Epidermolytic palmoplantar keratoderma cosegregates with a keratin 9 mutation in a pedigree with breast and ovarian cancer.

Epidermolytic palmoplantar keratosis (EPPK) cosegregates with breast and ovarian cancers in a large French pedigree, raising the possibility that a single genetic mutation might cause these conditions and offering a potential lead to the identification of a hereditary breast/ovarian cancer gene. We have performed linkage analysis and show that the EPPK locus lies on the long arm of chromosome 17 near the type I keratin gene cluster and the proposed breast cancer gene (BRCA1). The type I keratin 9 gene has been partially sequenced in four affected individuals. A single base mutation within the rod domain of the protein cosegregates with EPPK in all affected individuals tested. Although inheritance of this mutation is likely responsible for EPPK, it is unlikely to be the cause of the breast and ovarian cancer.

Inhibition of tumor growth by the peptidoglycan from Bacillus megaterium.

When injected in admixture with tumor cells, the peptidoglycan extracted from Bacillus megaterium inhibited the growth of a chemically induced fibrosarcoma in syngeneic rats. In some instances, the tumor growth was totally suppressed. Animals rejecting mixed inocula exhibited a tumor-specific immunity. Peptidoglycan was not cytotoxic for tumor cells; it rendered macrophages nonspecifically cytotoxic.

Cytotoxic responses induced by peptidoglycans with or without in vivo antitumor activity.

Cytotoxic responses mediated by effector cells stimulated in vivo were studied after ip injection in mice of peptidoglycans extracted from gram-positive bacteria. A comparison was done between Staphylococcus aureus peptidoglycan (PGS), which possessed an antitumor effect, and Micrococcus lysodeikticus peptidoglycan, which was ineffective against tumors. Both peptidoglycans induced similar effects on the modulation of T-cell cytotoxic response. Both were able to stimulate splenic and peritoneal nonspecific cytotoxicity against YAC-1 lymphoid tumor cells, but only PGS could induce cytotoxicity and cytostasis against solid tumor target cells.

Peptidoglycans extracted from gram-positive bacteria: expression of antitumor activity according to peptide structure and route of injection.

The antitumor effect of peptidoglycans of various structures extracted from different gram-positive bacteria was studied, on chemically induced fibrosarcomas, in C3H/He, C57BL/6, and (C57BL/6 X C3H/He)F1 mice. When given sc admixed with tumor cells, only some peptidoglycans (those extracted from Bacillus megaterium and Staphylococcus aureus) enhanced tumor resistance in syngeneic and semiallogeneic hosts, whereas other peptidoglycans (those extracted from Micrococcus lysodeikticus and Corynebacterium poinsettiae) possessed no antitumor effect. When tumor cells were given ip, administration of peptidoglycans by the same route was either without effect on tumor growth or it induced tumor enhancement. Enhancement could be observed with all of the peptidoglycans tested. The antitumor effect when given sc and the ability to stimulate the proliferation of B-lymphocytes were shared by the same two peptidoglycans, while the other two peptidoglycans were devoid of both activities. It appears that these biological activities depend on the structure of the peptide moiety of peptidoglycans and that mitogenic and antitumor responses are stimulated by similar structures.

Stimulation of one particular subset of natural killer cells by peptidoglycans extracted from gram-positive bacteria.

Peritoneal nonspecific cytotoxicity was stimulated by ip injection into C57BL/6 or (C57BL/6 X C3H/He)F1 mice of Staphylococcus aureus peptidoglycan (PGS), which possessed an antitumor effect, and of Micrococcus lysodeikticus peptidoglycan (PGM), which was ineffective against tumors. The natural killer (NK) cell populations elicited by both peptidoglycans had the same phenotype: Thy 1.2+, T200+, Ly 5.1+, Qa5+, and Ly 2.2-. In vivo treatment with sheep anti-mouse beta interferon serum abrogated this effect. The fluids from phorbol myristate acetate-stimulated murine EL 4 thymoma cells enhanced this particular NK activity, and thus PGM-induced effector cells became able to kill solid tumor cells. In conclusion, both PGS and PGM elicited the same particular subset of NK cell populations, and the peptidoglycan structure can play an important role in the intensity of this response.

Response of small-cell lung cancer xenografts to chemotherapy: multidrug resistance and direct clinical correlates.

BACKGROUND: Patients with small-cell lung carcinomas (SCLCs) initially respond to combination chemotherapy. Only a few benefit in terms of long-term survival because most relapse. Such outcome may be attributable to development of multidrug resistance. PURPOSE: The response of SCLC to chemotherapy was examined in terms of (a) patient survival, (b) drug sensitivity of tumors in patients and of tumor xenografts in nude mice, and (c) expression of multidrug resistance gene MDR1 and GST-pi gene. METHODS: Tumor samples obtained from seven untreated patients and from one patient both before and after chemotherapy were transplanted into nude mice. The patients were treated with a combination of cyclophosphamide (C'), cisplatin (C), doxorubicin (A), and etoposide (V) (C'CAV) or C'AV and radiotherapy. Drug sensitivity of SCLCs was tested in nude mice that had received tumor xenografts from these seven patients. The expression of MDR1 and GST-pi genes was assessed in the mRNA extracted from xenografts by Northern blot analysis. P-glycoprotein was quantified by enzyme immunoassay. RESULTS: The patients' responses to C'CAV closely correlated with those of the corresponding xenografts. The tumors of the two patients who showed long-term survival after C'CAV completely regressed when they were transplanted into nude mice and subsequently treated with C'CAV. Despite initial complete response, the remaining five patients died during year 1. A high percentage of mice receiving the tumor grafts from these five patients showed only partial tumor regression after C'CAV treatment. The MDR1 transcript was detected in all five of these xenografts. Four of five xenografts were from untreated patients, and the fifth was from a treated patient. MDR1 mRNA expression was absent in the tumor of this fifth patient before chemotherapy, but both the mice receiving the corresponding xenograft and the patient showed expression of MDR1 after C'CAV treatment. MDR1 mRNA expression was absent in the tumor xenografts obtained from two patients with long-term survival. Expression of P-glycoprotein correlated with MDR1 mRNA expression. All xenografts except one expressed the GST-pi gene. CONCLUSIONS: The absence of MDR1 gene expression during chemotherapy for SCLC indicates a favorable prognosis, gene expression is often coincident with ineffective chemotherapy, and tumor xenografts can be appropriately used to predict response to chemotherapy. IMPLICATIONS: Failure of chemotherapy to control SCLC seems to be related to an acquired multidrug resistance involving the MDR1-mediated mechanism. Therapeutic benefit could therefore be expected from chemotherapy combined with inhibitors of MDR1.

Mapping of the 7q31 subregion common to the small chromosome 7 derivatives from two sporadic papillary renal cell carcinomas: increased copy number and overexpression of the MET proto-oncogene.

Molecular cytogenetic analysis of several sporadic papillary renal cell carcinomas and of their xenografts in immunodeficient mice had previously allowed us to delimit a minimal overrepresented region of chromosome 7 shared by all of them to band 7q31. We have refined the location of the overlapping region to the junction of the subbands 7q31.2 and 7q31.3 by reverse painting with two differently labelled probes prepared from the small chromosome 7 derivatives microdissected from the cells of two distinct tumours. This small region was shown to contain the MET proto-oncogene, present at three to four copies per cell as determined by Southern blot analysis. The increased copy number of the MET gene was found to be associated with its overexpression at the mRNA level. However, no change in MET copy number or expression level was observed in the cells from two xenografted tumours serially transplanted into immunodeficient mice, as compared to those from the corresponding initial tumours. Our results indicate that expression of the MET proto-oncogene above a critical threshold is required for the maintenance of the tumorigenic phenotype of at least some papillary renal cell carcinomas, but does not further increase during tumour progression.

Non-Hodgkin's lymphomas and myeloid disorders: deletions associated with t(2;5) and t(3;5) detected by FISH.

The nucleophosmin (NPM) gene is involved in two recurrent translocations in hematological malignancies: t(2;5) (p23;q35) in anaplastic large cell lymphoma (ALCL) and t(3;5)(q25.1;q34-35) in myelodysplasia and acute non-lymphocytic leukemia (ANLL). Using eight YACs encompassing the 5q34-q35 region, we could easily detect these two translocations. In both types of translocation, probable unexpected deletions were also discovered downstream of the breakpoint at 5q35.

Preliminary evaluation of two new rapid immunoturbidimetric D-dimer assays in patients with clinically suspected venous thromboembolism (VTE).

The practical utility and diagnostic accuracy of two new rapid, automated and quantitative immunoturbidimetric D-dimer methods have been evaluated in a population of 123 randomly selected patients with suspected VTE. The STA Liatest D-dimer and MDA D-dimer methods are based on the photo-optical measurement of the rate of agglutination of antibody-coated latex particles. The VIDAS D-dimer automated Elisa was used as the reference method. Diagnosis was confirmed in 51 patients (29 PE, 19 DVT, 3 DVT+PE). The immunoturbidimetric methods compared favorably with the VIDAS Elisa as judged from the correlation coefficients of linear regression lines (r = 0.82, MDA vs VIDAS; r = 0.75, STA vs VIDAS) and areas under the curve of ROC plots (VIDAS 0.83; STA 0.83; MDA 0.81). At a discriminant value of 500 ng/mL, all three D-dimer assays showed high sensitivity (96-98%), high NPV (93-97%) and moderate specificity (39-42%). Reproducibility of results around the cut-off is an important aspect of the diagnostic utility of D-dimer assays. CV's of duplicate determinations in this critical zone showed average values of 5.4% and 17.0% for MDA and STA, respectively. These data demonstrate that such rapid and automated latex-based methods for the quantitative measurement of D-dimer hold promise as reliable and cost-efficient approaches for the exclusion of VTE. Prospective patient management studies will be required to confirm this.

Response of a multidrug-resistant human small-cell lung cancer xenograft to chemotherapy.

Small-cell lung carcinomas (SCLC) are highly responsive to various chemotherapies. However only a minority of patients benefit from long survival. SCLC patients treated at the Institut Gustave Roussy received a combined chemotherapy (CCAV) including cisplatin, cyclophosphamide (Cpa), Adriamycin (doxorubicin; Adm) and vepeside (VP16). We report here the intrinsic sensitivity of a small-cell lung carcinoma, designated SCLC-6, grafted in nude mice. This xenografted tumour was derived from an untreated patient. The CCAV regimen given to the patient donor of the tumour sample resulted in a complete response followed by recurrence and death, 8 months after the initial cure. The expression of P-glycoprotein encoded by the MDR1 gene was detected with the C219 antibody on the membrane of SCLC-6 tumour cells. When given to SCLC-6-tumour-bearing nude mice, CCAV induced a strong inhibition of tumour growth (84% of growth inhibition, 20 days after start of the treatment), but no cure. Intensification of CCAV doses did not improve the response. The efficacy of individual agents of the CCAV, given at maximal tolerated doses was analysed. Only cisplatin (10 mg/kg) and Cpa (3 x 50 mg/kg) inhibited SCLC-6 growth (79% and 100% inhibition respectively), VP16 (3 x 24 mg/kg) was poorly efficient (42%) and Adm (10 mg/kg) not at all. Two-drug combinations such as cisplatin plus VP16 or cisplatin plus Cpa inhibited tumour growth (81% and 70%, respectively). Curiously, the efficacy of Cpa, given in combination with cisplatin was less than that of Cpa alone. Repeated treatments with CCAV administered to mice at each in vivo passage of the tumour induced a loss of chemosensitivity, which was observed until the ninth passage. An improvement of the therapeutic response was obtained by adding a headline reverser of multi-drug resistance, verapamil (25 mg/kg), to CCAV (81% versus 63% inhibition). MDR1-related resistance appeared to play a role in the failure of SCLC-6 chemotherapy; frequent recurrences after treatment with cisplatin and Cpa, two drugs that are not recognized by the P-glycoprotein, indicated that other modes of resistance were simultaneously active.

Long-term French experience in INR standardization by a procedure using plasma calibrants.

The International Normalized Ratio (INR) has not lowered the interlaboratory differences in prothrombin time (PT) values to the extent expected, mainly because of the instrument-dependency of the International Sensitivity Index (ISI) and other factors (eg, accurate determination of the ISI, the normal value used in the PT ratio). The procedure (PPC) using plasma calibrants (reference lyophilized plasmas with assigned activity) has been evaluated since 1977 in nine French national external quality assessment surveys (NEQAS) involving approximately 4,000 laboratories and numerous local thromboplastin technique combinations. The PPC was compared with the conventional procedure (using the manufacturer's ISI), and the efficiency of antivitamin K-calibrated (AK Cal) plasmas from patients receiving oral anticoagulants vs artificially depleted plasma calibrants was also evaluated. The PPC efficiently standardized PTs with AK Cal plasmas, reducing interlaboratory variability (eg, coefficient of variation, 12% to 6% for survey 92 D) and reagent-instrument effects. However, AK Cal plasmas have drawbacks, such as limited supply, cost, and batch-to-batch variability. The artificially depleted plasma calibrants were less efficient, but usable if carefully prepared. The value of this simple procedure is that local practices are considered in the determination of PT, thus correcting for coagulometer effects and avoiding use of the manufacturer's ISI and need for a normal control plasma. These large-scale French surveys have demonstrated the validity of PPC through 15 years of experience and have shown that it offers the best compromise available in PT standardization.

Evolution of chromosomal alterations and biologic features in two small cell lung carcinoma cell lines established from one patient during the course of the disease.

Two small cell lung cancer (SCLC) cell lines were established from metastases of a patient during the course of the disease. SCLC 74A was derived from biopsy material obtained at the time of diagnosis and SCLC 74B was from a biopsy specimen of a relapsed tumor obtained after treatment. A transition occurred from SCLC 74A, an intermediate form with 5% large cells to SCLC 74B, a standard mixed form with 20% of large cells, with a decrease in neuroendocrine markers and a substantial increase in P-glycoprotein, a multidrug resistance marker. For both cell lines, R-banding and FISH indicated a del(1)(p35pter) also found in other neural-crest-derived tumors, the loss of regions with suspected tumor suppressor genes at 3p, 5q, and 17p, and a recurrent translocation of the 6q24-6qter region to 10p14. Further genetic modifications in SCLC 74B affected chromosomes 2, 3, 5, 10, 11, 14, and 15. The main observations were a der(2)t(2;5)(p16;q?); a der(3;11)(q10;p10) in SCLC 74A which became der(3;14)(q10;p10) and der(11;14)(p10;q10) in SCLC 74B; and the insertion of the 5q13-5q31 region in the der(10)t(6;10). The finding of the same structural abnormalities in both cell lines suggests a monoclonal origin for both metastases. Hypotetraploid cells were in the same proportion as large cells whose number was a characteristic feature of each cell line. They possessed twice the same chromosomal alterations observed in the hypodiploid cells. This suggests a permanent process of tetraploidization.

Patterns of specific genomic alterations associated with poor prognosis in high-grade renal cell carcinomas.

A series of 13 sporadic renal cell carcinomas was analyzed for the specific chromosome rearrangements after serial xenografting into immunodeficient mice. Seven tumors displayed genetic traits of the conventional subtype and 5 showed genetic features of the papillary subtype. In all the xenografted conventional tumors, we observed loss of 3p, as well as loss of the 9p21 region and of the long arm of chromosome 14, both considered as markers of a poor prognosis. In the xenografted papillary tumors, a duplication of chromosome arm 8q was observed concomitant with the duplication of the 7q31 region. The association of the 7q31 and 8q22 approximately qter duplicated regions was also observed for one conventional tumor. The latency of tumor take was found to be reduced and the median time to passage statistically shorter for all tumors which presented the associated duplication of the 7q31 and 8q22 approximately qter regions. The proto-oncogene NOV (nephroblastoma overexpressed gene) maps to 8q24.1 and is overexpressed in some Wilms tumors. It could be an interesting candidate gene, since its level of expression and release in the culture medium was found to be increased in all of the fast growing tumors analyzed.

MHC class I antigen expression and alterations induced by rIFN gamma in tumor hybrid cell immunogenicity.

Hybridization of a poorly immunogenic tumor cell with an allogeneic cell was performed in order to improve tumor immune response; several variants derived from one hybrid tumor cell were studied. We compared the immunogenicity of these variants and their allogeneic and syngeneic class I antigen expression before and after IFN gamma treatment. Allogeneic class I antigens were weakly expressed in all variants; IFN treatment enhanced their expression similarly in both immunogenic and nonimmunogenic variants. Syngeneic class I antigen expression differed among variants: IFN treatment induced changes in their expression which corresponded to a posttranscriptional event and which could, at least partly, explain the modifications observed in their immunogenicity.

HBV and HIV serological markers: the National External Quality Assessment Scheme in France.

Participation in external quality assessment (EQA) has been mandatory in France as from 1978. The Ministry of Health has responsibility for maintenance of a list of laboratories and contracts EQA schemes to be organised and supervised by scientific groups. Serological markers for viral hepatitis B and anti-HIV were included in surveys, four times per annum, as from 1985. The number of laboratories participating increased from 178 for anti-HIV and 499 for viral hepatitis markers in 1985 to 3421 for anti-HIV and 2384 for viral hepatitis markers at the present time. The percentage of correct results varies depending on the method and specimen from 98% to 99% for anti-HIV and from 95% to 97% for HBsAg; the false negative rate has been as high as 14% for anti-HBs.

[Comparative evaluation of unfractionated and low molecular weight heparins. Results of the French Etalonorme interlaboratory quality control]. / Evaluation comparative des héparines non fractionnées et de bas poids moléculaire. Résultats du contrôle de qualité interlaboratoire français Etalonorme.

Four recent interlaboratory surveys have been conducted by the French National "Etalonorme" Group of Quality Control in Haematology, involving about 3,800 laboratories working in coagulation. They have been concerned with human lyophilised plasma samples heparinized in vitro either with unfractionated heparins (UFH), or with low molecular weight heparins (LMWH) (Fragmin 89 D, and Fraxiparin 90 A). The doses added simulated therapeutic situations or were on the border-line of over- or underdosage: UFHs 89 B3/B4, 0.14 and 0.22 IU/ml; 89 C3/C4, 0.3 and 0.4 IU/ml respectively; LMWHs: D3-A3, 0.6; and D4-A4, 1.2 IU/ml. Automated partial thromboplastin times (APTT) were prolonged at these high doses of LMWH, due to noticeable residual anti-IIa activity. A relationship between APTT and heparinaemia was observed with both types of heparin; sensitivities of cephalin reagents to heparin were also noted, but they were different for UFH compared to LMWH. Dispersion on APTT results was still high (CV: 12-23% for the UFH samples, 12-19% for the four LMWH samples). Dispersion on heparin determinations was higher for UFH than for LMWH, even for lower anti-IIa activity, but they involved more heterogeneous assays. The anti-Xa activity for LMWH samples was mainly determined using clotting assays (70% of the laboratories) with larger dispersions (CV: 32-37%), than those observed on amidolytic assays (CV: 28%); however, these amidolytic assays were performed in 90% of the laboratories with only two reagents available from a single manufacturer. The clotting techniques, more than the colorimetric assays, seemed to underestimate the high heparinaemias.(ABSTRACT TRUNCATED AT 250 WORDS)

[Comparative evaluation of 3 automated optical coagulation detection systems]. / Evaluation comparée de trois automates de coagulation à détection optique.

The Organon Teknika-General Diagnostics Coag A Mate X2 (CAM X2), Ortho Koagulab 40A, MLA Electra 700 (E 700), were simultaneously evaluated following the same protocol, on the same samples. Compared with the former photo-optical-based automated devices, these newer advanced instruments present a high throughput in fibrin endpoint detection, permitting fibrinogen determination with reliable results; they also present a higher degree of automatism, greater flexibility in use and easy mastering, allied to elaborated expression of results. Statistical comparison shows equivalent performance characteristics. Repetabilities are excellent for Prothrombin Time (CV: 1-2.5%) with 5 thromboplastins and for the prothrombin complex factors assays: they are also good for Activated Partial Thromboplastin Time (APTT) with 4 reagents (CV: 0.5 to 3%) provided that kaolin be not used on the KLO and E700; the same limitation exists for specific factors assays. Fibrinogen assays display good results (CV: 3-4% and 6-8%) respectively for times of 11 sec. (/=/ 2.4 g/l) and 23 sec. (/=/ 1 g/l). Standard linearities are generally excellent whatever the test. The correlation with results obtained on other devices is good (0.96 less than R less than 1). The most fully automated is the KLO with reliable system of sample delivery, automatic calibration and quality control function. The E700 is the slowest instrument, especially for APTT and the least adapted to large homogenous series, but its original loading system with binary coded cuvettes makes it interesting for emergencies and mixed tests. Controls and adjustments of the pumptubing are particularly required on the CAM X2 which is, on the other hand, the quickest instrument with its 4 photo-optical cells, well adapted to handling a large series of samples.

Immunity to chemically induced rat sarcomas: study of the specificity of cytotoxic cells.

Spleen cells from WAG rats bearing methylcholanthrene-induced sarcomas were cytotoxic for homologous tumour cells and for some cell lines derived from other syngeneic tumours. Each tumour induced its own pattern of cross-reactive cytotoxicity. This cross-reactivity was not apparent by in vivo rejection tests. In tumour-bearing rats, specific cytotoxic cells could not be separated from cross-reactive cytotoxic cells. The effector cells did not adhere to plastic surface, they were retained neither on nylon wool nor on anti-Ig columns and were devoid of Fc receptors. After tumour excision the cells displaying specific cytotoxicity had the same properties and thus were probably T cells. In contrast, the cells exhibiting cross-reactive cytotoxicity were lost after passage through a nylon wool column or after the removal of Fc-receptor-bearing lymphocytes. These findings suggest that cross-reactivity could result from antibody-dependent cell-mediate cytotoxicity directed against shared antigens.

Interlaboratory quality assessment of lymphocyte phenotyping. Etalonorme 1990-1992 surveys.

A 3-year interlaboratory proficiency testing for lymphocyte subset phenotyping was initiated as part of the Etalonorme national quality control program. Specimens consisted of fresh whole blood and of lyophilised mononuclear cells (Cytotrol Coulter). The number of participating laboratories was 62 in 1990, 99 in 1991 and 129 in 1992. Statistical analysis indicated that results of phenotyping, expressed as percentages of positive cells, are not related to reagents, instruments or differences in methodology (like, eg time and temperature of incubation). The highest dispersion was observed for total lymphocyte counts and was found to correlate with the type of calibration of the instrument. The coefficients of variation for different lymphocyte subsets were similar if phenotyping was performed on whole blood or lyophilised cells and varied inversely with the percentage of positive cells in each specimen. The consistency of the results indicated that they could serve as a basis for clinical decisions.