Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Germline Variation Controls the Architecture of Somatic Alterations in Tumors

Studies have suggested that somatic events in tumors can depend on an individual''s constitutional genotype. We used squamous cell carcinomas (SCC) of the skin, which arise in high multiplicity in organ transplant recipients, as a model to compare the pattern of somatic alterations within and across individuals. Specifically, we performed array comparative genomic hybridization on 104 tumors from 25 unrelated individuals who each had three or more independently arisen SCCs and compared the profiles occurring within patients to profiles of tumors across a larger set of 135 patients. In general, chromosomal aberrations in SCCs were more similar within than across individuals (two-sided exact-test p-value ), consistent with the notion that the genetic background was affecting the pattern of somatic changes. To further test this possibility, we performed allele-specific imbalance studies using microsatellite markers mapping to 14 frequently aberrant regions of multiple independent tumors from 65 patients. We identified nine loci which show evidence of preferential allelic imbalance. One of these loci, 8q24, corresponded to a region in which multiple single nucleotide polymorphisms have been associated with increased cancer risk in genome-wide association studies (GWAS). We tested three implicated variants and identified one, rs13281615, with evidence of allele-specific imbalance (p-value = 0.012). The finding of an independently identified cancer susceptibility allele with allele-specific imbalance in a genomic region affected by recurrent DNA copy number changes suggest that it may also harbor risk alleles for SCC. Together these data provide strong evidence that the genetic background is a key driver of somatic events in cancer, opening an opportunity to expand this approach to identify cancer risk alleles.     

Micronucleus frequency in peripheral blood lymphocytes and exfoliated buccal cells of untreated cancer patients

Some genetic diseases may increase the cellular instability. Since most human tumors have some genetic base, this study was undertaken for the genetic instability in cancer patients by micronucleus analysis, a mutation-screening test, which is more practical and economic technique than metaphase analysis carried out for chromosomal aberrations. Genetic changes were assessed in untreated cancer patients (lung, stomach and colon cancer) by different genotoxical screening methods; the cytokinesis-block micronucleus test and the buccal mucosa cell micronucleus test. The evaluation of micronuclei number in peripheral blood lymphocytes and buccal cells showed a genomic instability in somatic cells. There was a significant increase in the number of micronuclei in cancer patients prior to the initiation of chemotherapy, and/or radiotherapy compared with healthy human subjects. Furthermore, there was no significant difference between smokers and non-smoking groups or male and female groups. These results suggest that cancer in humans is characterized by an increase of chromosomal damage and thus, the micronucleus assay carried out here may be useful in routine cytogenetic studies of cancer.     

Relative risks of radiation-associated cancer: comparison of second cancer in therapeutically irradiated populations with the Japanese atomic bomb survivors

In this paper the radiation-associated relative risks of second primary cancer incidence in groups treated for first primary cancer by radiotherapy are compared with radiation-associated relative risk estimates in the Japanese atomic bomb survivor cancer incidence data. For four cancer sites, namely lung cancer, bone cancer, ovarian cancer and leukaemia, the relative risks in the comparable (age at exposure, time since exposure, sex matched) subsets of the Japanese data are significantly greater than those in the majority of second cancer studies. Even when the differences between the relative risks in the Japanese atomic bomb survivors and the medical series do not approach conventional levels of statistical significance, relative risks tend to be higher in the Japanese data than in the second cancer studies. At least for leukaemia, the discrepancy between the Japanese and second cancer risks can be largely explained by cell- sterilisation effects. There are few indications of modification of radiation-associated second cancer relative risk among those treated with adjuvant chemotherapy, nor are there strong indications of modification of radiation- associated relative risk by heritable genetic factors. If anything, there is evidence that second cancer relative excess risks are lower among those patients with cancer-prone disorders than among non-susceptible patients. However, the higher underlying cancer risk in some of these medically exposed populations should also be considered, in particular for those with cancer-prone conditions, so that the absolute excess risk is sometimes higher than in the Japanese data. Received: 14 May 1999 / Accepted in revised form: 17 September 1999     

Micronucleus frequency in peripheral blood lymphocytes and exfoliated buccal cells of untreated cancer patients

Some genetic diseases may increase the cellular instability. Since most human tumors have some genetic base, this study was undertaken for the genetic instability in cancer patients by micronucleus analysis, a mutation-screening test, which is more practical and economic technique than metaphase analysis carried out for chromosomal aberrations. Genetic changes were assessed in untreated cancer patients (lung, stomach and colon cancer) by different genotoxical screening methods: the cytokinesis-block micronucleus test and the buccal mucosa cell micronucleus test. The evaluation of micronuclei number in peripheral blood lymphocytes and buccal cells showed genomic instability in somatic cells. There was a significant increase in the number of micronuclei in cancer patients prior to the initiation of chemotherapy, and/or radiotherapy compared with healthy human subjects. Furthermore, there was no significant difference between smokers and non-smoking groups or male and female groups. These results suggest that cancer in humans is characterized by an increase of chromosomal damage and thus, the micronucleus assay carried out here may be useful in routine cytogenetic studies of cancer. The text was submitted by the authors in English.     

Hypothesis on the possible relevance of the immunogenic cell death in the treatment of gestational trophoblastic neoplasms

The genetic background and the antigenic landscape of cancer cells play a critical role in the response to immunotherapies. A high tumor antigenicity, together with an increased adjuvanticity potentially induced by a peculiar type of cell death, namely immunogenic cell death (ICD), could foster the response to immunogenic therapies. The gestational trophoblastic neoplasm (GTN) is a one-of-a-kind cancer in the oncological landscape due to its exclusive genomic makeup. The prognosis of GTN is significantly better than non-gestational trophoblastic neoplasm (nGTN). Due to its peculiar genetic inheritance, GTN potentially constitutes a singular archetype in the immuno-oncological field.     

用多引物PCR分析γ射线引起人外周血淋巴细胞hprt基因突变

正常人外周血用~（６０）Ｃｏ射线分别进行１～８Ｇｙ照射后,在含６－ＴＧ的培养基上克隆和筛选人淋巴细胞ｈｐｒｔ突变细胞。细胞的突变频率与γ射线的照射剂量呈正相关．获得４２个ｈｐｒｔ突变细胞株,用８对ｈｐｒｔ寡核苷酸引物进行多聚酶链反应（ＰＣＲ）从细胞粗提物中分别扩增ｈｐｒｔ各个外显子,分析突变细胞的ｈｐｒｔ基因突变,约６０％（２５／４２）的ｈｐｒｔ基因突变为基因缺失,其中１３个突变是ｈｐｒｔ基因全部缺失,而１２个是部分ｈｐｒｔ基因外显子缺失,约有４０％（１７／４２）的突变无明显的ＰＣＲ扩增变化．同时显示ｈｐｒｔ基因外显子的缺失突变与辐射剂量有关,实验结果提示,此方法有可能用于估计辐射剂量和进行辐射远后效果观察,进而揭示辐射致突的分子机理．     

Reduced mRNA and protein expression of the genomic caretaker RAD9A in primary fibroblasts of individuals with childhood and independent second cancer

Background

The etiology of secondary cancer in childhood cancer survivors is largely unclear. Exposure of normal somatic cells to radiation and/or chemotherapy can damage DNA and if not all DNA lesions are properly fixed, the mis-repair may lead to pathological consequences. It is plausible to assume that genetic differences, i.e. in the pathways responsible for cell cycle control and DNA repair, play a critical role in the development of secondary cancer.

Methodology/Findings

To identify factors that may influence the susceptibility for second cancer formation, we recruited 20 individuals who survived a childhood malignancy and then developed a second cancer as well as 20 carefully matched control individuals with childhood malignancy but without a second cancer. By antibody microarrays, we screened primary fibroblasts of matched patients for differences in the amount of representative DNA repair-associated proteins. We found constitutively decreased levels of RAD9A and several other DNA repair proteins in two-cancer patients, compared to one-cancer patients. The RAD9A protein level increased in response to DNA damage, however to a lesser extent in the two-cancer patients. Quantification of mRNA expression by real-time RT PCR revealed lower RAD9A mRNA levels in both untreated and 1 Gy γ-irradiated cells of two-cancer patients.

Conclusions/Significance

Collectively, our results support the idea that modulation of RAD9A and other cell cycle arrest and DNA repair proteins contribute to the risk of developing a second malignancy in childhood cancer patients.     

Chromosomal aberrations,DNA strand breaks and gene mutations in nasopharyngeal cancer patients undergoing radiation therapy

Nasopharyngeal cancer (NPC) is a common disease in the south part of China, and its incidence is increasing in the southwest of China in recent years. Radiation therapy is the main therapeutic method for NPC in China. In this study, genetic changes were assessed in randomly selected nine NPC patients receiving radiation therapy by different genotoxical screening methods, the cytokinesis-block micronucleus test (CB-MNT), the buccal mucosa cell micronucleus test (BMC-MNT), the undivided lymphocyte micronucleus test (UL-MNT), chromosomal aberration (CA) test, the comet assay and the hprt gene mutation test (HPRT). Patients were used as self-control before receiving radiation therapy. Apart from the UL-MNT, all the methods detected genetic damages in NPC patients, though with different sensitivities. CB-MNT is the best biological indicator for evaluating genetic damage induced by radiation therapy in NPC patients; followed by CA and HPRT, while the BMC-MNT is simplest method as a potential biological indicator.     

Hypothesis on the possible relevance of the immunogenic cell death in the treatment of gestational trophoblastic neoplasms

The genetic background and the antigenic landscape of cancer cells play a critical role in the response to immunotherapies. A high tumor antigenicity, together with an increased adjuvanticity potentially induced by a peculiar type of cell death, namely immunogenic cell death (ICD), could foster the response to immunogenic therapies. The gestational trophoblastic neoplasm (GTN) is a one-of-a-kind cancer in the oncological landscape due to its exclusive genomic makeup. The prognosis of GTN is significantly better than non-gestational trophoblastic neoplasm (nGTN). Due to its peculiar genetic inheritance, GTN potentially constitutes a singular archetype in the immuno-oncological field.     

Cancer molecular epidemiology: new horizons of prophylaxis

Perspectives of malignant neoplasm prophylaxis based on molecular biology achievements are discussed. Gene variants critical to development of hereditary cancer syndromes, genes modulating malignant neoplasm development risk without hereditary cancer syndrome development, and genes determining tendency of individuals for different malignant neoplasm progress risk increasing lifestyle factors are examined. Molecular epidemiology by using large scale population analysis of cancerogenesis linked genetic polymorphisms prevalence allows determination of risk groups at the most earlier stages of cell transformation or even before the onset of cell malignization and development of goal-based prophylaxis measures based on polymorphism and corresponding cancer type. Epidemiologic analysis of this type allows for earlier diagnostics in risk groups, therapy efficacy increase, disability decrease. Specific therapy on molecular level may be possible in the future.     

Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.     

Genetic determinants of lung cancer: Understanding the oncogenic potential of somatic missense mutations

BackgroundTreatment of lung cancer is getting more personalized nowadays and medical practitioners are moving away from conventional histology-driven empirical treatments, platinum-based chemotherapy, and other invasive surgical resections and have started adopting alternate therapies in which therapeutic targets are patient's molecular oncogenic drivers.AimThe aim of the current study is to extract meaningful information from the online somatic mutation data (retrieved from cBioPortal) of 16 most significantly mutated oncogenes in non-small-cell lung cancer (NSCLC), namely EGFR, NRAS, KRAS, HER2 (ERBB2), RET, MET, ROS1, FGFR1, BRAF, AKT1, MEK1 (MAP2K1), PIK3CA, PTEN, DDR2, LKB1 (STK11) and ALK, for improving our understanding of the pathobiology of the lung cancer that can aid decision-making on critical clinical and therapeutic considerations.MethodsUsing an integrated approach comprising 4 steps, the oncogenic potential of 661 missense non-synonymous single nucleotide polymorphisms (nsSNPs) in 16 genes was ascertained using 2059 NSCLC (1575 lung adenocarcinomas, 484 lung squamous cell carcinomas) patients' online mutation data. The steps used comprise sequence/structure homology-based prediction, scoring of conservation of mutated residues and positions, prediction of resulting molecular and functional consequences using machine-learning and structure-guided approach.ResultsOut of a total of 661 nsSNPs analyzed, a set of 29 nsSNPs has been identified as conserved high confidence mutations in 10 of 16 genes relevant to the under study. Out of 29 conserved high confidence nsSNPs, 4 nsSNPs (EGFR N1094Y, BRAF M620I, DDR2 R307L, ALK P1350T) have been found to be putative novel rare genetic markers for NSCLC.ConclusionsThe current study, the first of its kind, has provided a list of deleterious non-synonymous somatic mutations in a selected pool of oncogenes that can be considered as a promising target for future drug design and therapy for patients with lung adenocarcinomas and squamous cell carcinomas.     

Hsp90 is essential in the filarial nematode Brugia pahangi

The development of a compound with activity against filarial nematodes (a 'macrofilaricide') has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.     

Familial risks of squamous cell carcinoma of the head and neck: retrospective case-control study.

OBJECTIVE: To determine the contribution of inheritance to the incidence of squamous cell carcinoma of the head and neck. DESIGN: Historical cohort study. First degree relatives of cases with squamous cell carcinoma of the head and neck made up the exposed cohort and first degree relatives of spouses of cases made up the comparison unexposed cohort. SETTING: Ear, nose, and throat clinic in a large metropolitan teaching hospital. SUBJECTS: 1429 first degree relatives of 242 index cases of squamous cell carcinoma of the head and neck; as controls, 934 first degree relatives of the spouses of 156 index cases. MAIN OUTCOME MEASURES: Relative risk of developing squamous cell carcinoma in first degree relatives of cases compared with risk in first degree relatives of spouses. RESULTS: The adjusted relative risk for developing head and neck cancer if the index case had squamous cell carcinoma of the head and neck was 3.79 (95% confidence interval 1.11 to 13.0). There were no significantly increased risks associated with a family history of cancer at other sites. The adjusted relative risk for squamous cell carcinoma of the head and neck was 7.89 (1.50 to 41.6) in first degree relatives of patients with multiple primary head and neck tumours. CONCLUSIONS: These data suggest that genetic factors are important in the aetiology of head and neck cancer, in particular for patients with multiple primary cancers. Given the prolonged exposure of these subjects to carcinogens, these genetic factors may have a role in modifying carcinogen activity or in host resistance to carcinogens. Inherited factors may be important in persons with environmentally induced cancers.     

Evaluation of adjuvant chemotherapy in patients with colorectal cancer in Primorsko-Goranska and Istarska County--a twenty years retrospective study

Colorectal cancer is the second most common malignant disease in developed countries, with about one million new cases worldwide every year, accompanied with high mortality rate. We examined the survival rate and recurrence (occurrence of distant metastases and/or local recurrence) of patients with colorectal cancer in Primorsko-Goranska and Istarska County who received adjuvant chemotherapy, compared to those who did not in the period since 1980. until 1999. This study involves 483 patients with colorectal cancer stages II and III of Primorsko-Goranska and Istarska County, which were underwent curative resections of colorectal cancer at the Clinical Hospital Centre Rijeka, and then treated with chemotherapy (288) or without Chemotherapy (195). We analyzed the five year survival rate and the recurrence of malignant disease in the adjuvant treatment group in comparison with not treated group with chemotherapy, depending on the stage of disease, degree of histological differentiation, patient age and location of cancer (colon or rectum). After follow-up of 60 months died 44.79% (129/288) of patients who received chemotherapy and 53.33% (104/195) of patients who did not receive chemotherapy. The relative risk of death (from any cause) in chemotherapy-treated group versus the group without chemotherapy was 0.82 (p < 0.008). Recurrence of malignant disease in the chemotherapy group was 38.54% (111/288), and in the group without chemotherapy was 46.15% (90/195). The relative risk of recurrence of malignant disease in the chemotherapy group versus the group without chemotherapy was 0.78 (p < 0.001). There was no difference in treatment efficacy regard to the localization of the tumor, but there were differences in efficiency with respect to disease stage, grade and age. Chemotherapy with 5-fluorouracil and leukovorin ameliorate the survival and reduces recurrence and distant metastases in patients with colorectal cancer stages II and III.     

Mutations and epimutations in the origin of cancer

Cancer is traditionally viewed as a disease of abnormal cell proliferation controlled by a series of mutations. Mutations typically affect oncogenes or tumor suppressor genes thereby conferring growth advantage. Genomic instability facilitates mutation accumulation. Recent findings demonstrate that activation of oncogenes and inactivation of tumor suppressor genes, as well as genomic instability, can be achieved by epigenetic mechanisms as well. Unlike genetic mutations, epimutations do not change the base sequence of DNA and are potentially reversible. Similar to genetic mutations, epimutations are associated with specific patterns of gene expression that are heritable through cell divisions. Knudson's hypothesis postulates that inactivation of tumor suppressor genes requires two hits, with the first hit occurring either in somatic cells (sporadic cancer) or in the germline (hereditary cancer) and the second one always being somatic. Studies on hereditary and sporadic forms of colorectal carcinoma have made it evident that, apart from genetic mutations, epimutations may serve as either hit or both. Furthermore, recent next-generation sequencing studies show that epigenetic genes, such as those encoding histone modifying enzymes and subunits for chromatin remodeling systems, are themselves frequent targets of somatic mutations in cancer and can act like tumor suppressor genes or oncogenes. This review discusses genetic vs. epigenetic origin of cancer, including cancer susceptibility, in light of recent discoveries. Situations in which mutations and epimutations occur to serve analogous purposes are highlighted.     

The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells

Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2(-/-) mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of -G and -A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.