Detection of gastrin mRNA in fresh human colonic carcinomas by reverse transcription-polymerase chain reaction.

To investigate the hypothesis that gastrin might be synthesized by tumour tissues in cancer of the colon, samples from six human colon tumours, one hepatic metastasis, four normal colonic mucosal samples and two antral and one fundic gastric mucosal samples from nine patients were analysed to determine whether gastrin mRNA was present. RNA was extracted from surgical specimens by ultracentrifugation on a CsCl cushion, purified using the guanidinium thiocyanate method, reverse-transcribed and amplified by polymerase chain reaction. Gastrin mRNA was detected in each colonic carcinoma sample (including the hepatic metastasis), while no such signal was observed in normal colon biopsies. Positive and negative controls (gastric antrum and fundus respectively) gave the expected results. In each of the positive samples, the chemiluminescent revelation of amplified products after Southern blotting corresponded to gastrin mRNA without the intron. These findings demonstrate the ability of primary and metastatic human colonic tumours to produce gastrin mRNA. Since malignant cell lines have been reported to produce gastrin peptide, and since gastrin receptors were present in some cases, our results support the idea that gastrin may be involved in an autocrine mechanism.

Expression of variant forms of fibroblast growth factor receptor 2 mRNA in human breast carcinomas.

FGF (fibroblast growth factors) and FGF receptors may play a role in stroma-epithelium relationships in the mammary gland. Dysregulation of their interactions may be important in mammary carcinogenesis. Isoforms of the FGFR2 receptor, differing in the structure of the extracellular region, are expressed in the mammary gland and may diversely affect stroma-epithelium relationships. We determined the mRNA variants encoding these isoforms in human cell lines and breast carcinomas. All possible combinations of variants were found. No correlation was observed between the presence of a particular variant and the expression of any FGF gene tested, or the status of any histoclinical parameter.

Cumulative regional allelotyping of human breast carcinomas.

We have analyzed losses of heterozygosity (LOH) at markers from chromosomes 1, 2, 4 and 5 in a panel of 53 consecutive breast carcinomas. Together with a parallel analysis of LOH at chromosome 3, this allowed the identification of twenty-one regions of LOH. In contrast, in a comparative analysis of chromosome X, no region of loss could be defined. Cumulative regional allelotyping of 38 tumors with 71 markers from the five autosomes (37% of the genome) enabled the study of the respective distribution of regions of frequent loss, to identify associations of regions of LOH, and to distinguish tumors frequently affected by LOH such as lobular carcinomas.

Detection of five different human papillomavirus types in cervical lesions by in situ hybridization. A study of 110 cases using sulfonated probes.

Several findings suggest an etiologic relationship between genital tract squamous cell carcinoma and certain types of human papillomavirus (HPV). Detection of these HPV types in cervical lesions considered as preneoplastic states (ie, cervical intraepithelial neoplasia or CIN) is extremely important but difficult because the morphology of these states is highly heterogeneous and clinical course is rarely predictable. In situ hybridization (ISH) is the only technique allowing correlation between HPV type and tissue or cell morphology. In this report, 110 biopsy specimens from uterine cervix lesions were studied: 66 CIN, 10 invasive carcinoma, 28 metaplasia, and six condyloma acuminata. A new ISH technique based on direct modification of DNA probes by sulfonation was used. The hybridized DNA was revealed first by a specific monoclonal antibody against sulfonated DNA, and then by an alkaline phosphatase system. In order to determine the sensitivity level of this method, 14 biopsy specimens were also submitted to Southern blot hybridization. Five probes were used separately (HPV 6, 11, 16, 18, and 33) for each biopsy specimen. Results of ISH were correlated with morphologic criteria such as number of koilocytes and mitoses. Oncogenic HPV was found exclusively in CIN. The number of labeled cells varied with CIN grade. These data suggest that, whatever the grade, CIN represents a unique preneoplastic process, and that HPV replication depends on the squamous maturation of the pathologic epithelium.

Detection of gastrin mRNA by in situ hybridization using radioactive- and digoxigenin-labelled probes: a comparative study.

The hormone gastrin is mainly produced by the G cells of the antral mucosa and plays a major role in the regulation of digestive mucosal growth. Since it permits identification of cell types containing mRNA, in situ hybridization (ISH) appears to be an interesting method for studying gastrin-producing tissues. In this study, in situ detection of gastrin mRNA has been carried out on frozen sections of four human normal antral mucosa samples and of six colonic carcinomas removed from patients with high levels of plasma gastrin, using a gastrin oligonucleotidic DNA probe. We have compared the results provided respectively by the [35s] labelling and the digoxigenin labelling of the synthetic probe. Positive cells were found in each normal sample analysed with radioactive- as well as digoxigenin-labelled antisense probes. The total number of cells expressing gastrin mRNA appeared slightly higher with the [35s]-labelled probe, while the digoxigenin-labelled probe gave a better definition of positive signals. In contrast, neither radioactive nor cold probes gave positive signals in the six colonic carcinoma samples, although gastrin expression had been demonstrated in these tumours using a reverse transcriptase-PCR method. These results show that, although ISH does not seem sensitive enough to allow the detection of very low levels of gastrin expression, it would appear to be a reliable method for visualizing gastrin mRNA in human antral mucosa.

Search for rearrangements and/or allelic loss of the fas/APO-1 gene in 101 human lymphomas.

The authors analyzed the possible occurrence of rearrangements and/or allelic loss of the fas/APO-1 gene in a representative series of human lymphomas, including 101 cases of Hodgkin's disease (HD) and non-Hodgkin's lymphomas (NHL). The rationale for this study was double. Chromosome 10 alterations, frequently observed in lymphoma subtypes, encompass the chromosomal localization of fas/APO-1. In addition, Ipr mouse mutants, which present with a generalized lymphoproliferative disease, were shown to exhibit alterations of fas/APO-1 structure and expression. In this retrospective study, the authors performed gene dosage of fas/APO-1 by Southern blots. No fas/APO-1 alterations were observed in the 31 HD cases. Among 70 T-cell and B-cell NHL, allelic loss of fas/APO-1 was observed in three cases. Two cases with different clinical, phenotypic, and histologic presentations showed a rearrangement of fas/APO-1. A third case showed amplification. Thus fas/APO-1 alterations do occur in human lymphomas, although at a relatively low frequency.

Expression of FGF1 and FGFR1 in human melanoma tissues.

Fibroblast growth factor 2 (FGF2) has been implicated in the pathogenesis of malignant melanoma (MM), but the role of other FGFs and their receptors (FGFRs) is not elucidated. To determine whether FGF1 and FGFR1 may be involved in MM growth in vivo, we have studied the expression of the FGF1 and FGFR1 genes in 77 fresh MM biopsy samples, using RT-PCR analysis. Samples of benign nevi, normal skin and carcinoma cell lines were included as controls. Using RT-PCR analysis, expression of FGF1 and FGFR1 was observed in 69/77 and 68/77 cases, respectively. Immunohistochemical detection of the FGFR1 protein was positive in reactive stromal cells and at a much lower level in neoplastic cells. In situ hybridization experiments demonstrated FGFR1 mRNA mainly located in the stromal component. Southern blot analysis of genomic DNA prepared from MM tumors did not show any structural alteration of the FGFR1 gene. There was no correlation between FGF1/FGFR1 expression and the usual clinicopathological parameters of MM. We conclude that FGF1 and FGFR1 are frequently co-expressed in MM, a situation that may contribute to aberrant autocrine and paracrine pathways. Due to the absence of correlation with clinico-pathological parameters, this expression cannot be used as a marker of prognosis in the management of MM patients.

[Gastric adenocarcinoma with an endocrine component. Report of nine cases]. / Adénocarcinomes gastriques à composante endocrine. A propos de neuf cas.

Nine gastric adenocarcinomas containing endocrine cells were retrospectively studied to evaluate anatomo-clinical and immunohistochemical features, and the out-come of the patients. These gastric tumors were bulky with frequently lymph node metastasis. The immunohistochemical analysis had shown that all tumors contain EC cells, two tumors D cells, one tumor PP cells, but neither G cells nor A cells were detected. Five-years survival rate of these gastric tumors was slightly but not significantly increased as compared to usual adenocarcinomas (45.5% vs 36.5%). However, one can raise the question of possible control of local hormonal secretions on tumoral growth, particularly the anti-trophic part of the somatostatin.

Molecular analysis of the NPM-ALK rearrangement in Hodgkin's disease.

The fusion gene NPM-ALK occurs in a subset of anaplastic large cell lymphomas (ALCLs), as a result of a chromosomal translocation, t(2;5) (p23;q35). It has been suggested that Hodgkin's disease (HD) and ALCL share a common histogenesis because of pathological and phenotypical similarities. In order to check this hypothesis, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect the hybrid NPM-ALK gene in 30 tumour samples, including 22 lymph node biopsies from HD and eight ALCL specimens. The threshold level of sensitivity was shown to reach at least 1/10(4) by dilution experiments using cell lines as positive and negative controls. The expected 177 bp product indicative of the NPM-ALK rearrangement was identified in Karpas 299 and SUDHL-1 cell lines and in two out of eight ALCLs. The 22 HD cases were negative, even after two successive tests. Thus, since the ALCL-specific genetic alteration was absent in our series of HD cases, the present study does not support the hypothesis that HD and ALCL are histogenetically related entities.

Frequent expression of FAS/APO-1 in Hodgkin's disease and anaplastic large cell lymphomas.

FAS/APO-1 (CD95) is a membrane glycoprotein belonging to the tumour necrosis factor/nerve growth factor receptor family, and which can trigger apoptosis in some lymphoid cell lines. Immunohistochemistry combined with Northern blotting allowed determination of the pattern of FAS/APO-1 expression in a series of Ki-1 [CD30] positive lymphoid malignancies, including 27 Hodgkin's disease and eight anaplastic large cell lymphomas. CD30 negative tumours used as controls included 27 B-cell non-Hodgkin's lymphomas. 14 T-cell non-Hodgkin's lymphomas, four reactive lymphadenitis, and non-lymphoid tissues. Immunohistochemistry, performed on frozen sections, revealed a strong FAS/APO-1 expression in 25 out of 27 (92%) Hodgkin's disease cases, predominantly in Reed Sternberg cells; 50 to 100% of the neoplastic cells in eight out of (100%) anaplastic large cell lymphoma cases were positive. In contrast, positive FAS/APO-1 immunostaining was observed only in 22 out of 41 (53%) CD30 negative non-Hodgkin's lymphomas. Northern blot analysis detected variable amounts of the FAS/APO-1 transcript in the immunohistochemistry-positive samples. These results suggest possible hyper-expression of FAS/APO-1 (CD95) in Hodgkin's disease and anaplastic large cell lymphomas.

Unrestricted T-cell receptor V-region gene repertoire in tumor-infiltrating lymphocytes from human breast carcinomas.

BACKGROUND: Several lines of evidence indicate that a more favorable prognosis is correlated with the degree of lymphocyte infiltration within breast carcinomas. The characterization of these T-cell infiltrates appears necessary to explore the possible existence of an in situ immunologic response of the host against neoplastic cells. Previous studies have indeed suggested that the T-cell receptor (TCR) repertoire of tumor-infiltrating T-lymphocytes (TIL) should be restricted if they act specifically against tumor-related antigens. METHODS: To address this question, the expression of variable (V) region genes of the TCR-alpha and beta chains in intratumoral lymphocytes from five breast carcinoma biopsy specimens and one normal mammary gland specimen was analyzed using the polymerase chain reaction (PCR). The neoplastic samples included one medullary carcinoma and four ductal infiltrating carcinomas selected on the basis of a rich lymphoid stromal component. Primers specific for 18 different V alpha and 21 V beta families were used for PCR. RESULTS: In every case, TIL showed an unrestricted pattern of TCR V-region gene expression, similar to the repertoire observed in peripheral blood lymphocytes and in the normal breast tissue. CONCLUSIONS: According to these nonquantitative PCR experiments, the apparent lack of selection of a limited number of T-cell specificities in the affected tissues does not favor the existence of an in vivo lymphoid recruitment based on TCR recognition of neoplastic antigenic determinants. Further studies, however, leading to an accurate quantification of immunologically relevant T-cell clones and including larger series of cases still are necessary before it will be possible to draw a final conclusion.

PCR-mismatch analysis of p53 gene mutation in Hodgkin's disease.

Expression of the p53 protein can be detected by immunohistochemistry in Reed-Sternberg (RS) cells, the presumed neoplastic component of Hodgkin's disease (HD) lesions. At present, there is no clear molecular evidence that p53 positive immunostaining in HD correlates with the presence of mutations or other structural alterations of the p53 gene. To address this question, 49 cases of HD have been investigated for p53 expression by immunohistochemistry, using the DO1 monoclonal antibody on paraffin sections. Thirty-seven out of 49 cases (75 per cent) exhibited positive immunostaining restricted to RS cells and variants. Among these 37 positive cases, ten cases were selected on the basis of a rich content of RS cells showing virtually 100 per cent DO1 positivity. A PCR-mismatch strategy was chosen for the detection of p53 mutations. The threshold level of sensitivity was assessed on positive cell-line controls. A proportion of 10-15 per cent p53 mutated cells mixed in a normal population could be identified. Total genomic DNA was extracted from the ten selected HD cases and PCR amplification of exons 5-8 of the p53 gene was performed. Heteroduplex mismatch analysis revealed no structural alterations of the p53 gene in any case. In view of these findings, it appears unlikely that the sensitivity of the method by itself can fully explain the negative results, although this possibility cannot be completely ruled out. Thus, it is conceivable that p53 positive immunostaining in HD may not necessarily imply genomic alterations in the classic 'hot spot' regions and may be related to another mechanism of p53 protein stabilization.

Loss of heterozygosity at loci from chromosome arm 22Q in human sporadic breast carcinomas.

Forty-four breast carcinomas were studied for loss of heterozygosity (LOH) at 25 microsatellite markers distributed almost evenly along chromosome arm 22q. LOH at at least one marker were observed in 66% tumors, while 6 regions of consistent LOH were identified. The size of each region ranged between 3 and 6 cM, and the distance between each region was estimated to be 8 to 12 cM. Even if not all these regions contain a bona fide tumor-suppressor gene, it is possible that several loci from chromosome arm 22q may be involved in breast carcinogenesis.

Expression of FGF and FGF receptor genes in human breast cancer.

The family of FGF growth factors is involved in several biological processes and might play an important role in tumorigenesis. We have studied the respective expression of 8 of the 9 characterized FGF genes, and of the 4 known FGF receptor genes, in a panel of 10 tumor-cell lines and 103 breast-tumor samples, using RT-PCR and Northern-blot analyses. FGF1 and FGF2 were expressed in almost all samples, while expression of FGF5, FGF6, FGF7, and FGF9 was more restricted. FGFR1, FGFR2 and FGFR4 were expressed at high levels in respectively 22%, 4% and 32% of tumors. FGFR3 expression was not detected. The transcript encoding an FGFR1 isoform with 2 immunoglobulin-like domains was the most prevalent.

Fibroblast growth factor gene expression in AIDS-Kaposi's sarcoma detected by in situ hybridization.

Biopsy samples from five acquired immune deficiency syndrome (AIDS)-Kaposi's sarcomas and one non-AIDS-associated Kaposi's sarcoma were assayed by in situ RNA hybridization onto paraformaldehyde-fixed, paraffin-embedded skin sections for the presence of two fibroblast growth factor gene transcripts, FGFB and FGF5. FGF5 gene expression was detected in the characteristic Kaposi's sarcoma spindle-shaped cells in the five samples from human immunodeficiency-positive (HIV+) patients. FGFB transcripts were detected in Kaposi's sarcoma cells as well as in epidermis of HIV- and HIV+ patients. These results complement the observations about growth factor gene expression done on Kaposi's sarcoma-derived cell lines, which thus appear to be representative of what happens in vivo. Furthermore, they demonstrate a contrasting expression pattern of FGF5 and FGFB genes, both involved in the growth factor pathogenic cascade leading to Kaposi's sarcoma.

Expression of the FGFR1 gene in human breast-carcinoma cells.

Fibroblast growth factors (FGF) constitute a family of at least 9 members which act through high-affinity tyrosine-kinase receptors encoded by 4 distinct genes. In humans, the FGFR1 gene is located in chromosomal region 8p12. Its amplification and expression were examined in a panel of 110 breast carcinoma samples by Southern- and Northern-blot analyses. FGFR1 was amplified in 9% and overexpressed in about 15% of the tumors studied. In situ hybridization experiments were performed on tissue sections of normal breast and tumors with a high level of FGFR1 expression. In both normal and tumoral tissues, FGFR1 RNA was detected in the epithelial cells. Overexpression of FGFR1 seems to be associated with small, well-differentiated diploid tumors.

Predominant expression of the long isoform of Bcl-x (Bcl-xL) in human lymphomas.

Bcl-x is a member of the bcl-2 family of proteins which are characterized by their ability to modulate apoptosis. Alternative splicing results in two distinct bcl-x mRNAs encoding a long isoform, bcl-xL, which acts as a bcl-2 agonist; and a short isoform, bcl-xS, which inhibits bcl-2 effects. The aim of the study was to determine whether bcl-x is expressed in lymphoma tissues and to characterize the respective production of bcl-xs and bcl-xL. We investigated the expression of bcl-x mRNA in a series of 50 non-Hodgkin's lymphomas (NHL) and Hodgkin's disease (HD) cases using a RT-PCR method in order to amplify both transcripts simultaneously, and to estimate their relative abundance. The rearrangements of the bcl-2 gene were analysed by RT-PCR expression of the hybrid bcl-2-lgH mRNA. In addition, 20 PCR-positive NHL cases and three HD cases were analysed by immunohistochemistry using bcl-x polyclonal antisera. RT-PCR showed bcl-x expression in 43/45 NHLs and 5/5 HD cases. The bcl-xL transcript was predominant in all positive cases and was associated with variable amounts of bcl-xS. There was no significant correlation between the profile of bcl-xL/bcl-xS expression and the histological and immunological subtyping. Bcl-x immunodetection was positive in the neoplastic cell component in all analysed cases, but the degree of staining was highly variable between cases. Expression of the hybrid bcl-2-IgH gene was detected by RT-PCR in five cases of follicular NHL and in one case of HD, but this group of tumours did not display a particular profile of bcl-xL/bcl-xS expression. We conclude that bcl-x is commonly expressed by malignant cells in various types of malignant lymphomas, with a predominance of the bcl-xL transcript. Since the corresponding bcl-xL isoform can block the cell death machinery and potentialize bcl-2 effects, it may be involved in some pathways of lymphomagenesis.