Consensus on precision medicine for metastatic cancers: a report from the MAP conference.

Recent advances in biotechnologies have led to the development of multiplex genomic and proteomic analyses for clinical use. Nevertheless, guidelines are currently lacking to determine which molecular assays should be implemented in metastatic cancers. The first MAP conference was dedicated to exploring the use of genomics to better select therapies in the treatment of metastatic cancers. Sixteen consensus items were covered. There was a consensus that new technologies like next-generation sequencing of tumors and ddPCR on circulating free DNA have convincing analytical validity. Further work needs to be undertaken to establish the clinical utility of liquid biopsies and the added clinical value of expanding from individual gene tests into large gene panels. Experts agreed that standardized bioinformatics methods for biological interpretation of genomic data are needed and that precision medicine trials should be stratified based on the level of evidence available for the genomic alterations identified.

PML induces a novel caspase-independent death process.

PML nuclear bodies (NBs) are nuclear matrix-associated structures altered by viruses and oncogenes. We show here that PML overexpression induces rapid cell death, independent of de novo transcription and cell cycling. PML death involves cytoplasmic features of apoptosis in the absence of caspase-3 activation, and caspase inhibitors such as zVAD accelerate PML death. zVAD also accelerates interferon (IFN)-induced death, suggesting that PML contributes to IFN-induced apoptosis. The death effector BAX and the cdk inhibitor p27KIP1 are novel NB-associated proteins recruited by PML to these nuclear domains, whereas the acute promyelocytic leukaemia (APL) PML/RAR alpha oncoprotein delocalizes them. Arsenic enhances targeting of PML, BAX and p27KIP1 to NBs and synergizes with PML and IFN to induce cell death. Thus, cell death susceptibility correlates with NB recruitment of NB proteins. These findings reveal a novel cell death pathway that neither requires nor induces caspase-3 activation, and suggest that NBs participate in the control of cell survival.

BRCA1 and BRCA2 are necessary for the transcription-coupled repair of the oxidative 8-oxoguanine lesion in human cells.

The breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are likely to participate in DNA lesion processing. Oxidative lesions, such as 8-oxoguanine, occur in DNA after endogenous or exogenous oxidative stress. We show that deficiency for either BRCA1 or BRCA2 in human cancer cells leads to a block of the RNA polymerase II transcription machinery at the 8-oxoguanine site and impairs the transcription-coupled repair of the lesion, leading to a high mutation rate. Expression of wild-type BRCA1 from a recombinant adenovirus fully complements the repair defect in BRCA1-deficient cells. These results represent the first demonstration of the essential contribution of BRCA1 and BRCA2 gene products in the repair of the 8-oxoguanine oxidative damage specifically located on the transcribed strand in human cells. This suggests that cells from individuals predisposed to breast and/or ovarian cancer may undergo a high rate of mutations because of the deficiency of this damage repair pathway after oxidative stress.

A 1-kb Alu-mediated germ-line deletion removing BRCA1 exon 17.

Although more than 100 different BRCA1 germ-line mutations have already been identified in breast and/or ovarian cancer families, we report for the first time a deleterious genomic rearrangement in BRCA1. A 1-kb deletion comprising exon 17 was found in a large breast and ovarian cancer family, leading to a frameshift in the mutant mRNA due to the absence of exon 17. This deletion is probably the result of a recombination between two closely related Alu sequences. It was not detected by conventional PCR-based methods involving the genomic screening of the 22 coding exons or reverse transcription-PCR because the transcript without exon 17 is unstable in lymphoblastoid cell lines. Therefore, rearrangements in the BRCA1 gene should be sought in breast/ovarian cancer families in which no mutations have been found by PCR-based methods in the coding region or in the splice sites.

The hamster polyomavirus transforming properties.

The hamster papovavirus (HaPV) is a polyomavirus isolated from hair follicle tumor arising spontaneously in newborn hamsters which can also induce lymphoma and leukemia. This tissue specificity displayed in vivo can be bypassed in vitro since HaPV carries the full transforming properties of a polyomavirus (immortalization and transformation). We report here the phenotypic characterization of cells that were selected as immortalized or transformed and express constitutively the HaPV early genes. The viral genome is integrated in the host DNA and the early region is actively transcribed and translated.

Mutations within the hamster polyomavirus large T antigen domain involved in pRb binding impair virus productive cycle and immortalization capacity.

Hamster polyomavirus (HaPV) causes lymphoma and leukemia when injected into newborn Syrian hamsters and achieves full transformation of rodent fibroblasts in vitro. It offers a comprehensive model to study at a molecular level the contributions of the viral oncogenes to neoplastic transformation in vitro and in the animal. We have investigated the ability of HaPV large T antigen to form a complex with the product of the retinoblastoma gene (pRb) in vitro. In this report, we demonstrate that HaPV large T antigen can indeed complex the pRb polypeptide. In order to investigate to what extent this interaction might contribute to tumor induction by the virus, we have introduced two different point mutations within the putative pRb-binding sequence of large T antigen, and as a preliminary to in vivo experiments we have studied their effects in vitro on some biological activities relevant to tumor induction. We show that the substitution (Glu-134-->Lys) obliterates pRb binding, suggesting that Glu-134 participates in the interaction between pRb and large T antigen, whereas the substitution (Glu-135-->Lys) has no effect. The Lys-134 mutation is strongly deleterious to the immortalization capacity of the viral genome, whereas the Lys-135 mutation has no effect. Neither of the two mutations affects the capacity of the viral genome to induce foci formation in the rat established cell line F111. These results indicate that the interaction between large T and pRb is required in the immortalization process but irrelevant to transformation. Both mutants show at least partial impairment of replication and productive cycle.

Oncogenic potential of a mutant human thyrotropin receptor expressed in FRTL-5 cells.

An abnormal stimulation of the cAMP pathway has been recognized as the primary event in various pathological situations that lead to goitrogenesis or thyroid tumors. Thyroid adenomas are monoclonal neoplasms that become independent of thyroid stimulating hormone (TSH) in their secretory function and growth. Mutated forms of the TSH receptor (TSHR) and the adenylyl cyclase-activating Gs alpha protein, which confer a constitutive activity on these proteins, have been observed in human adenomas. The FRTL-5 rat thyroid cell line is a permanent but untransformed line; the growth of which depends on the presence of TSH, and at least in part, on the stimulation of the cAMP pathway. In order to compare the oncogenic potential of the activated mutant Gs alpha protein and the constitutively activated TSHR, we have transfected FRTL-5 cells with an expression vector bearing either the cDNA of the Gs alpha gene carrying the A201S mutation or the cDNA of the TSH receptor carrying the M453T mutation recently identified in a case of congenital hyperthyroidism. The expression of these two cDNAs was driven by the bovine thyroglobulin gene promoter. We show that, although the expression of both the Gs alpha or TSHR mutant proteins leads to TSH-independent proliferation and to constitutive cAMP accumulation in FRTL-5 cells, only the mutant TSHR is able to induce neoplastic transformation, as demonstrated by growth in semi-solid medium and tumorigenesis in nude mice.

Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2.

There is now evidence to suggest that BRCA1 and BRCA2 are involved in the response of cells to DNA damage and cell cycle checkpoint control. This report examines the death pathways of human cells with various BRCA1 and BRCA2 genotypes after exposure to gamma-rays. A lack of functional BRCA1 and BRCA2 led to defective repair of DNA double-strand breaks in irradiated cells. This impairment resulted in a relaxation of cell cycle checkpoints, production of micronuclei, and a loss of proliferative capacity. Heterozygous BRCA1 and BRCA2 mutations also led to enhanced radiosensitivity, with an impaired proliferative capacity after irradiation. The existence of a phenotype related to radiosensitivity in BRCA1+/- and BRCA2+/- cells raises the question of the response of heterozygous women to radiation.

Early gene expression in lymphoma-associated hamster polyomavirus viral genomes.

Hamster polyomavirus (HaPV) is the causal agent of hair follicle epithelioma in hamsters belonging to a colony bred in Berlin-Buch. These tumors shed virus particles that are assembled in the keratinized layer of the epidermis. By contrast, HaPV induces lymphomas after inoculation into newborn hamsters from a distinct colony bred in Potsdam. These lymphoid tumors accumulate massive amounts of episomal viral genomes characterized by deletions that alter specifically the regulatory and the late coding sequences. Assuming that these alterations of the regulatory region may affect the transcription of the viral oncogenes in the tumor cells, the transcriptional activity of the wild-type and deleted early promoters have been studied in vitro in transient chloramphenicol acetyltransferase (CAT) expression assays. These assays performed in various cell types demonstrate that both versions of the HaPV early promoter carry a weak constitutive activity. Simultaneous expression of the HaPV early gene products leads to a strong stimulation of CAT activity with a concomitant activation of the replication of the plasmid constructs. The results obtained with origin-defective CAT vectors indicate that the replication contributes significantly to the stimulating effect of the early gene products. Indeed, transfection of massive amounts of CAT vectors that are unable to replicate can simulate the dosage effect of replication and also leads to measurable CAT activities. Under these conditions, the wild-type promoter is more active than the deleted version, indicating that sequences within the deletion carry a distinct stimulatory effect on transcription. This conclusion is supported by the observation that the lymphoma cells contain a low level of early transcripts, indicating that the deleted episomal viral templates accumulated in these tumors carry a weak transcriptional activity.

Apoptosis is antagonized by large T antigens in the pathway to immortalization by polyomaviruses.

The viability of rat embryo cells immortalized by thermosensitive mutants of SV40 or polyoma Large T antigen is impaired at the non-permissive temperature thus demonstrating that the immortal phenotype is dominantly maintained by Large T antigens. We have observed that exposing these cells to the restrictive temperature not only induces growth arrest but also causes apoptotic cell death. We present evidence supporting the model that polyomaviruses may indeed establish immortality by antagonizing the lethal effects of tumor suppressor genes via physical interactions between their products and Large T antigens. In the case of SV40-immortalized cells REtsAF, shift-up to 39.5 degrees C dissociates Large T antigen/p53 complexes releasing wild-type p53 molecules capable of inducing apoptotic cell death. In polyomavirus-immortalized cells, apoptosis may result from an alternative pathway mediated by other unidentified negatively acting molecules.

p53 compound heterozygosity in a severely affected child with Li-Fraumeni syndrome.

The Li-Fraumeni Syndrome (LFS) is a rare, dominantly inherited syndrome that features high risk of cancers in childhood and early adulthood. Affected families tend to develop bone and soft tissue sarcomas, breast cancers, brain tumors, leukemias, and adrenocortical carcinomas. In some kindreds, the genetic abnormality associated with this cancer phenotype is a heterozygous germline mutation in the p53 tumor suppressor gene. Recently, we identified one patient who presented in early childhood with multiple primary cancers and who harbored three germline p53 alterations (R156H and R267Q on the maternal allele and R290H on the paternal allele). To classify the biologic effects of these alterations, functional properties of each of the p53 mutants were examined using in vitro assays of cellular growth suppression and transcriptional activation. Each amino acid substitution conferred partial or complete loss of wild-type p53 function, but the child completed normal embryonic development. This observation has not been previously reported in a human, but is consistent with observations of normal embryogenesis in p53-deficient mice.

Overexpression of MDM2, due to enhanced translation, results in inactivation of wild-type p53 in Burkitt's lymphoma cells.

Numerous studies have indicated that inactivation of p53 is one of the essential requirements for the unrestrained growth of tumoral cells. When the status of the p53 gene was examined in various types of lymphoid malignancies, mutations in p53 have been predominantly detected in Burkitt's lymphoma (BL) cells, therefore suggesting that alteration of p53 could specifically contribute to the malignant phenotype of these tumoral cells. In addition to mutations, functional inactivation of p53 can also occur through interaction of the wild-type gene product with various viral or cellular proteins. The cellular MDM2 protein, for example, is able to inhibit p53 tumor suppressor function by concealing its transactivation domain. Mdm2 gene amplification has been described in several types of sarcomas, resulting in overexpression of the MDM2 protein. In this study, we have examined the status of MDM2 and p53 in 20 BL cell lines. Four were found to contain wild-type p53 and to overexpress MDM2 protein. Within these BL cells, both molecules are physically associated since they can be co-precipitated and p53 is inactivated as cells neither arrest in G1 nor enter apoptosis following gamma-radiation. We also report that the high level of the MDM2 protein in BL cells is neither associated with an amplification of the mdm2 gene nor with an elevated level of RNA or an increased protein stability, but is rather due to an enhanced translation ability of the mdm2 RNA. These results indicate that in certain BL cells, overexpression of MDM2 protein regulated at the posttranscriptional level, induces an escape from p53-controlled cell growth.

SV40 induced cellular immortalization: phenotypic changes associated with the loss of proliferative capacity in a conditionally immortalized cell line.

Immortalization of rodent embryo fibroblasts by SV40 is dominantly maintained by the large T antigen. The aim of this work is to characterize some of the events associated with the loss of proliferative capacity in a rat cell line, called REtsAF, which is conditionally immortalized by the tsA58 allele of SV40 large T antigen. DNA replication is arrested less than 24 h after the shift to the restrictive temperature (39 degrees C). This arrest occurs without specificity relative to the cell cycle stage, which suggests that a function essential throughout the cell cycle is affected. A two-dimensional SDS polyacrylamide gel electrophoresis analysis of proteins shows that, although the global rate of protein synthesis is only slightly affected at 39 degrees C, the rate of accumulation of specific proteins is either increased or decreased. Finally we present biochemical and electron microscopy data showing that alterations of the mitochondria occur upon shift to 39 degrees C.

Thyroid pathologies in transgenic mice expressing a human activated Ras gene driven by a thyroglobulin promoter.

Four transgenic mice carrying the human activated c-Ha-Ras gene, the expression of which was driven into the thyroid gland by a bovine thyroglobulin promoter, have been produced. The M1 and M2 mice developed papillary thyroid carcinomas and the M2 mouse also developed a lung carcinoma, however none of them transmitted the transgene. Both the M3 and the M4 mice gave rise to transgenic lines. M3 progeny mice develop a goitre with morphological aspects of hyperplasia as well as a thymus hyperplasia. M4 developed a papillary thyroid carcinoma and a lung carcinoma. Lung tumors but not thyroid tumors were observed in M4 adult transgenic progeny. In this M4 line, thyroid dysgenesis leading to growth retardation and premature death was observed upon serial backcross that enhanced the DBA/2J genetic background. The development of thyroid tumors in M1, M2, M4 transgenic mice demonstrates the oncogenic potential of activated Ras gene in the thyroid gland. The M4 line raises interesting questions relative to the interference between the Ras-mediated signal transduction pathway and thyroid morphogenesis.

Germ-line exclusion of a single p53 allele by premature termination of translation in a Li-Fraumeni syndrome family.

Germline p53 mutations have been detected in approximately half of the families affected by the Li-Fraumeni syndrome (LFS), in which they are believed to represent the genetic status predisposing to multiple cancers. Failure to detect mutations in the other half of LFS families suggests that sequence analysis, which has been limited to the p53 gene coding region, have overlooked other genetic events lying outside of this region or/and that alterations in other gene(s) than p53 may also lead to the syndrome. In this report, we present the evidence that a single base pair deletion in the p53 coding sequence, leading to premature signal termination of translation, generates a null allele by preventing transport of mutant allele mRNAs into the cytoplasm. This allelic exclusion which confers a status of unizygote vis-à-vis the wild-type p53 gene to individuals who carry the mutant allele, leads to predisposition to multiple cancers in a Li-Fraumeni family. Thus, the loss of the wild-type p53 allele appears as the rate limiting step in tumor induction.

Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function.

We have investigated the relationship between the development of tumor resistance towards the cytotoxic action of tumor necrosis factor-alpha (TNF) and p53 function, using the TNF-sensitive MCF7 human breast adenocarcinoma cell line and two TNF-resistant sublines, MCF7/R-A1 and MCF7/Adr. Use of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing shows that MCF7 has a wild-type p53 gene, whereas both TNF-resistant sublines exhibit mutant p53. This includes a point mutation R280K in MCF7/R-A1 cells, and a point mutation at the splicing acceptor site on the upstream border of exon 5 resulting in a 21 pb deletion in MCF7/Adr cells. These mutations result in loss of p53 capacity to transactivate FASAY (functional assay in yeast). In contrast to what is observed for parental MCF7 cells, treatment of resistant sublines with TNF or gamma-irradiation fails neither to induce the expression of the p53-regulated gene products p21waf1/CIP1 and MDM2, nor to arrest the cells in the G1 phase of the cell cycle. Disruption of p53 wild-type function in MCF7 cells by transfection with human papillomavirus type-16 E6 gene, leads to abrogation of the cytotoxic, but not the cytostatic activity of TNF. Altogether, our results strongly suggest that wild-type p53 is involved in cytotoxic action of TNF, and point out that loss of p53 function contributes to resistance of tumor cell to TNF-induced killing.

DNA screening for breast/ovarian cancer susceptibility based on linked markers. A family study.

BACKGROUND: Linkage to chromosome 17q has been identified in hereditary breast cancer and hereditary breast/ovarian cancer syndrome. A hereditary breast/ovarian cancer syndrome kindred was identified that yielded a highly significant lod score (4.20) when 17q markers were studied, enabling us to identify those who probably carried the cancer-associated gene among the high-risk members of the family. METHODS: High-risk members of the hereditary breast/ovarian cancer syndrome kindred were offered counselling on the basis of 17q markers. Family members responding positively received one-to-one genetic counseling in a structured setting. Subjects were educated before disclosure, and the immediate impact of this information was assessed after disclosure. RESULTS: We provided genetic counseling on the basis of linkage findings to 32 relatives (four men and 28 women). Women who were told they were linkage positive expressed an increased motivation for surveillance and prophylactic surgery. Most women who were told they were linkage negative indicated that they would not proceed with prophylactic surgery but would continue careful surveillance. To date, there has been no evidence of serious emotional disturbances resulting from this disclosure. We believe that this experience can be used by cancer geneticists and physicians in developing protocols for genetic counseling in cancer-associated hereditary disorders. CONCLUSIONS: Physicians must understand current developments in cancer genetics and linkage so that they can be applied to genetic counseling and treatment of high-risk patients.

Molecular cloning of the hamster papovavirus genome in Escherichia coli plasmid vector pBR322.

The complete genome of the hamster papovavirus (HaPV) which was isolated from virions found in multiple skin tumors of Syrian hamsters was cloned in Escherichia coli using the plasmid vector pBR322. The cloned viral DNAs were identified by digestion of the recombinant DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested uncloned DNAs. The cloned HaPV DNAs showed the same migration pattern as the corresponding fragments from the restricted uncloned DNAs, indicating that no major insertions or deletions occurred during cloning and plasmid propagation. The electrophoretic data were confirmed by Southern blot hybridization.

BRCA1 carries tumor suppressor activity distinct from that of p53 and p21.

The loss of BRCA1 function appears as an essential step in breast and ovarian epithelial cells oncogenesis and is consistent with the concept that BRCA1 acts as a tumor suppressor gene. However, the mechanism underlying this activity is not understood. In 1996, a retroviral vector was used for BRCA1 delivery to demonstrate that the transfer of BRCA1 inhibits breast and ovarian cancer cell growth. Since this early observation, the tumor growth inhibitory activity of BRCA1 in vivo has not been further documented. Here we re-address this issue and report experiments designed to evaluate the potential of adenovirus-mediated BRCA1 delivery to suppress the growth of cells with various status of endogenous BRCA1 in comparison with p53 and p21. Delivery of wild-type BRCA1 by an adenovirus vector in breast and ovarian tumor cells, decreases in vitro proliferation and tumorigenicity. Similarly, in vivo administration of BRCA1 provokes tumor growth retardation or regression comparable to that obtained with p53 or p21. The antitumor effect of BRCA1 is not observed upon transfer of a mutant lacking the 542 C-terminal residues. The p53- or p21-mediated antiproliferative activities are likely to bear on their capacity to induce apoptosis and/or interfere with cell cycle checkpoint. By contrast, the data presented here show that neither of these mechanisms can account for the BRCA1-mediated antitumor activity and suggest the activation of an alternative route.