Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines.

The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e.g., cancer-derived cells), obtaining a knockout usually requires lengthy screening and characterization procedures. Given the more precise type of modifications that can be introduced upon homology-directed repair (HDR), we have developed HDR-based gene-targeting strategies that greatly facilitate the process of knockout generation in cell lines. To generate reversible knockouts (R-KO), a selectable promoter-less STOP cassette is inserted in an intron, interrupting transcription. Loss-of-function can be validated by RT-qPCR and is removable, enabling subsequent restoration of gene function. A variant of the R-KO procedure can be used to introduce point mutations. To generate constitutive knockouts (C-KO), an exon is targeted, which makes use of HDR-based gene disruption together with NHEJ-induced indels on non-HDR targeted allele(s). Hence the C-KO procedure greatly facilitates simultaneous inactivation of multiple alleles. Overall these genome-editing tools offer superior precision and efficiency for functional genetic approaches. We provide detailed protocols guiding in the design of targeting vectors and in the analysis and validation of gene targeting experiments.

IFNL3 (IL28B) polymorphism does not predict long-term response to interferon therapy in HBeAg-positive chronic hepatitis B patients.

UNLABELLED: The impact of IFNL3 (IL28B) polymorphism on response to interferon (IFN) treatment in patients infected with hepatitis B virus (HBV) is controversial. We aimed to investigate whether IFNL3 polymorphism (rs12979860) influences the long-term response of chronic hepatitis B (CHB) treatment to conventional IFN. DESIGN: Ninety-seven HBeAg-positive patients treated with IFN were evaluated in this study. Associations were investigated between IFNL3 genotypes and (i) HBeAg seroconversion at the end of treatment (EOT), (ii) sustained virological response (SVR) and (iii) HBsAg seroconversion through long-term follow-up (LTFU). Patients were followed for a median of 14 years. The majority of patients were infected with HBV genotype A (69.6%) and were Caucasian (77.9%). Ninety-five patients were genotyped at rs12979860. Similar IFNL3 distribution was observed among the different ethnicities (P = 0.62) or across HBV genotypes A through G (P = 0.70). Thirty-six patients experienced HBeAg seroconversion at EOT; HBeAg seroconversion rates were 37.0 and 35.5% in patients with CC and CT/TT genotypes, respectively (P = 0.82). Among the 44 patients (45%) who achieved a SVR, SVR rates were 48.9 and 39.6% in patients with CC and CT/TT IL28B genotypes, respectively (P = 0.80). HBsAg seroconversion occurred through LTFU in 28 patients. HBsAg seroconversion rates were 25.5 and 31.2% in patients with CC and CT/TT genotypes, respectively (P = 0.51). No significant relationship between IFNL3 rs12979860 and fibrosis stage was observed (P = 0.85). IFNL3 genotype was neither associated with SVR, nor with HBeAg seroconversion and long-term HBsAg seroconversion in HBeAg-positive CHB patients responding to IFN therapy.

[PSA testing, biopsy and cancer and benign prostate hyperplasia in France]. / Dosage du PSA, biopsie, cancer et hypertrophie bénigne de la prostate en France.

INTRODUCTION: Prostate-specific antigen (PSA) testing is high in France. The aim of this study was to estimate their frequency and those of biopsy and newly diagnosed cancer (PCa) according to the presence or absence of treated benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: This study concerned men 40 years and older covered by the main French national health insurance scheme (73 % of all men of this age). Data were collected from the national health insurance information system (SNIIRAM). This database comprehensively records all of the outpatient prescriptions and healthcare services reimbursed. This information are linked to data collected during hospitalisations. RESULTS: The frequency of men without diagnosed PCa (10.9 millions) with at least one PSA test was very high in 2011 (men aged 40 years and older: 30 %, 70-74 years: 56 %, 85 years and older: 33 % and without HBP: 25 %, 41 % and 19 %). Men with treated BPH totalized 9 % of the study population, but 18 % of the men with at least one PSA test, 44 % of those with at least one prostate biopsy and 40 % of those with newly managed PCa. Over a 3-year period, excluding men with PCa, 88 % of men with BPH had at least one PSA test and 52 % had three or more PSA tests versus 52 % and 15 % for men without BPH. One year after PSA testing, men of 55-69 years with BPH more frequently underwent prostate biopsy than those without BPH (5.4 % vs 1.8 %) and presented PCa (1.9 % vs 0.9 %). CONCLUSIONS: PSA testing frequencies in France are very high even after exclusion of men with BPH, who can be a group with more frequent managed PCa. LEVEL OF EVIDENCE: 4.

Single agent and combination studies of pralatrexate and molecular correlates of sensitivity.

BACKGROUND: Pralatrexate is a dihydrofolate reductase (DHFR) inhibitor with high affinity for reduced folate carrier 1 (RFC-1) and folylpolyglutamate synthetase (FPGS), resulting in extensive internalization and accumulation in tumour cells. Pralatrexate is approved in the US for the treatment of relapsed or refractory peripheral T-cell lymphoma and is being investigated in various malignancies. Here, we evaluated molecular correlates of sensitivity to pralatrexate and explored combinations with a variety of anticancer agents. METHODS: Antiproliferative effects of pralatrexate were evaluated in 15 human-cancer cell lines using the MTT assay. Gene expression was evaluated using qRT-PCR. RESULTS: Pralatrexate and methotrexate had a similar pattern of cytotoxicity, pralatrexate being more potent. Pralatrexate potentiated the effects of platinum drugs, antimetabolites and EGFR inhibitors. Dose- and time-dependent cytotoxicity of pralatrexate correlated with high mRNA expression of FPGS. Acquired resistance to pralatrexate was associated with decreased RFC-1 expression, whereas methotrexate resistance correlated with increased DHFR expression, suggesting different mechanisms of acquired resistance. CONCLUSION: Pralatrexate was more potent than methotrexate in a panel of solid tumour lines. Our findings support the further clinical development of pralatrexate in combination with certain cytotoxics and targeted therapies, and suggest that RFC-1, FPGS and DHFR may be potential biomarkers of outcome.

Sequential therapy with adefovir dipivoxil and pegylated interferon alfa-2a for HBeAg-negative patients.

To assess the impact of sequential therapy with adefovir dipivoxil (ADV) and pegylated interferon alfa-2a (PEG-IFN) on virological (serum HBV-DNA) and serological (serum HBsAg) response in 20 consecutive HBeAg-negative patients. Patients received ADV for 20 weeks, then ADV and PEG-IFN for 4 weeks and lastly PEG-IFN for 44 weeks. Serum HBV-DNA and HBsAg were assessed at baseline, during therapy (weeks 20, 44 and 68) and follow-up (weeks 92 and 116). Sustained virological response (SVR) was defined as serum HBV-DNA <10 000 copies/mL (partial) or <70 copies/mL (complete) 24 weeks after stopping treatment. A serological response was defined as a serum HBsAg decrease ≥1 log(10) IU/mL at the end of treatment. Baseline median serum HBV-DNA and HBsAg levels were 7.6 log(10) copies/mL and 3.8 log(10) IU/mL, respectively. Ten patients (50%) achieved SVR, six of them had partial response and four complete response. Four patients (20%) achieved serological response. Complete SVRs showed a major and steep decline in HBsAg level with a median decrease of 0.5, 1.6 and 2.0 log(10) IU/mL at treatment week 20, 44 and 68, respectively. Partial SVRs showed a slight and slow decline in serum HBsAg level (0.1, 0.4, and 0.6 log IU/mL at weeks 20, 44 and 68, respectively). On-treatment serum HBsAg decrease had a high accuracy to predict SVR (AUROC = 0.88). Our results suggest that sequential therapy might be an interesting strategy for HBeAg-negative patients. Serum HBsAg kinetics seem to be an accurate tool to predict SVR. Large clinical trials are needed to explore this strategy with more potent analogues.

SPRED1 germline mutations caused a neurofibromatosis type 1 overlapping phenotype.

OBJECTIVE: Germline loss-of-function mutations in the SPRED1 gene have recently been identified in patients fulfilling the National Institutes of Health (NIH) diagnostic criteria for neurofibromatosis type 1 (NF1) but with no NF1 (neurofibromin 1) mutation found, suggesting a neurofibromatosis type 1-like syndrome. METHODS: 61 index cases with NF1 clinical diagnosis but no identifiable NF1 mutation were screened for SPRED1 mutation. RESULTS: We describe one known SPRED1 mutation (c.190C>T leading to p.Arg64Stop) and four novel mutations (c.637C>T leading to p.Gln213Stop, c.2T>C leading to p.Met1Thr, c.46C>T leading to p.Arg16Stop, and c.1048_1060del leading to p.Gly350fs) in five French families. Their NF1-like phenotype was characterised by a high prevalence of café-au-lait spots, freckling, learning disability, and an absence of neurofibromas and Lisch nodules in agreement with the original description. However, we did not observe Noonan-like dysmorphy. It is noteworthy that one patient with the p.Arg16Stop mutation developed a monoblastic acute leukaemia. CONCLUSIONS: In our series, SPRED1 mutations occurred with a prevalence of 0.5% in NF1 patients and in 5% of NF1 patients displaying an NF1-like phenotype. SPRED1 mutated patients did not display any specific dermatologic features that were not present in NF1 patients, except for the absence of neurofibromas that seem to be a specific clinical feature of NF1. The exact phenotypic spectrum and the putative complications of this NF1 overlapping syndrome, in particular haematological malignancies, remain to be further characterised. NIH diagnostic criteria for NF1 must be revised in view of this newly characterised Legius syndrome in order to establish a specific genetic counselling.

Gene expression and hepatitis C virus infection.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with about 170 million people infected worldwide. Up to 70% of patients will have persistent infection after inoculation, making this disease a significant cause of morbidity and mortality. The severity of disease varies widely, from asymptomatic chronic infection to cirrhosis and hepatocellular carcinoma. Since the discovery of HCV, the treatment of hepatitis C has considerably improved. Recently, combination of pegylated interferons with ribavirin gives a response rate of about 55%. Treatment is indicated in patients with moderate or severe fibrosis. The tolerability of combination treatment is relatively poor, with a frequent flu-like syndrome and an impaired quality of life. In addition to viral and environmental behavioural factors, host genetic diversity is believed to contribute to the spectrum of clinical outcomes in HCV infection. The sequencing of the human genome, together with the development of high-throughput technologies that measure the function of the genome, have afforded unique opportunities to develop profiles that can distinguish, identify and classify discrete subsets of disease, predict the disease outcome or predict the response to treatment. This paper reviews the published literature on gene expression associated with HCV infection (HCV infection, fibrosis progression), and also according to response to treatment.

Insulin resistance and geographical origin: major predictors of liver fibrosis and response to peginterferon and ribavirin in HCV-4.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) genotype 4 (HCV-4) is increasing in prevalence in Western countries. However, little is known about the severity of the disease and response to treatment. The aim of this study was to assess the predictors (logistic regression) of severe fibrosis (METAVIR score F3-F4), and sustained virological response (SVR) to peginterferon and ribavirin in 226 consecutive HCV-4 patients (Egyptians 40%, Europeans 35% and Africans 24%). PATIENTS AND METHODS: Insulin resistance was assessed using the homeostasis model (HOMA-IR). Serum HCV-RNA level (bDNA) and subtypes of HCV (LiPA) were determined for all patients. RESULTS: Insulin resistance (HOMA-IR >3) was present in 105 patients (46%), and was associated with: age >45 years (OR, 2.614; 95% CI, 1.316 to 5.194), body mass index (BMI) >25 kg/m(2) (OR, 2.105; 95% CI, 1.048 to 4.229), serum HCV-RNA >800 000 IU/ml (OR, 3.143; 95% CI, 1.503 to 6.574), severe fibrosis (OR, 2.657; 95% CI, 1.214 to 5.818), and steatosis >30% (OR, 2.488; 95% CI, 1.105 to 5.602). Severe fibrosis was present in 67 patients (29%) and was associated with Egyptian origin (OR, 5.872; 95% CI, 2.747 to 12.553), excessive alcohol intake (OR, 5.311; 95% CI, 1.287 to 21.924), and HOMA-IR >3 (OR, 3.864; 95% CI, 1.859 to 8.034). 108 patients received a 48 week course of peginterferon plus ribavirin. SVR (undetectable serum HCV-RNA (TMA) 24 weeks after treatment stopping) was achieved in 59 patients (55%) and was associated with Egyptian origin (OR, 13.119; 95% CI, 3.089 to 55.706), HOMA-IR <2 (OR, 5.314; 95% CI, 1.953 to 14.459), and non-severe fibrosis (OR, 8.059; 95% CI, 2.512 to 25.855). CONCLUSION: Insulin resistance and geographical origin are major predictors of liver fibrosis and response to peginterferon and ribavirin in HCV-4 patients. Insulin resistance is frequently encountered in these patients, and correlated independently with serum HCV-RNA.

SOS1: a new player in the Noonan-like/multiple giant cell lesion syndrome.

Noonan-like/multiple giant cell lesion syndrome is a rare condition with phenotypic overlap with Noonan syndrome (NS) and cherubism. PTPN11 gene mutations were described in several individuals with this phenotype, and it is recently considered as a variant phenotype of NS. Gain-of-function mutations in the SOS1 gene were recently described as the second major cause of NS. Here, we report for the first time the involvement of SOS1 gene in a family with the Noonan-like/multiple giant cell lesion phenotype.

Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C.

BACKGROUND AND AIMS: The gold standard treatment of chronic hepatitis C (CHC) is combined pegylated interferon and ribavirin. Considering side effects and treatment cost, prediction of treatment response before therapy is important. The aim of this study was to identify a liver gene signature to predict sustained virological response in patients with CHC. METHODS: Group A (training set) comprised 40 patients with CHC including 14 non-responders (NRs) and 26 sustained virological responders (SVRs). Group B (validation set) comprised 29 patients including 9 NRs and 20 SVRs. Eleven responder-relapsers were also included. A total of 58 genes associated with liver gene expression dysregulation during CHC were selected from the literature. Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of these 58 selected genes in liver biopsy specimens taken from the patients before treatment. RESULTS: From the Group A data, three genes whose expression was significantly increased in NRs compared with SVRs were identified: IFI-6-16/G1P3, IFI27 and ISG15/G1P2. These three genes also showed significant differences in their expression profiles between NRs and SVRs in the independent sample (Group B). Supervised class prediction analysis identified a two-gene (IFI27 and CXCL9) signature, which accurately predicted treatment response in 79.3% (23/29) of patients from the validation set (Group B), with a predictive accuracy of 100% (9/9) and of 70% (14/20) in NRs and SVRs, respectively. The expression profiles of responder-relapsers did not differ significantly from those of NRs and SVRs, and 73% (8/11) of them were predicted as SVRs with the two-gene classifier. CONCLUSION: NRs and SVRs have different liver gene expression profiles before treatment. The most notable changes occurred mainly in interferon-stimulated genes. Treatment response could be predicted with a two-gene signature (IFI27 and CXCL9).

Tenascin-C expression relates to clinicopathological features in pilocytic and diffuse astrocytomas.

AIMS: Tenascin-C (TN-C) is an extracellular matrix brain glycoprotein for which conflicting in vitro and in vivo results are reported in the literature dealing with its involvement in astrocytoma aggressiveness, in particular astrocytoma invasion. In view of these conflicting results and the lack of data available on low-grade astrocytomas, the present study focuses on pilocytic World Health Organization (WHO) grade I, and diffuse WHO grade II astrocytomas, that is, two histological entities associated with very different invasive abilities. METHODS: Using real-time reverse transcription polymerase chain reaction and immunohistochemistry, we analysed the TN-C expression in normal brain tissue as well as in a series of 54 pilocytic and 53 grade II astrocytomas. CONCLUSIONS: Our data on normal brain showed that while TN-C is largely expressed in supratentorial white matter, it was largely absent in infratentorial white matter. Paralleling these observations, we showed that TN-C expression in low-grade astrocytomas similarly varies according to tumour site. Cox regression analysis evidenced that TN-C provided an independent prognostic value which is enhanced in the case of grade II astrocytomas for which positive TN-C expression is associated with a higher risk of recurrence. We also analysed TN-C expression specifically in vascular areas of low-grade astrocytomas without demonstrating any prognostic value for this additional feature. RESULTS: Similarly to normal brain, low-grade astrocytomas exhibit variations in TN-C expression with site, and this expression is associated with an independent prognostic value in terms of recurrence.

Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy.

OBJECTIVE: Neurofibromatosis type 2 (NF2) affects about one in 25,000 to 40,000 people. Most NF2 patients have private loss-of-function mutations scattered along the NF2 gene. Here, we present our NF2 investigation strategy. MATERIAL AND METHODS: We report a comprehensive NF2 mutation analysis of 221 NF2 French patients: 134 unrelated typical NF2 patients fulfilling the Manchester criteria and 87 unrelated patients presenting symptoms that partially fulfilled the Manchester criteria. RESULTS: A NF2 mutation was identified in 56 of the 221 patients, giving a global mutation detection rate of 25%. This rate reached 37% (49/134) for typical NF2 patients fulfilling the Manchester criteria and only 8% (7/87) for patients presenting symptoms suggestive of NF2. Six of these seven patients were under 25 of age. Our approach showed that 77% of NF2 identified variants were detected by coding exons sequencing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements (23% of NF2 identified variants corresponding to complete deletion or partial deletion/duplication of NF2). CONCLUSION: High mutation detection rate can be achieved if well phenotyped NF2 patients are studied with multiple complementary and optimized techniques. NF2 somatic mosaicism detection was improved by frozen tumor samples molecular analysis.

The angiopoietin pathway is modulated by PAR-1 activation on human endothelial progenitor cells.

OBJECTIVES: The importance of protease-activated receptor-1 (PAR-1) in blood vessel development has been shown in knock-out mice. As endothelial progenitor cells (EPCs) express functional PAR-1, we examined whether PAR-1 stimulation by the peptide SFLLRN interfered with the angiopoietin pathway, that is EPC commitment, proliferation and migration. METHODS AND RESULTS: Given the strong PAR-1 expression on CD34+ cells, we tested the effect of SFLLRN 75 micromol L(-1) on the emergence of EPCs from cord blood. PAR-1 activation did not modify the number of colonies or the day of emergence, in keeping with the lack of induction of angiopoietin 1 gene expression. Conversely, SFLLRN treatment of EPCs induced angiopoietin 2 gene expression and protein synthesis. Experiments with polyclonal blocking antibodies showed that angiopoietin 2 was involved in the proliferative effect of PAR-1 activation. PAR-1 activation also enhanced migration toward angiopoietin 1 in a Boyden chamber assay. CONCLUSIONS: Our study demonstrates that PAR-1-induced proliferation of EPCs involves angiopoietin 2. PAR-1 also enhances EPC migration toward angiopoietin 1. These findings might explain the role of thrombin in neovascularization via the angiopoietin pathway.

Identification of CGA as a novel estrogen receptor-responsive gene in breast cancer: an outstanding candidate marker to predict the response to endocrine therapy.

The estrogen receptor (ER) status of breast tumors is used to identify patients who may respond to endocrine agents such as tamoxifen. However, ER status alone is not perfectly predictive, and there is a pressing need for more reliable markers of endocrine responsiveness. Here, we identified the well-known CGA gene (coding for the alpha subunit of glycoprotein hormones) as a new ERalpha-responsive gene in human breast cancer cells. We used a real-time quantitative reverse transcription-PCR assay to quantify CGA mRNA copy numbers in a large series of breast tumors. CGA overexpression (> 10 SD above the mean for normal breast tissues) was observed in 44 of 131 (33.6%) breast tumor RNAs, ranging from 20 to 16,500 times the level in normal breast tissues; the highest levels of CGA gene expression were close to those observed in placenta. Significant links were observed between CGA gene overexpression and Scarff-Bloom-Richardson histopathological grade I+II (P = 0.015), and progesterone (P = 0.0009) and estrogen (P < 10(-7)) receptor positivity, which suggested that CGA is a marker of low tumor aggressiveness. We observed CGA mRNA overexpression in 44 of 90 (48.9%) ERalpha-positive tumors and in none of the 41 ERalpha-negative tumors. Immunohistochemical studies demonstrated that human chorionic gonadotropin alpha protein was strictly limited to ERalpha-positive tumor cells. Overexpression of the CGA gene was not accompanied by overexpression of the CGB gene. Our results also suggest that CGA could be a more reliable marker than PS2 and PR for ERalpha functionality and, thus, for endocrine responsiveness. Moreover, the CGA marker has the added value of dichotomizing ERalpha-positive patients into two subgroups of similar size. Specific antibodies directed to secreted human chorionic gonadotropin alpha protein are commercially available, thus facilitating the future application of this marker to the clinical management of breast cancer.

Evaluation of androgen, estrogen (ER alpha and ER beta), and progesterone receptor expression in human prostate cancer by real-time quantitative reverse transcription-polymerase chain reaction assays.

Steroid hormones can have profound effects on prostate tumor development making it important to define steroid receptor expression in prostate tissues. For this purpose, androgen receptor (AR) and estrogen receptor (ER alpha and ER beta) expression was quantified in 12 clinically localized and 11 hormone-refractory sporadic prostate tumors, using real-time quantitative reverse transcription-PCR assays. To gain more insight into hormone-responsiveness, estrogen-regulated progesterone receptor (PGR) and androgen-regulated prostatic acid phosphatase (PAP) mRNA levels were also quantified. There is a decrease in expression of ER beta in both clinically localized and hormone-refractory tumors relative to normal prostate tissues. Moreover, hormone-refractory tumors display a decreased expression of ER alpha and an increased expression of AR. There is a positive association between ER alpha, ER beta, and PGR expression (P < 0.0001) and a negative association between AR and the androgen-regulated gene PAP expression in hormone-refractory tumors. Taken together, these data indicate that, although increased expression of the AR gene might play a key role in endocrine treatment failure, it cannot be considered as the sole actor of this unresolved dilemma, and abnormalities in ER alpha and/or ER beta expression may also modulate the growth response of prostate cancer to hormone withdrawal. Our results also suggest that ER alpha and ER beta expression status could be used to identify advanced prostate tumor patients who may respond to antiestrogen therapy.

Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay.

MYC gene overexpression was identified recently as a downstream step at the end of the Wnt/APC/beta-catenin pathway dysregulation observed in colorectal cancer (T-C. He et al., Science (Washington DC), 281: 1509-1512, 1998). It thus appears that an excess of c-myc protein is a primary cause of numerous cancers. In breast cancer, MYC has been studied mostly at the DNA level because of the poor quality of available antibodies against the protein product. The renewed interest in MYC calls for a sensitive and accurate method for analyzing MYC overexpression in breast tumors. We have developed a real-time quantitative reverse transcription-PCR assay based on TaqMan fluorescence methodology to quantify the MYC mRNA copy number. We validated the method on a large series of breast tumors. MYC gene overexpression was observed in 29 of 134 (22%) breast tumor RNAs, ranging from 3.2 to 19 times the level in normal breast tissues. These data imply that dysregulated MYC gene expression is potentially involved in the pathogenesis of breast cancer, especially by favoring local cell proliferation. We also found that MYC gene overexpression was rarely due to an increased MYC gene copy number in breast cancer. This new, simple, rapid, and semiautomated method will be useful for screening cancer patients for MYC overexpression and will prove a powerful tool in large, randomized, prospective, cooperative group trials and in the MYC-based therapy project.

Malignant transformation of nontrophoblastic cells is associated with the expression of chorionic gonadotropin beta genes normally transcribed in trophoblastic cells.

The beta subunit of human chorionic gonadotropin (hCGbeta) is encoded by four nonallelic CGbeta genes. An assay was developed for distinguishing type I CGbeta allelic genes beta7 and beta6, which possess a GCC codon corresponding to an alanine at position 117 of hCGbeta, from type II CGbeta genes beta8, beta5, and beta3 and its allele beta9, which possess a GAC codon corresponding to an aspartic acid at the same position. In normal trophoblast, hCGbeta is encoded by type II CGbeta genes, whereas normal nontrophoblastic tissues of differing histological origin (breast, prostate, skeletal muscle, bladder, adrenal glands, thyroid, colon, and uterus) express only type I CGbeta genes. We studied the expression of CGbeta genes in 86 tumor specimens collected from patients with breast, bladder, prostate, and thyroid cancer and found that up to 61% of these nontrophoblastic tumors expressed type II CGbeta genes. Experiments performed on tumor tissues and their normal counterparts confirmed that the malignant transformation of nontrophoblastic cells is associated with the expression of type II CGbeta genes. These findings provide the basis for a simple test (the CG117 assay) that may be useful for the diagnosis of the most frequent malignancies.

Expression of human chorionic gonadotropin beta subunit genes in superficial and invasive bladder carcinomas.

Increased serum levels of human chorionic gonadotropin beta subunit (hCG beta) were described previously in patients with bladder cancer. To obtain insight into such production of hCG beta, the expression of hCG beta 7, 8, 5, and 3 genes in bladder carcinomas and normal urothelia was investigated by reverse transcription PCR. Surprisingly, hCG beta mRNAs were detected in both normal urothelial and carcinomatous cells. However, tumor progression was characterized by different patterns of transcription of the hCG beta genes; the beta 7 gene was the only gene transcribed in normal urothelia and Ta tumors included in this study, whereas in addition to beta 7, genes beta 5, 8, and 3 were transcribed in T1 to T4 tumors. Moreover, transcription levels of the latter three genes increased with the stage of the disease. These observations showed that dramatic modifications in the expression of hCG beta genes accompany progression of bladder carcinomas.

Germ-line deletion involving the INK4 locus in familial proneness to melanoma and nervous system tumors.

Joint predisposition to malignant melanoma and nervous system tumors (NSTs) is a puzzle. Several melanoma susceptibility genes have been identified, including p16, a clustered tumor suppressor. However, the molecular bases of inherited proclivity to NSTs in the absence of a recognizable genetic syndrome are unknown. We analyzed two families with joint proneness to melanoma and NSTs in view of genetic linkage and identification of the causal molecular lesions. Highly informative linkage markers were used for segregation analyses of the predisposition alleles in the two pedigrees. Characterization of the molecular lesions required hemizygosity mapping based on microsatellite markers physically mapped to contigs of the 9p21 region and a Southern blot approach using several PCR-generated probes. Both families were found to be allelic and linked to p16 markers. In the family segregating the melanoma/NST syndrome, a large germ-line deletion ablated the whole p16, p19, and p15 gene cluster (or INK4 locus), whereas a more circumscribed molecular lesion disrupting p16 and p19 but leaving p15 unaltered segregated with the melanoma-astrocytoma syndrome (MIM 155755). Our results suggest that multiple cancer susceptibility in these two families ensues from contiguous tumor suppressor gene deletion. Indeed, known phenotypes associated with germ-line p16 mutations and an apparent correlation between the deletion span and tumor spectrum in the two families suggest a new model of cancer pathogenesis based on the inactivation of contiguous tumor suppressor genes, an alternative to the established pleiotropic effects of single-gene disruption.

Quantification of estrogen receptor alpha and beta expression in sporadic breast cancer.

The recent cloning of a second estrogen receptor (ER), designated ERbeta, has prompted a reevaluation of the role of ERs in breast cancer. We have developed and validated a real-time RT-PCR assay to quantify ERalpha and ERbeta gene expression at the mRNA level in a series of 131 patients with unilateral invasive primary breast cancer. Although ERbeta expression showed wide variations in tumor tissues, its range (nearly three orders of magnitude) was smaller than that of ERalpha (nearly four orders of magnitude), suggesting that ERbeta is more tightly controlled than ERalpha. We observed a negative correlation between ERalpha and ERbeta expression. 'ERalpha-negative' tumors (containing very low ERalpha mRNA levels) were associated with SBR histopathological grade III, RB1 underexpression and ERBB2 overexpression, confirming that ERalpha negativity delineates poorly differentiated tumors. The amount of ERalpha mRNA (but not that of ERbeta mRNA) increased with age and was consequently higher in postmenopausal patients' tumors. Expression of ERalpha (but not that of ERbeta) also correlated strongly with progesterone receptor (PR) and PS2 expression, suggesting that ERalpha has stronger transcriptional activity than ERbeta towards genes containing an ERE (estrogen response element) in their promoters. Interestingly, we found a negative correlation between the expression of ERbeta (but not ERalpha) and CCND1, which contains an AP1 element but not an ERE in its promoter. Taken together, these data confirm that ERalpha and ERbeta play different roles in breast cancer, partly by mediating the transcription of various genes via different types of DNA enhancer. PR and PS2 seem to be mainly ERalpha-responsive genes, whereas CCND1 may be mainly ERbeta-responsive. Our findings also underline the need for a reliable method, providing full range of quantitative values, to determine ERalpha and ERbeta status in the clinical setting.