Cancer-prone Family Research Articles

Resolving pathogenicity classification for the CDH1 c.[715G>A] (p.Gly239Arg) Variant.

Hereditary Diffuse Gastric Cancer (HDGC) syndrome is associated with CDH1 germline likely pathogenic/pathogenic variants. Carriers of CDH1 germline likely pathogenic/pathogenic variants are predisposed to diffuse gastric cancer and lobular breast cancer. This study aims to classify the CDH1 c.[715G>A] missense variant identified in a diffuse gastric cancer prone family by performing splicing studies. RT-PCR and subsequent cloning experiments were performed to investigate whether this variant completely disrupts normal splicing. This variant preferentially abolishes normal splicing through activation of a cryptic 3' acceptor splice site within exon 6 of CDH1, presumably leading to a premature protein truncation within first extracellular domain repeat of E-cadherin protein. Our results contributed to evidence necessary to resolve pathogenicity classification of this variant, indicating that this variant is to be classified as pathogenic.

A germline mutation of the KIF1Bβ gene on 1p36 in a family with neural and nonneural tumors

Recently, the KIF1B beta gene on 1p36, a region commonly deleted in neural crest cancers, was found to be a proapoptotic factor for sympathetic precursors. KIF1B beta mutations were detected in pheochromocytomas and neuroblastomas, two sympathetic lineage tumors, suggesting a role for this gene in cancer. Here, we studied five individuals from a three-generation cancer-prone family with a KIF1B beta germline variant and seven of their tumors, both of neural crest and nonneural origin. Genetic studies including sequencing, copy number analysis and fluorescence in situ-hybridization (FISH) showed retention of both KIF1B beta alleles in all neural crest-derived tumors in this family, consistent with haploinsufficiency or methylation of the wild-type allele. In contrast, the lung adenocarcinoma from one mutation carrier had somatic loss of the wild-type allele in agreement with a classical two-hit inactivation. Global transcription analysis of KIF1B beta mutant pheochromocytomas revealed that these tumors are transcriptionally related to pheochromocytomas with RET and NF1 mutations but independent from SDH- and VHL-associated tumors. Furthermore, KIF1B beta-mutant tumors are uniquely enriched for pathways related to glutamate metabolism and the oxidative stress response. Our data start to delineate the signals that are disrupted by KIF1B beta dysfunction in pheochromocytomas and suggest that loss of this gene may also be permissive to the development of nonneural crest malignancies. This may imply the existence of a tissue-specific gene dosage requirement for its tumorigenesis.

Muir–Torre syndrome caused by partial duplication of MSH2 gene by Alu-mediated nonhomologous recombination

We describe a 54-year-old man with a pedicled tumour on the neck. The surgical specimen revealed a sebaceous carcinoma. He belonged to a cancer-prone family susceptible to gastrointestinal cancer. Systemic evaluation for latent malignancies revealed early-stage colonic adenocarcinoma. These findings were compatible with Muir-Torre syndrome (MTS). Microsatellite instability was detected in the sebaceous carcinoma, suggesting a DNA mismatch repair gene mutation. Moreover, duplication of exon 7 generated a nonsense codon at codon 427 of the MSH2 gene causing truncation of MSH2 protein. Immunohistochemical analysis showed diminished MSH2 protein levels in the sebaceous carcinoma and colonic adenocarcinoma. To date, there have been no reports showing duplication of exon 7 of the MSH2 gene in MTS or hereditary nonpolyposis colorectal cancer kindreds. Furthermore, the present case indicates that the dermatologist plays an important role in the diagnosis of MTS and evaluation for latent malignancies.

Familial sarcoma: challenging pedigrees.

Partially due to the rare occurrence of soft tissue and osteogenic sarcomas in the general population, scant attention has been given to their hereditary etiology. Their overall poor prognosis might be ameliorated through an understanding of their environmental and hereditary causal factors, and/or their interactions, thereby contributing to earlier diagnosis and even the development of molecularly based targeted therapy. The authors selected 10 sarcoma-prone families from their extensive hereditary cancer-prone family resource and focused on their challenging diagnostic, surveillance, and management features. The family study protocol included the compilation of a detailed family history of malignant disease of all anatomic sites and the collection of all available primary medical and pathology documents for verification. Genetic counseling was provided before DNA collection and at disclosure of results. These families displayed marked phenotypic and genotypic heterogeneity. In one of these families, 16 relatives had sarcomas, with 2 of the 16 each having 2 metachronous sarcomas; to our knowledge, this represents the greatest number of sarcomas reported in any family described to date. Two familial atypical multiple-mole melanoma syndrome kindreds with the CDKN2A mutation showed the association of sarcoma with malignant melanoma, whereas one family had several pancreatic carcinomas. Other families with sarcoma had hereditary nonpolyposis colorectal carcinoma with MSH2 mutation, hereditary breast carcinoma with BRCA1 mutation, and p53 mutation in a Li-Fraumeni syndrome. Sarcoma-prone families reported in the current study were selected carefully to depict clinicopathology and compliance features, the understanding of which could elucidate the etiologic role of genetic factors in concert with the phenotypic and genotypic heterogeneity encountered in such families. The lack of a population-based data set for these families posed a limitation.

Gene-regulating chemoprevention against cancer--as a model for "molecular-targeting prevention" of cancer

Recent progress in molecular biology and genetics has improved understanding of the mechanisms of carcinogenesis. However, there are few effective methods for prevention or therapies against cancer based on such elucidated molecular mechanisms of carcinogenesis. We therefore tried to develop novel methods of cancer prevention and therapy based on them. For example, the tumor-suppressor gene p53 is mutated in about 50% of human malignancies or in a cancer-prone family with Li-Fraumeni syndrome. It is known that p53 stimulates the promoter activities of p21/WAF1, gadd45 and bax genes to enhance their expression as a transcriptional factor, resulting in cell cycle arrest, DNA repair and apoptosis, respectively. Therefore, chemical compounds or food factors that can stimulate these genes might compensate for part of the p53 function. As a model of our hypothesis, we found that histone deacetylase inhibitors such as butyrate and trichostatin A dramatically stimulate the p21/WAF1 gene promoter through the Sp1 sites, resulting in cell cycle arrest. We therefore hypothesized that a strategy for up-regulating p53-target genes such as p21/WAF1, gadd45 and bax might be useful for cancer prevention or therapy, and termed this method "Gene-regulating chemoprevention" or "Gene-regulating chemotherapy" against cancer. In fact, butyrate, a short chain fatty acid, exists in colon lumen as a metabolite of dietary fiber, and is believed to be preventive against colon cancer. In conclusion, we proposed that "Gene-regulating chemoprevention" and "Gene-regulating chemotherapy" may be new promising strategies for cancer prevention or therapy, and histone deacetylase inhibitors are good candidates for these strategies. "Gene-regulating chemoprevention" is a particularly suitable model for "Molecular-targeting prevention", which we have proposed recently. We believe that "Molecular-targeting prevention" will become one of the most important concepts in the 21st century for general prevention of a variety of common hereditary or non-hereditary common diseases.

Hereditary colorectal cancer: clinical consequences of predictive molecular testing.

The continuing increase in knowledge about the genetic basis of carcinogenesis has led to diverse efforts to exploit this knowledge clinically, primarily in the form of predictive genetic testing. In conjunction with family history, gene tests are intended to improve individual cancer risk assessment. The objectives of predictive molecular testing are to identify the disease-causing germline mutation in an index person who has already developed the disease and to distinguish asymptomatic mutation carriers from non-mutation carriers within a given cancer-prone family. At present, genetic testing for colorectal cancer risk, primarily in form of DNA sequencing, is applied in familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). In these inherited colorectal tumor syndromes determining the genetic status may result in an individually tailored surveillance program and prophylactic treatment. The implications of genetic testing for the clinical management of disease, both of mutation and non-mutation carriers, in familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer families are discussed.

Classification of Familial Adenomatous Polyposis: A Diagnostic Nightmare

Classification of Familial Adenomatous Polyposis: A Diagnostic Nightmare

Hereditary gynecologic cancer.

It is estimated that approximately 1.3 million new cases of cancer will occur in the United States in 1997 (785,800 males, 596,600 females) [1]. Given a conservative estimate that 5% of these cases are hereditary, at least 65,000 patients will merit DNA testing annually. Using this same 5% estimate, then of the 181,600 cases of carcinoma of the breast and the 26,800 cases of carcinoma of the ovary that will occur in the United States during this same year, approximately 9080 patients with breast cancer and 1340 patients with ovarian carcinoma will fit a hereditary etiology. Barring new germline mutations, this means that each individual will be a member of a cancer-prone family. When viewed as annual incidence rates of hereditary cancer, it is clear that the prevalence of hereditary breast and ovarian carcinoma in the United States will truly be enormous. Similar calculations can be made for virtually all forms of cancer that will strike women.

Germline p53 mutation at codon 133 in a cancer-prone family.

We identified four families in which we suspected the presence of genetic factors predisposing them to cancer. We examined one family with features suggesting Li-Fraumeni syndrome for the presence of a germline p53 mutation in 13 of its members. To detect germline p53 mutations we performed polymerase chain reaction/nonradioisotopic single-strand conformation polymorphism and DNA sequencing analysis on exons 4-9 of the p53 gene. Mutated polymerase chain reaction-restriction fragment length polymorphism analysis was also performed on exon 5 to confirm the mutation identified by the sequencing analysis. A novel germline p53 mutation was identified at codon 133 (ATG-->AGG) in exon 5, resulting in the substitution of arginine for methionine, in all four cancer-affected individuals and in three apparently healthy individuals. We also analyzed tumor specimens for additional p53 mutations in the wild-type alleles using the same methods. However, heterozygosity was retained, and no other additional mutations in the wild-type allele were identified in any of the tumor tissues. It is possible that additional mutations in the wild-type allele are not always necessary for the loss of tumor suppressor functions. This study presents serious clinical and ethical problems about the predictive value of identifying germline p53 mutations in presymptomatic carriers. However, accurate predictive testing will be very useful in identifying unaffected individuals who are at increased risk of developing cancer and in detecting cancer at an early stage.

The P53-MDM2 Interaction in a Cancer-Prone Family, and the Identification of a Novel Therapeutic Target

One approach to developing novel anti-cancer agents is to identify and characterise targets that directly regulate cell growth and are dysfunctional in the disease state. One such target is the interaction between the p53 tumour suppressor and the oncogene product of the murine double minute gene, MDM2. MDM2 is known to bind wild-type p53 and block the transcriptional activation of p53-dependent genes. We have previously described a cancer-prone family with elevated levels of wild-type p53, and now show that MDM2 is also over-expressed in the proband from this family. Interestingly, the overexpression of MDM2 is independent of other p53-regulated genes such as p21WAF1. The present work and a review of recent insights into the p53-MDM2 interaction, and p53 transcriptional activity, identify a new target site for the rational development of novel anti-cancer agents.

Faulty DNA polymerase delta/epsilon-mediated excision repair in response to gamma radiation or ultraviolet light in p53-deficient fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome.

Dermal fibroblast strains cultured from affected members of a cancer-prone family with Li-Fraumeni syndrome (LFS) harbor a point mutation in one allele of the p53 tumor suppressor gene, resulting in loss of normal p53 function. In this study we have examined the ability of these p53-deficient strains to carry out the long-patch mode of excision repair, mediated by DNA polymerases delta and epsilon, after exposure to 60Co gamma radiation or far ultraviolet (UV) (chiefly 254 nm) light. Repair was monitored by incubation of the irradiated cultures in the presence of aphidicolin (apc) or 1-beta-D-arabinofuranosylcytosine (araC), each a specific inhibitor of long-patch repair, followed by measurement of drug-induced DNA strand breaks (reflecting non-ligated strand incision events) by alkaline sucrose velocity sedimentation. The LFS strains displayed deficient repair capacity in response to both gamma rays and UV light. The repair anomaly in UV-irradiated LFS cultures was manifested not only in the overall genome, but also in the transcriptionally active, preferentially repaired c-myc gene. Using autoradiography we also assessed unscheduled DNA synthesis (UDS) after UV irradiation and found this conventional measure of repair replication to be deficient in LFS strains. Moreover, both apc and araC decreased the level of UV-induced UDS by approximately 75% in normal cells, but each had only a marginal effect on LFS cells. We further demonstrated that the LFS strains are impaired in the recovery of both RNA and replicative DNA syntheses after UV treatment, two molecular anomalies of the DNA repair deficiency disorders xeroderma pigmentosum and Cockayne's syndrome. Together these results imply a critical role for wild-type p53 protein in DNA polymerase delta/epsilon-mediated excision repair, both the mechanism operating on the entire genome and that acting on expressed genes.

Presence of a heterozygous substitution and its relationship to DT-diaphorase activity.

A point mutation in the mRNA of NADP(H): quinone oxidoreductase 1 (NQO1, DT-diaphorase) is believed to be responsible for reduced enzyme activity in the adenocarcinoma BE cell line. The present study examined nine cultured human non-cancerous fibroblast cell strains, five of which were from members of a single cancer-prone family, which demonstrated widely varying activity levels of DT-diaphorase (41 - 3462 nmol min-1 mg-1 protein), to determine if genetic alteration of the NQO1 or NOQ2 gene was involved in determining enzyme activity. All cell strains expressed NQO1 and NQO2 mRNA as measured by a quantitative polymerase chain reaction amplification technique. No relationship was found between the level of mRNA expressed and the enzyme activity in the cells. Sequencing of the entire complementary DNA from the cell strains revealed only a single base substitution at nucleotide 609 in one allele encoding NQO1 in every cell strain from members of the cancer-prone family, except for one cell strain which expressed only the T at nucleotide 609 in both alleles. Subsequent examination of genomic DNA from 44 individuals revealed that this base substitution is present in approximately 50% of the population. The presence of the T at nucleotide 609 in the NQO1 locus does not appear to be directly causal for altered DT-diaphorase activity.

What does it mean to be a cancer gene carrier? Problems in establishing causality from the molecular genetics of cancer.

This article addresses the following question: What does it mean to be a cancer gene carrier? The existence of families prone to cancer has prompted an intense search for predisposing heritable gene mutations. Genes that impart susceptibility to colorectal, breast, and ovarian cancers have been recently identified. It is doubtful, however, that the action of a single mutant gene totally accounts for the development of malignant disease. The mutant gene likely causes cancer in these family members only in conjunction with other genes, environmental factors, or both. Furthermore, although an individual carrier of a mutant gene within a cancer-prone family has an increased risk of malignancy, nutritional, pharmacologic, or other interventions may still confer protection. Extrapolations from cancer-prone families to the general population are even more problematic. The excess risk of malignancy among carriers of mutant genes who are not members of cancer-prone families is unknown. Large-scale epidemiologic studies are needed to determine the magnitude (or even the existence) of such excess risk.

Breast cancer and importance of zygosity determination in triplet sisters

This study is the first to examine the genetic risk in a breast cancer-prone family wherein two of three triplet sisters and their mother manifested breast cancer. The unaffected triplet, the proband, was found to be monozygotic with her deceased sister through DNA testing of tumor blocks and dizygotic to her living affected sister. Genetic counseling implications are discussed.

FAMILIAL TESTICULAR CARCINOMA: IN SEARCH OF GENETIC TRIGGERS

Two cases of testicular tumours in non-twin brothers of a cancer-prone family are described. Cytogenetic studies of these two patients and human leucocyte antigen (HLA) typing of the family failed to identify any genetic defects. The authors propose using linkage analysis for further genetic studies but would require additional families for this to be performed.

Cytogenetic Response to G 2 -Phase X Irradiation in Relation to DNA Repair and Radiosensitivity in a Cancer-Prone Family with Li-Fraumeni Syndrome

Noncancerous skin fibroblasts from six family members with Li-Fraumeni syndrome, five with cancer of diverse tissue origin and one with a premalignant neoplasm, showed a high frequency of chromatid aberrations, 94 to 119 breaks and 58 to 95 gaps per 100 metaphase cells arrested with colcemid 0.5 to 1.5 h after X irradiation (1.75 x 10(-2) C/kg). This response results from deficient repair of the radiation-induced DNA damage. In contrast, skin fibroblasts from two unrelated normal controls and a spouse showed 19 breaks and 17 to 19 gaps per 100 cells. Whereas all six members of the cancer-prone family had a radioresistant phenotype, only four had an inherited p53 mutation. Fibroblasts from a radioresistant family member showed the same extent of chromatid damage directly (0 to 0.5 h) after G2-phase X irradiation as those from the radiosensitive control spouse. We conclude, therefore, that radiosensitivity, as determined by cell killing in asynchronous populations of skin fibroblasts, is unrelated to chromosomal sensitivity to G2-phase X irradiation. However, the persistence of a high frequency of chromatid breaks and gaps at 0.5 to 1.5 h after G2-phase X irradiation, a manifestation of deficient DNA repair, is associated with proneness to cancer in this family.

Hyperresistance to 4-nitroquinoline 1-oxide cytotoxicity and reduced DNA damage formation in dermal fibroblast strains derived from five members of a cancer-prone family

Dermal fibroblasts cultured from members of a family presenting multiple polyps and sarcomas were compared with fibroblast strains from unrelated healthy donors for sensitivity to killing by four genotoxic agents. Cells from the sister of the male proband (strain 3437T), mother (strain 3703T), two of his paternal aunts (3701T and 3704T) and one paternal uncle (3702T) displayed marked resistance (1.8 to 4.3 times greater than the normal mean) to 4-nitroquinoline 1-oxide (4NQO), a procarcinogen whose DNA-damaging properties encompass those of both far (254 nm) ultraviolet (UV) light and ionising radiation. These same 4NQO-resistant cells, however, responded normally to reproductive inactivation by UV light, 60Co gamma radiation or the alkylating agent methylnitrosourea, signifying that the abnormal resistance of these cells to 4NQO is not associated with aberrant DNA metabolism. In keeping with this conclusion, exposure to a given dose of 4NQO produced decreased amounts of DNA damage and stimulated lower levels of repair DNA synthesis in all five 4NQO-resistant strains than in normal controls. Moreover, exogenous radiolabelled 4NQO accumulated to a lesser extent in the 4NQO-resistant than in the normal fibroblasts. Cell sonicates of strains 3437T, 3701T and 3702T exhibited reduced capacities (40-60% of normal) to catalise the conversion of 4NQO to the proximate carcinogen 4-hydroxyaminoquinoline 1-oxide. However, the 4NQO-resistant strains 3703T and 3704T carried out 4NQO bioreduction at normal rates. Our data therefore indicate that enhanced resistance to 4NQO cytotoxicity in 3437T, 3701T and 3702T is a consequence of anomalies in both intracellular accumulation and enzymatic reduction of 4NQO, whereas 4NQO resistance in 3703T and 3704T appears to result solely from reduced intracellular drug accumulation.

Cellular approaches to bioreductive drug mechanisms

Mitomycin C is being used as an adjunct to ionizing radiation in the treatment of some solid tumors. A rationale for this is that radioresistant hypoxic cells in solid tumors will have enhanced sensitivity to this bioreductively activated drug, compared to aerobic cells. The role of oxygen concentration and enzymatic drug reduction in bioreductive drug activation have been investigated. Techniques are reviewed for the in vitro determination of the oxygen concentration dependency of bioreductive drug activation. One of these techniques, an open cell suspension system using Chinese hamster ovary cells, is described. Results are shown that indicate that the oxygen concentration dependency of toxicity of mitomycin C and one of its analogues profiromycin, though qualitatively complementing the oxygen dependency of ionizing radiation toxicity, are not quantitatively optimal. Using a mitomycin C resistant human cell strain (3437T) from a cancer prone family, a possible role for DT-diaphorase, an oxygen insensitive 2-electron transfer enzyme, is suggested. A correlation between a low level of DT-diaphorase in 3437T cells and mitomycin C resistance under aerobic exposure conditions is seen. Under hypoxic exposure conditions this resistance is lost, suggesting 1-electron transfer enzymes control hypoxic cell bioreductive activation. An activation role for DT-diaphorase in mitomycin C toxicity in the treatment of solid tumors is contrasted to a potential detoxification role for the enzyme with other xenobiotics in the cancer prone family phenotype.

Studies on the mechanism of resistance to mitomycin C and porfiromycin in a human cell strain derived from a cancer-prone individual

The mechanism of aerobic resistance to the quinone-containing anti-tumour agents mitomycin C (MMC) and porfiromycin (PM) has been investigated using non-transformed human cells. One of the cell strains used (3437T) was derived from an afflicted member of a cancer-prone family. This cell strain had been shown previously to be six times more resistant to the cytotoxic effects of these agents under aerobic but not hypoxic conditions when compared to a cell strain derived from an unrelated, normal donor (GM38). Differences could not be detected in the ability of cell sonicates prepared from either cell strain to produce alkylating species under aerobic conditions using a 4-( p-nitrobenzyl)pyridine assay. However, using 3H-labelled PM to monitor rapid drug uptake and subsequent accumulation due to drug metabolism, results were obtained indicating that the resistant cell strain (3437T) was deficient in an enzymatic pathway capable of metabolizing these compounds under aerobic but not hypoxic conditions. Dicumarol, an inhibitor of the quinone reductase DT-diaphorase (EC 1.6.99.2), decreased aerobic drug accumulation and cytotoxicity in the control cell strain, but did not alter the lack of accumulation noted in the resistant cell strain. Under hypoxic conditions, dicumarol increased cytotoxicity and drug accumulation in both cell strains. The mechanism of this enhanced cytotoxicity remains unclear. These results suggested that the resistant cells were deficient in the enzyme DT-diaphorase, a potential activator of PM. Enzymatic assays confirmed this and revealed no alterations in cytochrome P450 reductase (EC 1.6.2.4) activity or glutathione content. No protein characteristic of DT-diaphorase was detected in the resistant cell strain using a polyclonal rabbit-anti-rat antibody raised against this enzyme. Southern blot analysis using a rat DT-diaphorase cDNA probe demonstrated differences between the normal and resistant cell strains in the restriction fragment patterns. The present results are consistent with the hypothesis that decreased DT-diaphorase levels are causally associated with PM and MMC resistance in these cells under aerobic exposure conditions.

Aberrant DNA topoisomerase II activity, radioresistance and inherited susceptibility to cancer

Inherited susceptibility to a wide variety of neoplasias (Li-Fraumeni syndrome), has been shown in studies of one cancer-prone family, to have an intriguing association with an aberrant c-raf-1 gene and inheritance of a radioresistant phenotype in their non-cancerous skin fibroblasts. This association together with observations that DNA topoisomerases, when defective, can introduce errors into DNA and that these enzymes are perturbed in vitro by serine/threonine kinases similar to raf encoded proteins, prompted investigation of DNA topoisomerase activity of the family's fibroblasts. Since radioresistance was transferred to murine cells (NIH-3T3) when the aberrant c-raf-1 gene from this family was transfected, we also examined transformants containing this and other oncogenes. V-raf/c-myc and EJ-ras transformants were examined, the former because the family's skin fibroblasts also have 3-8-fold elevated myc expression (not apparently relevant to radioresistance) and the latter because ras, like raf, conveys radioresistance. The family members' fibroblasts and the three transfected murine lines, showed a similar perturbation of a spermidine and ATP-dependent DNA catenation activity (typical of DNA topoisomerase II). There was a significant positive correlation (r = 0.93; P = 0.0026) between the degree of activation of topoisomerase II and one measure of radioresistance (the Dq value). Relaxation of DNA supercoiling (topoisomerase I activity and other DNA nicking enzymes) was not abnormal. Cytotoxicity assays and evaluation of the influence of topoisomerase II inhibitors on DNA/protein complex formation, corroborated the existence of a qualitative topoisomerase II defect in the family's cells and transfectants. Although the contention that the qualitative topoisomerase II abnormalities observed here may be associated with malfunction is highly speculative, these findings may be relevant to the mechanism of oncogenesis, not only in this family, but with raf and ras type oncogenes.