellular carcinoma

Summaryp53 alterations are considered to be predictive of poor prognosis in hepatocellular carcinoma (HCC) and may induce a humoral response. Anti-p53 serum antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) using purified recombinant human p53 on 130 European HCC patients before treatment and during the clinical course of the disease. p53 immunohistochemistry was performed on tumours from the 52 patients who underwent surgery, and DNA sequencing analysis was initiated when circulating anti-p53 antibodies were detected. Nine (7%) HCC patients had anti-p53 serum antibodies before treatment. During a mean period of 30 months of follow-up, all the negative patients remained negative, even when recurrence was observed. Of the nine positive patients, eight were still positive 12–30 months after surgery. The presence of anti-p53 serum antibodies was correlated neither with mutation of the p53 gene nor the serum alpha-fetoprotein levels and clinicopathological characterics of the tumours. However, a greater incidence of vascular invasion and accumulation of p53 protein were observed in the tumours of these patients (P < 0.03 and P < 0.01 respectively) as well as a better survival rate without recurrence (P = 0.05). In conclusion, as was recently shown in pancreatic cancer, anti-p53 serum antibodies may constitute a marker of relative ‘good prognosis’ in a subgroup of patients exhibiting one or several markers traditionally thought to be of bad prognosis.

Co expression of SCF and KIT in gastrointestinal stromal tumours (GISTs) suggests an autocrine/paracrine mechanism.

KIT is a tyrosine kinase receptor expressed by several tumours, which has for specific ligand the stem cell factor (SCF). KIT is the main oncogene in gastrointestinal stromal tumours (GISTs), and gain-of-function KIT mutations are present in 70% of these tumours. The aim of the study was to measure and investigate the mechanisms of KIT activation in 80 KIT-positive GIST patients. KIT activation was quantified by detecting phosphotyrosine residues in Western blotting. SCF production was determined by reverse transcriptase–PCR, ELISA and/or immunohistochemistry. Primary cultures established from three GISTs were also analysed. The results show that KIT activation was detected in all cases, even in absence of KIT mutations. The fraction of activated KIT was not correlated with the mutational status of GISTs. Membrane and soluble isoforms of SCF mRNA were present in all GISTs analysed. Additionally, SCF was also detected in up to 93% of GISTs, and seen to be present within GIST cells. Likewise, the two SCF mRNA isoforms were found to be expressed in GIST-derived primary cultures. Thus, KIT activation in GISTs may in part result from the presence of SCF within the tumours.

High-throughput somatic mutation profiling in pulmonary sarcomatoid carcinomas using the LungCarta™ Panel: exploring therapeutic targets

BACKGROUND Pulmonary sarcomatoid carcinomas (SC) are tumors characterized by poor prognosis and resistance to conventional platinum-based chemotherapy. This study sought to describe the mutational profile of SC using high-throughput genotyping technology. PATIENTS AND METHODS We used mass spectrometry to test 114 surgical biopsies from 81 patients with SC for 214 mutations affecting 26 oncogenes and tumor suppressor genes. RESULTS In total, 75 (92.6%) patients were smokers. Within the total 81 tumors, 67 distinct somatic alterations were identified, with 56 tumors (69.1%) harboring at least one mutation. The most frequent mutations were KRAS (27.2%), EGFR (22.2%), TP53 (22.2%), STK11 (7.4%), NOTCH1 (4.9%), NRAS (4.9%), and PI3KCA (4.9%). The EGFR mutations were almost always rare mutations (89%). In 32 tumors (39.5%), two or more mutations co-existed, with up to four mutations in a single case. In six different cases, comparative genetic analysis of different histological areas from the same tumor (giant, spindle, or epithelial component) revealed a 61% concordance rate for all the mutations with a 10% detection threshold, compared with 91.7% with a 20% detection threshold. CONCLUSION Our results demonstrated a high mutation rate and frequent co-mutations. Despite SC tumors exhibiting a high histological heterogeneity, some intratumoral molecular homogeneity was found. Now with newly developed targeted therapies, SC patients may be eligible for new target mutations, and can now therefore be screened for clinical trials.

Real-Time Quantification of AFP mRNA to Assess Hematogenous Dissemination After Transarterial Chemoembolization of Hepatocellular Carcinoma

Objective To determine whether the number of hepatocytes containing AFP mRNA shed into the bloodstream during transarterial chemoembolization (TAE) affects the incidence and pattern of recurrence of hepatocellular carcinoma (HCC). Patients and Methods We developed a Taqman procedure to quantify AFP mRNA prospectively in 52 consecutive patients before and after TAE. Results are expressed in hepatocytes /mL. Results Thirteen of the patients (24.5%) were positive for AFP mRNA (42 ± 19 hepatocytes/mL) before TAE and 13 (24.5%) (80 ± 32 hepatocytes/mL) after TAE; the difference was not significant. The presence of AFP mRNA in the bloodstream before TAE was associated with larger nodules (85.2 ± 73.8 mm versus 34.8 ± 26.1 mm; P = 0.006). Six of the patients were excluded from the analysis because they underwent curative surgery or were lost to follow-up. The circulating levels of AFP mRNA released in the 46 remaining patients after TAE did not affect metastasis-free survival. A significant number of extrahepatic metastases were found in patients exhibiting at least 1 AFP mRNA-positive blood sample either before or after TAE. However, the TAE procedure did not increase the risk of extrahepatic recurrences. Conclusion Cells containing AFP mRNA are inconsistently released into the circulation during TAE. The amount of these cells released does not affect the recurrence of HCC.