Rare germline large rearrangements in the BRCA1/2 genes and eight candidate genes in 472 patients with breast cancer predisposition.

Hereditary breast cancers account for up to 5-10 % of breast cancers and a majority are related to the BRCA1 and BRCA2 genes. However, many families with breast cancer predisposition do not carry any known mutations for BRCA1 and BRCA2 genes. We explored the incidence of rare large rearrangements in the coding, noncoding and flanking regions of BRCA1/2 and in eight other candidate genes--CHEK2, BARD1, ATM, RAD50, RAD51, BRIP1, RAP80 and PALB2. A dedicated zoom-in CGH-array was applied to screen for rearrangements in 472 unrelated French individuals from breast-ovarian cancer families that were being followed in eight French oncogenetic laboratories. No new rearrangement was found neither in the genomic regions of BRCA1/2 nor in candidate genes, except for the CHEK2 and BARD1 genes. Three heterozygous deletions were detected in the 5' and 3' flanking regions of BRCA1. One large deletion introducing a frameshift was identified in the CHEK2 gene in two families and one heterozygous deletion was detected within an intron of BARD1. The study demonstrates the usefulness of CGH-array in routine genetic analysis and, aside from the CHEK2 rearrangements, indicates there is a very low incidence of large rearrangements in BRCA1/2 and in the other eight candidate genes in families already explored for BRCA1/2 mutations. Finally, next-generation sequencing should bring new information about point mutations in intronic and flanking regions and also medium size rearrangements.

[Diagnostic modalities of visceral metastases during follow-up of patients with stage I-II melanoma]. / Malades suivis pour mélanome de stade I-II: modalités diagnostiques des métastases viscérales.

INTRODUCTION: Although not recommended in France at the consensus conference of 1994, routine monitoring of patients with stage I melanoma using imaging techniques is commonly carried out. The aim of this retrospective regional study was to define methods for diagnosing transition to the metastatic stage of melanoma. PATIENTS AND METHODS: This was a retrospective study based on questionnaires among dermatologists in the Champagne-Ardenne and southern Aisne regions of France. For each patient with stage IV melanoma between 1987 and 2002, data were collected concerning the primary melanoma (date of diagnosis, clinical picture, histopathologic features), stage of melanoma prior to diagnosis of metastatic melanoma and characteristics of the metastases (date, number, type, site and modern discovery: clinical signs or routine imaging). RESULTS: One hundred and eight patients (63 men and 45 women; mean age: 59 years) were included in the study. The predominant site of the primary melanoma was the trunk for men (n=31) and the lower limbs for women (n=16) and the mean Breslow index was 4.31 mm (SD=4.22), with histologic ulceration being present in 40% of cases. The mean time to transition to stage IV after discovery of the primary tumour was 2.8 years (SD=2.95). The modes of discovery of metastases comprised clinical examination (functional signs or physical examination) in 58 cases and routine imaging in 50 cases, with no significant differences based on whether patients were initially in stage I-II or in stage III. DISCUSSION: This study shows that over half of patients progressing to stage IV melanoma had a suspicious sign or clinical symptom, once again highlighting the importance of clinical monitoring. In contrast, many organ metastases, particularly pulmonary, were discovered by routine imaging examinations carried out as part of patient follow-up, although this is not currently recommended practice in France. CONCLUSION: The role of powerful imaging examinations such as scans, with constantly improving resolution, still remains to be defined in the follow-up of patients with stage I-II melanoma, and further prospective studies are thus required.

The contribution of germline rearrangements to the spectrum of BRCA2 mutations.

BACKGROUND: Few germline BRCA2 rearrangements have been described compared with the large number of germline rearrangements reported in the BRCA1 gene. However, some BRCA2 rearrangements have been reported in families that included at least one case of male breast cancer. OBJECTIVE: To estimate the contribution of large genomic rearrangements to the spectrum of BRCA2 defects. METHODS: Quantitative multiplex PCR of short fluorescent fragments (QMPSF) was used to screen the BRCA2 gene for germline rearrangements in highly selected families. QMPSF was previously used to detect heterozygous deletions/duplications in many genes including BRCA1 and BRCA2. RESULTS: We selected a subgroup of 194 high risk families with four or more breast cancers with an average age at diagnosis of < or = 50 years, who were recruited through 14 genetic counselling centres in France and one centre in Switzerland. BRCA2 mutations were detected in 18.6% (36 index cases) and BRCA1 mutations in 12.4% (24 index cases) of these families. Of the 134 BRCA1/2 negative index cases in this subgroup, 120 were screened for large rearrangements of BRCA2 using QMPSF. Novel and distinct BRCA2 deletions were detected in three families and their boundaries were determined. We found that genomic rearrangements represent 7.7% (95% confidence interval 0% to 16%) of the BRCA2 mutation spectrum. CONCLUSION: The molecular diagnosis of breast cancer predisposition should include screening for BRCA2 rearrangements, at least in families with a high probability of BRCA2 defects.

[Skin cancer diagnosis by dermatologists in the region of Champagne-Ardenne: a prospective study]. / Etude prospective de l'incidence des cancers cutanés dépistés en pratique dermatologique dans la région Champagne-Ardenne.

INTRODUCTION: The exact incidence of skin cancers is not well established in France as in other european countries, except for cutaneous melanoma. It is the same for the part taken by dermatologists in the screening of skin malignancies. We have therefore planned a prospective study in the Champagne-Ardenne region to determine the incidence of the various types of skin cancers diagnosed by dermatologists. PATIENTS AND METHOD: A prospective study was performed within a 4-month period at the end of 1998 with the participation of the dermatologists of the Champagne-Ardenne and the south of Aisne. The new incident cases of skin cancers confirmed histologically were recorded using a questionnaire including the type of tumour, demographic data of the patient and type of practice of the dermatologist. Parallely, the pathology laboratories of the same region were asked about the number of squamous and basal cell carcinomas and melanomas diagnosed histologically during the same period of time. RESULTS: The rate of participation of dermatologists was 86 p. 100. During the study period, 898 skin cancers were diagnosed in 794 patients (408 women and 386 men; mean age 70.1 years) including 643 basal cell carcinomas, 123 squamous cell carcinomas, 48 Bowen diseases, 53 melanomas, 7 lymphomas and 24 other tumours. More than 80 p. 100 of patients were primarily seen in private practice. Ten of 13 pathology laboratories of the region replied to the inquiry. From these pathological data, 585 basal cell carcinomas, 125 squamous cell carcinomas and 44 melanomas were recorded during the same period of time. DISCUSSION: This study shows that the oncologic clinical practice of dermatologists in our region is considerable. Taking into account both participation and exhaustivity rates, one can estimate at more than 3,500 the annual number of skin cancers diagnosed by dermatologists in our region, with an expected distribution of the various types of cancers. The essential role of the dermatologists in screening skin malignancies is confirmed by data provided by pathology laboratories. This role is a french characteristic and cannot by applied to other industrialized countries.

Quantitative determination of c-erbB-2 in human breast tumours: potential prognostic significance of low values.

The purpose of this prospective multicentric study was to quantify the c-erbB-2 protein and investigate its relationship with DNA amplification and with various prognostic parameters of breast cancer. A total of 1062 primary operable human breast tumours were collected from six French anticancer centres. The c-erbB-2 protein was measured using an enzymoimmunoassay using two monoclonal antibodies directed against the extracellular domain of the protein. The results were expressed in arbitrary units/mg membrane protein (AU) after adjustment for the anticancer centre. A significant association was found between the dosage of the protein and DNA amplification (P = 0.0001). A value of 200 AU was found to maximise sensibility and specificity and was chosen as a cut-off for over-expression. Significant associations were found between c-erbB-2 values and oestrogen receptor (ER) (P = 0.01), progesterone receptor (PgR) (P = 0.0001) and histological grading (P = 0.01). The extreme high values (above the mean plus one standard deviation, S.D.) were significantly more numerous in ER- (P = 10(-16)), PgR- (P = 10(-14)) and grade III (P = 10(-8)) tumours. The extreme low values (below the mean minus one S.D.) were significantly more numerous in ER- (P = 10(-9)) and PgR- (P = 0.02) tumours. This prospective study confirms that high c-erbB-2 protein values are linked to poor prognostic factors and shows for the first time that low values are also linked to hormone receptor negative tumours, suggesting that these low values might also have a negative prognostic significance.

[Detection of metastatic activity of the MCF-7 multidrug resistant cell line cultured in spheroids]. / Mise en évidence d'une activité métastasiante dans la lignée MCF-7 multidrogue résistante cultivée en sphéroïdes.

The occurrence of solid tumors spreading through the body is a major concern for the clinicians. Moreover, in numerous cases, metastases exhibit a multidrug resistant (MDR) pattern. This dual characteristic still remains supported by few biological explanations. The purpose of our study was to compare invasive properties of sensitive and MDR MCF-7 cells. Spheroids were chosen as experimental model since they exhibit a number of characteristics (i.e. tridimensional structure) close to the growth of an in vivo tumor. MDR spheroids formed more compact structures compared to sensitive ones. In every experiment, spheroids made from sensitive cells were more resistant to doxorubicin than the same cells grown as monolayers, a characteristic not observed with MDR cells. On an other hand, a form of multicellular resistance appeared in spheroids of sensitive cells, a fact which was not present in MDR spheroids. Incubation of MDR spheroids in Boyden's chambers put in evidence increased motility and invasive properties through Matrigel which were not present in sensitive MCF-7 cells. Zymograms of culture media and membrane extracts were performed in polyacrylamide gels. Two metalloproteases, progelatinases A et B were detected in culture media conditioned by monolayers and spheroids of both sensitive and resistant cells. In contrast, 2 unidentified serine proteases were detected only in media conditioned by spheroids of both cell types. An intense band of pro-MMP2 was present only in membrane extracts from MDR spheroids. Taken altogether, these results demonstrate that spheroids of MDR cells exhibit a number of properties which could lead to an increased ability to form metastases.

Sequential overexpression of LRP and MRP but not P-gp 170 in VP16-selected A549 adenocarcinoma cells.

Chemoresistance remains the major obstacle to successful therapy of lung cancer. In order to understand drug resistance mechanisms, the expression of three proteins involved in multidrug resistance (P-gp, MRP and LRP) was studied, using the non-small cell lung cancer (NSCLC) A549 cell line. In addition, 3 levels of resistance were obtained by continuous exposure of cells to etoposide (VP16), which led to a 22-fold increase of the resistance index. The wild-type A549 strongly expressed the LRP protein while MRP protein was found at a moderate level. Induction of resistance paralleled an increase of the expression of the mrp gene and a decrease of the lrp gene; the mdr1 gene was not expressed. Taken together, these results indicate that intrinsically resistant NSCLC cells exhibit a complex pattern of MDR proteins, still susceptible to evolve under treatment. Such a fact would have to be considered in clinical situations.

Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.

Ret and trk proto-oncogene activation in thyroid papillary carcinomas in French patients from the Champagne-Ardenne region.

OBJECTIVES: To investigate the presence of ret and trk proto-oncogene rearrangements in thyroid tumors. DESIGN AND METHODS: High-molecular-weight DNA was extracted from 36 thyroid tumors (1 multinodular goiter, 14 follicular adenomas, 16 papillary carcinomas, 1 lymph node metastasis of a papillary carcinoma, 1 follicular carcinoma, and 3 medullary carcinomas) and 22 adjacent tissues. Southern blot analysis was performed after digestion with EcoR1 or BamH1, using specific probes for ret and trk. RESULTS: Only 2 ret rearrangements were found in 2 papillary carcinomas (overall frequency: 6%; papillary carcinoma frequency: 13%). All normal or tumor samples were negative for the presence of a trk rearrangement. CONCLUSIONS: The previous data from the literature are highly conflicting, ranging from 0 to 30% of activation. Our results could be, therefore, classified as medium between these extreme values. It seems, therefore, that genetic and/or geographical factors could play a role in ret and trk proto-oncogene activation.

Characteristics of four Friend leukemia cell sublines resistant to adriamycin.

Resistance of Friend murine erythroleukemia cells was induced or selected by continuous stepwise exposure to adriamycin (ADM). The resistance index (R.I.) varied with different "mdr type" drugs, even when the compounds were closely related as ADM and daunorubicin (DNR). The cell uptake of anthracycline, evaluated by flow cytometry (FCM), was better correlated with the R.I. than the HPLC assessment. The quantitative cytology showed nuclear changes (size and color distribution); all the modifications were in part dependent on the degree of resistance. This study showed that the graded resistant sublines differed in their resistant phenotypes apart from their different degree of resistance.

[The multidrug resistance phenotype: some morphological and biological characteristics except efflux pump]. / Le phénotype de résistance multidrogue: quelques caractéristiques morphologiques et biologiques autres que la pompe à efflux.

Recent data from the literature together with personal results strongly suggest that multidrug resistance phenotype is overwhelming the sole expression of P170 glycoprotein efflux pump. Morphological alterations have been put in evidence in MDR cells after transmission and scanning electron microscopy. They include presence of osmiophilic vesicles and modifications of nuclear and nucleolar chromatin. Biological characteristics include the hypersecretory pattern of lysosomal enzymes from MDR cells. Such a fact could be more or less related to the increased occurrence of mdr1 RNA in metastasis, especially in breast cancers, compared to primary tumors. If the P170-mediated efflux is one of the key mechanism of MDR, a decreased influx of anticancer drugs cannot be excluded. Liposomes, for instance made of cardiolipin, are thus able to increase the intracellular drug uptake of vinblastine without any action upon efflux mechanism.

Expression of mdr1 gene in human breast primary tumors and metastases.

Expression of mdr1 gene has been evaluated in 34 tumor samples obtained from breast cancer patients who were classified according to their treatment, and clinical follow-up. No gene amplification was found. mdr1-RNA was never detected in 29 primary breast tumors including 5 samples from patients previously treated by 6 courses of 5-fluorouracil, epirubicin, cyclophosphamide (FEC). On the other hand, mdr1-RNA expression was detected in 1 local recurrence and 2 out of 3 metastases, all of them being treated and exhibiting a poor evolution. A second, untreated local recurrence remained negative. Clinical follow-up for 7 to 48 months in patients receiving chemotherapy showed that absence of mdr1-RNA could not be an accurate factor of satisfactory response to chemotherapy. But, all the patients with detectable mdr1-RNA exhibited a poor evolution and response to treatment. In conclusion, evaluation of mdr1-RNA seemed to be of little interest in primary breast tumors. However, the concomitant presence of an mdr1-RNA and a metastatic phenotype could give a new insight into the relationship between invasive and resistance properties of cancer cells. Such situations would need to be analyzed very carefully for a better utilization of chemotherapy.

[Serum enzymes and triglycerides in mice with a mammary tumor (Ca-755 adenocarcinoma)]. / Enzymes sériques et triglycérides chez les souris porteuses d'une tumeur mammaire (adénocarcinome Ca-755).

Investigations on mice inoculated with Ca-755 mammary adenocarcinoma have shown that the levels of serum LDH (especially isozymes resulting from the B sub-unit), TGO and TG are higher than those found in normal mice, while PAL (only bone isozyme) and TGP are lower. Comparisons were made with tumour age (exponential and plateau-growth phases), which appeared to be important for LDH and PAL bone isozyme, since LDH increased and PAL decreased with increasing age of the tumour. For the PAL and TG alterations, there could be assumed an effect of cachectin (TNF: tumour necrosis factor).

Expression of CR1 and CR2 complement receptors following Epstein-Barr virus infection of Burkitt's lymphoma cell lines.

Epstein-Barr virus (EBV) infection of human B lymphocytes involves a specific receptor closely associated with, or identical to, the C3d complement receptor, CR2. Thus, 25 out of 29 EBV-positive Burkitt's lymphoma (BL) cell lines but none of 15 EBV-negative BL lines were found to express C3 receptors. Furthermore, in vitro infection with EBV of six EBV-negative cell lines resulted in the expression of C3 receptors in association with that of EBV-determined nuclear antigen (EBNA). Rosette assays using erythrocytes coated with human C3b, C3bi, and C3d, inhibition of rosette formation with anti-receptor antibodies, and flow cytometry analysis of stained cells demonstrated that EBV-converted lines expressed C3b and C3d receptors, CR1 and CR2. Anti-receptor antibodies recognized an average of 40,700 anti-CR1 and 140,000 anti-CR2 binding sites on an EBV-converted line (BL41/B95), whereas no specific binding occurred on the corresponding EBV-negative (BL41) cells. Because CR1 and CR2 are involved in B-cell proliferation and/or differentiation, enhanced expression of C3 receptors following the interaction between EBV and B cells and/or subsequent infection of the cells by EBV may provide a basis for positive control of B lymphocyte proliferation by EBV.

[Cancer of the prostate: prostatic acid phosphatases. Biochemical properties and assay]. / Cancer de la prostate: phosphatases acides prostatiques. Propriétés biochimiques et détermination.

The first data on acidic phosphatase were published in the beginning of the 20th century. The applications to prostatic carcinoma were performed by Gutman in 1936. The enzyme is a glycoprotein which action mechanism is similar to a histidine-phosphatase. Acidic phosphatase has been found in numerous tissues and blood-stream cells. Its structure is dimeric and glycans average 10% of the M.W. The enzyme has been assayed in serum, using numerous substrates in the presence of specific inhibitors. The best results were obtained with thymolphthalein phosphate. Improvements were done by assays in bone marrow and by the introduction of immunological technics which lead to a better appreciation of tumoral invasion.

[Immunohistochemical study of plasmocytes and T-lymphocyte subpopulations in Hashimoto's thyroiditis]. / Etude immunohistochimique des plasmocytes et des sous-populations T lymphocytaires dans la thyroïdite de Hashimoto.

In Hashimoto's thyroiditis, there is a diffuse interfollicular infiltration of lymphocytes and plasmocytes and lymphoid collections with germinal centers. Eleven cases of Hashimoto's disease were studied with immunocytochemical methods for characterization of intracytoplasmic immunoglobulins (IgA, IgG, IgM, Kappa and lambda light chains). Most of the cells are plasmocytes stained positively for intracytoplasmic IgG; IgA positive cells were less frequent and IgM positive cells are rare. Kappa positive plasma cells are more numerous than lambda positive cells in the germinal centers and the interfollicular infiltration. By an indirect immunofluorescence technique in frozen tissue, we have studied with four monoclonal antibodies (OKT3, OKT4, OKT8, B1) the T cells subsets populations in two cases.