Lobular and ductal carcinomas of the breast have distinct genomic and expression profiles.

Invasive ductal carcinomas (IDCs) and invasive lobular carcinomas (ILCs) are the two major pathological types of breast cancer. Epidemiological and histoclinical data suggest biological differences, but little is known about the molecular alterations involved in ILCs. We undertook a comparative large-scale study by both array-compared genomic hybridization and cDNA microarray of a set of 50 breast tumors (21 classic ILCs and 29 IDCs) selected on homogeneous histoclinical criteria. Results were validated on independent tumor sets, as well as by quantitative RT-PCR. ILCs and IDCs presented differences at both the genomic and expression levels with ILCs being less rearranged and heterogeneous than IDCs. Supervised analysis defined a 75-BACs signature discriminating accurately ILCs from IDCs. Expression profiles identified two subgroups of ILCs: typical ILCs ( approximately 50%), which were homogeneous and displayed a normal-like molecular pattern, and atypical ILCs, more heterogeneous with features intermediate between ILCs and IDCs. Supervised analysis identified a 75-gene expression signature that discriminated ILCs from IDCs, with many genes involved in cell adhesion, motility, apoptosis, protein folding, extracellular matrix and protein phosphorylation. Although ILCs and IDCs share common alterations, our data show that ILCs and IDCs could be distinguished on the basis of their genomic and expression profiles suggesting that they evolve along distinct genetic pathways.

Kinetoplast DNA minicircles of phloem-restricted Phytomonas associated with wilt diseases of coconut and oil palms have a two-domain structure.

We report the cloning and sequencing of the first minicircle from a phloem-restricted, pathogenic Phytomonas sp. (Hart 1) isolated from a coconut palm with hartrot disease. The minicircle possessed a two-domain structure of two conserved regions, each containing three conserved sequence blocks (CSB). Based on the sequence around CSB 3 from Hart 1, PCR primers were designed to allow specific amplification of Phytomonas minicircles. This primer pair demonstrated specificity for at least six groups of plant trypanosomatids and did not amplify from insect trypanosomatids. The PCR results were consistent with a two-domain structure for other plant trypanosomatids.

P73 expression in basal layers of head and neck squamous epithelium: a role in differentiation and carcinogenesis in concert with p53 and p63?

P73, a p53-homologue gene, has been studied for its possible role in head and neck squamous epithelium (HNSE) differentiation and carcinogenesis. P73 RNA and protein were analysed in 50 biopsies, including well- and moderately-differentiated carcinomas, and 21 matched normal adjacent tissues. P73 immunohistochemical analyses revealed intense p73 nuclear staining in basal and parabasal cells of normal squamous epithelium, in contrast with complete absence of staining in the more superficial cell layers. Moderately-differentiated carcinomas demonstrated homogeneous and diffuse staining in all tumour cells, while only basal cells were stained in well-differentiated carcinomas as in normal tissue. No correlation was observed between p73 and p53 protein expression. Immunostaining for p63, another p53-related protein previously described as being involved in HNSE morphogenesis and overexpressed in head and neck squamous cell carcinomas (HNSCC), was found to be similar to p73 labelling in carcinomas, but spread to the more differentiated layers in normal epithelium. Biallelic expression of p73 was found in tumours as well as in matched normal tissues. Comparison of p73 transcript levels between tumours and normal tissues showed decreased mRNA expression in 5/17 (30%) tumours independently of the differentiation status. Mutation and loss of heterozygosity analyses of the p73 gene revealed wild type status and no deletion. Our results strongly suggest that: (i) p73 is associated with homeostasis and control of differentiation of head and neck squamous epithelium probably in concert with p53 and p63; (ii) down-regulation of p73 expression could participate in HNSE carcinogenesis.

No evidence of somatic FGFR3 mutation in various types of carcinoma.

Germline specific point mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3) are associated with autosomal dominant human skeletal dysplasia and craniosynostosis syndromes. Mutations identical to the germinal activating mutations found in severe skeletal dysplasias have been identified in certain types of cancer: at low frequency in multiple myeloma and cervix carcinoma and at high frequency in bladder carcinoma. We analysed, by SSCP and sequencing, the prevalence of FGFR3 mutations in 116 primary tumours of various types (upper aerodigestive tract, oesophagus, stomach, lung and skin). The regions analysed encompassed all FGFR3 point mutations previously described in severe skeletal dysplasia and cancers. No mutations were detected in the tumour types examined, suggesting that FGFR3 mutations are restricted to a few tumour types, the evidence to date suggesting that they are very specific to bladder carcinomas.

c-erbB-2 (HER-2/neu) gene amplification is a better indicator of poor prognosis than protein over-expression in operable breast-cancer patients.

Our aim was to compare the prognostic value of c-erbB-2 gene amplification analyzed by Southern blot with that of protein (p185) over-expression measured by immunohistochemistry in 172 patients with operable breast cancer (BC). Amplification and p185 over-expression were found in 31 (18%) and 51 (30%) BCs, respectively. All but 1 of the tumors showed both amplification and over-expression, while 21 (12%) tumors displayed over-expression without amplification. The risk of death associated with c-erbB-2 gene amplification and p185 over-expression was evaluated by multivariate analysis, taking into account tumor size, histoprognostic grade, hormone receptors and axillary node status. During a mean follow-up of 9.5 (+/-2) years, node involvement (p < 0.001), c-erbB-2 gene amplification (p = 0.02) and negative hormone receptors (p = 0.02) were found to be independent prognostic indicators of the risk of death. Over-expression of p185 with no amplification was not correlated with this risk. When the risk of death associated with c-erbB-2 amplification was studied according to chemo- and hormone therapy, no significant difference was observed between subgroups of subjects. Amplification was also associated (p = 0.02) with the risk of multifocal distant metastases (i.e., metastases detected concomitantly in at least 2 sites) and, thus, with BC aggressiveness. These data show the importance of c-erbB-2 gene amplification in predicting the long-term outcome of patients and in selecting eligible patients for c-erbB-2-targeted therapies.

Loss of heterozygosity, allele silencing and decreased expression of p73 gene in breast cancers: prevalence of alterations in inflammatory breast cancers.

The p73 gene is a p53 homologue located at 1p36-33, a region submitted to deletions in breast cancer (BC) and putatively imprinted. To study whether p73 was associated with breast carcinogenesis, loss of heterozygosity (LOH), allele expression and transcript levels were assessed in 59 BC, including 39 BC presenting no inflammatory symptoms (NBC) and 20 inflammatory BC (IBC). IBC is a rare but aggressive form of cancer with a very poor prognosis. Normal breast epithelium (BE) and lymphocytes from patients were used as controls. StyI polymorphism generating GC and/or AT alleles was used to select 22 heterozygous patients. p73 LOH was significantly higher in IBC than in NBC [five of eight cases (62%) versus two of 14 cases (14%); Fisher's exact test, P=0.05]. p73 was biallelically expressed in all BE. In contrast, 12 of 16 (75%) BC were monoallelically expressed, showing that allele silencing was significantly associated with breast carcinogenesis (P=0.012), AT being the preferential silent allele (10 out of 12 tumours). p73 mRNA levels in NBC and IBC were two- and threefold lower than in BE, respectively, suggesting that decreased expression could be related to tumour aggressiveness. In conclusion, LOH, allele silencing and decreased expression of the p73 gene may play a role in breast carcinogenesis.

[Oncogenic factors of metastatic dissemination in neuroblastoma]. / Facteurs oncogéniques de la dissémination métastatique du neuroblastome.

Disseminated neuroblastoma frequently show a very poor prognosis. N-myc gene amplification, 1p deletion and lack of CD44 gene expression, are all genetic factors associated with the disease's dissemination. Human neuroblastoma xenografts in nude mice has permitted to characterize, in disseminated neuroblasts, oncogenes overexpression, inactivation of tumor suppressor genes as well as detoxifying genes activation which contributes to increase cellular resistance to chemotherapy. These genetic abnormalities permit to propose a nosology of this very aggressive pediatric solid tumor. Hopefully, this genetic classification could be of great value for new therapeutic approaches.

[TSG101 and breast cancer: a correctly named tumor-suppressor gene?]. / TSG101 et cancer du sein: un gène suppresseur de tumeur bien nommé?

In January 1997, PCR genomic analysis of the TSG101 gene showed frequent large intragenic deletions. The investigators used a small cohort of sporadic breast cancers. Li et al. proposed human TSG101 as a breast tumor suppressor gene. Since then, several teams have worked on large series of breast cancers, using a variety of techniques. They have shown that intragenic deletion, if it exists at all, is rare. Is TSG101 a tumor gene or an impostor?

Cisplatin-induced apoptosis and p53 gene status in a cisplatin-resistant human ovarian carcinoma cell line.

Cisplatin-induced apoptosis and p53 gene status were analyzed in human ovarian carcinoma using a parental IGR-OV1 line and a derived cisplatin-resistant IGR-OV1/DDP subline. Compared with parental cells, cisplatin-resistant cells exhibited a 5-fold higher resistance index and a 2-fold longer doubling time. Cisplatin induced apoptosis in both cell lines, as assessed by cell morphology and the presence of a DNA ladder. However, high concentrations were necessary to induce apoptosis in resistant cells. These cells elicited a 5-fold decrease in the number of platinum atoms bound per nucleotide. IGR-OV1/DDP cells also exhibited enhanced drug efflux and a higher glutathione content. Our data suggest that the levels of cisplatin-DNA lesions are critical for drug sensitivity and apoptosis induction in this in vitro ovarian carcinoma model. Comparative analysis of the p53 gene in sensitive and resistant cells revealed the presence of the same heterozygous mutation in exon 5. A 2-fold increase in p53 mRNA and protein amounts was observed in resistant cells as assessed by Northern and Western blots, respectively. Immunocytochemical staining revealed a higher percentage of p53 stained nuclei in resistant cells. RT-PCR analysis of p53 transcripts showed that both wild-type and mutated alleles were transcribed in sensitive as well as in resistant cells. However, mutated transcripts were 1.5-fold more abundant than wild-type transcripts in sensitive cells, whereas they were 2-fold higher in resistant cells. In addition, mdm-2 protein was over-expressed in resistant cells. Our results address the question of the functionality of p53 protein and its possible role in apoptosis induction in this model. In resistant cells, p53 protein might be inactivated by 2 mechanisms: mutation and complexation with mdm-2 protein. Therefore, the presence of non-functional p53 in resistant cells might be involved in the relative failure of cisplatin-induced apoptosis in these cells.

[High incidence of p53 mutations in primary and metastatic head and neck tumors. Frequent protein overexpression in normal epithelium]. / Incidence élevée des mutations p53 dans les tumeurs primitives et métastases des voies aérodigestives supérieures et fréquente surexpression en protéine dans les épithéliums normaux.

Mutation of the p53 tumor suppressor gene is the most commonly observed gene alteration in human cancers. In order to identify new prognostic factors and tumor aggressiveness in squamous cell head and neck carcinomas, we analyzed 50 node metastases and 28 primary tumors including 13 matched specimens for p53 alterations. Mutations were found in 54 (69%) tumors, 76% of which were missense, 9% were nonsense and 15% were microdeletions or microinsertions. Twenty-five mutations were transitions mostly G-->A (40%) and 20 were transversions mostly G-->T (25%) thus confirming the role of tobacco carcinogens in the induction of these mutations. For eight patients mutations were observed in matched primary tumors and metastases, indicating clonal dissemination of tumor cells in most of these carcinomas. Furthermore the incidence of mutations was not different in primary tumors and node metastases indicating that this gene alteration was not related to the metastatic dissemination. No correlation was found between mutation and clinical parameters, the 8-year survival rates were not different (log rank test: P = 0.49) in patients with and without mutation. There was a good correlation between p53 mutation and protein overexpression (Fisher's exact test: P < 10(-4). Interestingly, immunostaining was also observed in basal cells from normal mucosa and in early lesions adjacent to the primary tumor in 11/15 specimens irrespective of the presence of mutation in the corresponding tumors. p53 protein overexpression may therefore constitute a biomarker for early stages of carcinogenesis of the head and neck epithelium.

High incidence of loss of heterozygosity and abnormal imprinting of H19 and IGF2 genes in invasive cervical carcinomas. Uncoupling of H19 and IGF2 expression and biallelic hypomethylation of H19.

The few imprinted genes characterized so far include the insulin-like growth factor-2 gene (IGF2) coding for a foetal growth factor and the H19 gene whose normal function is unknown but which is likely to act as an RNA with an antitumour effect. IGF2 is expressed by the paternal allele and H19 by the maternal allele. This reciprocal expression is quite interesting because both H19 and IGF2 genes are located close to each other on chromosome 11p15.5 in a region subject to loss of heterozygosity (LOH). Moreover, loss of imprinting (LOI) or biallelic expression has been proposed as an epigenetic mechanism for tumorigenesis in a variety of human cancers including Wilms' tumour. In this study we report the LOH, LOI and methylation status of H19 and IGF2 genes in 29 invasive cervical carcinomas of different clinical stages. Fourteen (48%) and 13 (45%) tumours were heterozygous for H19 and IGF2 respectively. LOH for H19 and IGF2 genes were found in 2 of 14 (14%) and 3 of 13 (23%) informative tumours, respectively. LOI of H19 and IGF2 was detected in 2 of 12 (17%) and 5 of 10 (50%) tumours with no LOH, respectively. More interestingly, monoallelic expression of the otherwise silent H19 allele (allele switch) was observed in 2 of 12 (17%) tumours and biallelic expression of IGF2 was detected in one specimen of normal cervix adjacent to the tumour. The expressing H19 allele, and to a lower degree also the silent allele, were hypomethylated in tumours suggesting that demethylation of both H19 alleles may be associated with an early step of imprinting alteration. In cervical cancer H19 and IGF2 expressions could be independently regulated. In conclusion, our data suggest that H19 and IGF2 genes, via deletions and/or abnormal imprinting, could play a crucial role in a large proportion (58%) of cervical cancers where they may be associated with disease progression.

p53 mediated tumor cell response to chemotherapeutic DNA damage: a preliminary study in matched pairs of breast cancer biopsies.

Wild type p53 plays a crucial role in maintaining genomic stability in both normal and tumor cells in vitro. When DNA damage occurs, p53 acts as a cell cycle checkpoint and induces a cellular response that aims at restoring genomic integrity. p53 may either allow the repair of damaged DNA by inducing a transient G1 arrest or may eliminate the damaged cells by triggering apoptosis. Mutant p53 fails to mediate any of these effects. From this, a p53 status-dependent response to therapy might be expected when tumors are treated with DNA-damaging genotoxic agents: Although wild type p53-harboring tumors have an intact checkpoint that might allow them to restore genomic integrity back to a pre-exposure level, mutant p53 tumors have a corrupted checkpoint that could lead to an accelerated loss of genomic stability. Until now, no studies have been described that examine such a p53-mediated effect in vivo. The authors tested this response model in vivo comparing 32 matched biopsy pairs from patients with breast cancer before and after rigorously standardized polychemotherapy. Four of the five drugs specifically induce a wild type p53-mediated checkpoint response. Tumor tissue from matched pairs of untreated and treated biopsies of the same patient were analyzed for treatment-associated changes of p53 protein expression by immunocytochemistry and, in a few available specimens, of p53 genotype changes by polymerase chain reaction-based DNA analysis. Treatment-associated changes of the p53 immunophenotype, which the authors speculate to reflect clonal selection, occurred in 39% (12 of 31) of the specimens. One specimen was not informative. Most tumors undergoing clonal selection originally harbored mutant p53 (nine of 12), and only three of 12 tumors were wild type. This study shows that exposure to genotoxic agents is commonly associated with a change in p53 immunophenotype. Although the limited material in this cohort prevented direct analysis of genetic instability, these results suggest that tumors with altered p53 may be genomically less stable and, therefore, may be more likely to undergo treatment-induced clonal changes than wild type tumors. This study also shows that the rigorous matched sample approach, although difficult to obtain, is an important tool that allows the in vivo assessment of the tumor response to genotoxic therapy in a controlled fashion.

High incidence of p53 alterations (mutation, deletion, overexpression) in head and neck primary tumors and metastases; absence of correlation with clinical outcome. Frequent protein overexpression in normal epithelium and in early non-invasive lesions.

We have analysed 78 head and neck carcinomas (50 node metastases and 28 primary tumors including 13 matched specimens) in 65 patients for p53 alterations. Mutations were found in 54 (69%) tumors. Of the 53 mutations within exons, 40 (76%) were missense, five (9%) nonsense and eight (15%) microdeletions or microinsertions. Twenty-five (47%) mutations were transitions mostly G-->A (40%) and 20 (38%) were transversions, mostly G-->T (25%), thus confirming the role of tobacco carcinogens in the induction of these mutations. The incidence of mutations was not different in primary tumors (68%) and node metastases (70%) indicating that this gene alteration was not related to the metastatic dissemination. For eight patients, mutations were observed in matched primary tumors and metastases, indicating clonal dissemination of tumor cells in most of these carcinomas. There was a good correlation between mutations and protein overexpression (Fisher's exact test P < 10(-4). Immunostaining was also observed in basal cells from normal epithelium and in early lesions adjacent to the primary tumor in 11/15 (73%) specimens irrespective of the presence of mutation in the corresponding tumors. These data confirm that p53 overexpression is an early event in the multistep process of epithelial cell carcinogenesis. Loss of heterozygosity for the TP53 locus was detected in 54% of tumors but no association was found with mutation (Fisher's exact test P = 0.14). No mdm-2 amplification was detected in any tumors. No correlation was found between mutation and clinical parameters, the 5-year survival rates were not different (log rank test P = 0.39) in patients with and without mutation. In conclusion, we have shown that p53 gene mutations and deletions and protein overexpression are frequent in the most aggressive head and neck carcinomas but are not associated with disease progression. The presence of protein in normal mucosa and in non-invasive lesions may constitute a biomarker for early stages of carcinogenesis.

Association of c-erbB2-gene amplification with poor prognosis in non-inflammatory breast carcinomas but not in carcinomas of the inflammatory type.

It is now accepted that c-erbB2-gene amplification is correlated with poor clinical outcome for patients, mainly when axillary nodes are invaded. We have confirmed this result by multivariate analysis in 178 patients with non-inflammatory breast cancer followed up for a mean period of 6.8 years (SD, 1.6 years). In addition, we have shown that c-erbB2 amplification, found in 30 (17%) specimens, was associated with a high risk of multiple metastases developing simultaneously. In contrast, for the 67 patients with inflammatory breast carcinoma, the most aggressive type of breast carcinoma, the c-erbB2 amplification detected in 24 (36%) specimens was not found to be associated with a higher risk of death, suggesting that the c-erbB2 gene plays a different role in the progression of these 2 types of breast cancer. Furthermore, our data stress the importance of the methodological approach used to determine gene amplification. Although Southern blot hybridization is a tumour- and time-consuming method not easy to adopt in routine clinical practice, this method remains a reference quantitative method.

Study of molecular markers of resistance to m-AMSA in a human breast cancer cell line. Decrease of topoisomerase II and increase of both topoisomerase I and acidic glutathione S transferase.

Resistance to 0.8 microM 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) was induced by stepwise increases of drug concentration in the human tumor cell line CALc18 originating from a breast adenocarcinoma. The resistant cell line CALc18/AMSA exhibited a resistance index of 10 and a cross-resistance to other topoisomerase II inhibitors. A 3-fold decrease in the levels of topoisomerase II decatenating activity was found in CALc18/AMSA cells. By contrast, topoisomerase I activity was increased by about 3-fold in resistant cells. Interestingly this line was hypersensitive to camptothecin, a specific inhibitor of topoisomerase I. Restriction endonuclease patterns of the topoisomerase I and topoisomerase II loci were found to be identical in CALc18/AMSA and CALc18 with no evidence of gene amplification and rearrangements. Alkaline elution of m-AMSA-treated cells showed that DNA single strand breaks and DNA-protein crosslinks were decreased in CALc18/AMSA. The DNA lesions also obtained in m-AMSA-treated nuclei indicated that no drug uptake modification occurred in both cells. Moreover, the in vitro m-AMSA-induced DNA cleavage per unit of decatenating activity and the inhibitory effects of antitumoral drugs on decatenation were not found to be different with topoisomerase II from sensitive or resistant cells. However the specific cleavage induced by m-AMSA/per mg of crude protein from resistant cells was 2 to 3 times decreased. Multidrug resistance gene transcripts were not detected while levels of acidic glutathione S transferase mRNA were found to be 8 to 10-fold greater in resistant than in sensitive cell line with no amplification of the gene. In conclusion, the diminution of topoisomerase II activity and the increase of both topoisomerase I and acidic glutathione S transferase transcripts could contribute to the resistant phenotype of these breast cancer cells.

Expression of multidrug-resistance (MDR1) gene in normal epithelia and in invasive carcinomas of the uterine cervix.

Total RNA from 92 invasive cervical cancers was analyzed for the presence of multidrug-resistance (MDR1) gene (also known as PGY1) transcripts. A 4.5-kilobase MDR1 transcript band was detected in 40 (43%) of 92 invasive cervical carcinomas and in 15 (68%) of 22 normal cervices. MDR1 levels were low [mean, 2.5 arbitrary units (U)] except in one liver metastasis (50 U) treated with a drug regimen including vincristine. Of eight carcinomas treated by radiotherapy and/or chemotherapy, seven (88%) exhibited MDR1 transcripts as compared with 24 (35%) of 69 untreated carcinomas (Fisher's exact test; P = .01). In conclusion, our data suggest that the MDR1 gene plays a role in drug resistance of certain cervical cancers, but also that other mechanisms may be involved.

Topoisomerase II-mediated DNA cleavage activity induced by ellipticines on the human tumor cell line N417.

Ellipticine derivatives have been shown to induce DNA strand breaks by trapping DNA-topoisomerase II (Topo II) in an intermediary covalent complex between Topo II and DNA which could be related to their cytotoxic effects. We report here that Celiptium and Detalliptinium, two ellipticine derivatives clinically used for their antitumoral properties against breast cancer, exhibit the highest in vitro activity on Topo II DNA cleavage reaction and decatenation among a series of 14 ellipticine derivatives. The in vitro cleavage site specificity in pBR 322 plasmid DNA and in a human c-myc gene inserted in a lambda phage DNA is identical for both ellipticines, but different from m-AMSA, another Topo II related antitumoral agent. Recently, it has been shown that the ellipticine derivative Celiptium presents a strong cytotoxic activity in vitro on different human tumors including small cell lung carcinoma (SCLC). However, the studies that involved Topo II as a target for ellipticine derivatives have been performed only by using animal tumor cell lines. Therefore we have studied the in vivo DNA cleavage activity of Celiptium and Detalliptinium on a human SCLC cell line, NCI N417, comparatively to that obtained with m-AMSA. The respective IC50 on cell growth are 9, 8 and 1 microM for Celiptium, Detalliptinium and m-AMSA, respectively. Using the alkaline elution technique, we have observed that Celiptium and Detalliptinium exhibit a weak cleavage activity on genomic DNA from whole cells. The ellipticines are about 50 times less potent than m-AMSA in inducing DNA strand breaks. Analysis of in vivo c-myc gene cleavage by Southern blot hybridization also demonstrates a lack of activity of the ellipticine derivatives as no gene cleavage could be detected up to 50 microM of the drug. With m-AMSA, c-myc gene cleavage is detected at a concentration of 0.2 microM, which indicates that this methodology is less sensitive in detecting DNA strand breaks than is the alkaline elution. Further studies of the drug effect on isolated nuclei by alkaline elution also show that the DNA cleavage activity of Celiptium and Detalliptinium is increased when compared to whole cells. Our data indicate that these two drugs have a weaker cytotoxic effect than m-AMSA on NCI N417 cell line, due to a limited access to the cell nucleus rather than to a lack of activity on Topo II as assessed by in vitro and isolated nuclei experiments.

ATP-independent DNA topoisomerase II as potential drug target in trypanosomes.

Two categories of trypanosomal type II topoisomerases have been isolated from trypanosomes: one is unique since it is able to realize DNA topoisomerization reactions in the absence of ATP, in contrast to the other enzyme and mammalian topoisomerase II. The biochemical properties of ATP-independent topoisomerase II from Trypanosoma cruzi are described in this report. The enzyme can decatenate trypanosome kinetoplast DNA networks, catenate supercoiled DNA molecules, unknot P4 phage DNA, and cleave double-stranded DNA. The enzyme is inhibited by various classes of drugs and is more sensitive than mammalian topoisomerase II. Therefore, trypanosome ATP-independent topoisomerase II provides a potential target for chemotherapy.