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Title : Cytogenetic aberrations in ovarian tumors using FISH. LIST OF AUTHORS:1. Dr. Shashi Gupta MD. Prof & Head, Deptt of Obst & Gynae SMGS Hospital, GMC, Jammu, India. 2. Dr. Vikram Mahajan M.D (Obst & Gynae) Deptt. Of Obs & Gynae, SMGS Hospital GMC Jammu, India. 3. Dr. Subhash Gupta Phd. Professor & Head, Human Genetics Research Cum Counselling Centre, Jammu University, India. 4. Dr. Charu Jandial M.D ( Obst & Gynae) SMGS Hospital GMC Jammu, India. 5. Dr. Surender Kumar MD. Associate Professor, Deptt of Obst & Gynae SMGS Hospital, GMC, Jammu, India

“Disclosure: None of the authors have a conflict of interest,” For Reperint request :Dr. Vikram Mahajan M.D (Obst & Gynae) Deptt. Of Obs & Gynae, SMGS Hospital GMC Jammu, India. e-mail:- [email protected] Correspondence:Dr. Vikram Mahajan M.D (Obst & Gynae) Deptt. Of Obs & Gynae, SMGS Hospital GMC Jammu, India. e-mail:- [email protected] Dr Shashi Gupta Prof & Head , Deptt of Obst & Gynae ,GMC , Jammu e-mail :- [email protected]

Word count abstract :- 149 Wordcount of Text:- 1792

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Condensation & Short version of the title:FISH on chromosome 12, 17 and X shows that these chromosomes are involved in initiation and progression of ovarian malignancies , further research is necessary. FISH on Ovarian tumor.

Abstract

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Objectives: The identification of recurrent specific cytogenetic findings in various malignancies has provided an improved means to diagnose and treat patients. To date, no characteristic markers have been found for ovarian cancer. FISH (Florescent in situ hybridization) can provide critical information about the genetic mechanisms underlying the initiation and progression of ovarian malignancies and thus, will help in better diagnosis and management of patients. Methods: Fluorescence in situ hybridization (FISH) using centromere enumeration probes (CEP) for chromosome number 12, 17 and X was done on twenty patients with ovarian tumors. The slides were then observed under fluorescence microscope for fluorescent signal. Result: Loss of chromosome 12, 17 and X was seen in both benign and malignant epithelial ovarian tumors. Gain of chromosome 12 was seen in granulosa cell tumors; while in mature cystic teratomas, no aberration was found.Conclusions: Chromosome 12 can serve as a marker chromosome for ovarian tumors. Keywords:- FISH , cytogenetics operations and ovarian tumors.

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Introduction Ovarian cancer is second most common malignancy of the female worldwide. It accounts for 4% of cancer in women. A woman’s risk of getting invasive ovarian cancer in her lifetime is about 1 in 71. (1) The future of cancer research is no longer limited to epidemiological data and clinical management, but rather encompasses a new dimension of understanding, that involves genetics of tumors themselves. Although 5-10% of ovarian cancers are familial, much is yet to be learned about molecular pathogenesis of these cancers by identifying the culprit genes in these cases (2). It has been shown that recurrent chromosomal rearrangements are strongly associated with the activation of oncogenes, acquisition of drug resistance and loss of tumor suppressor gene function. Chromosomal aberrations have also been shown to characterize many solid tumors including epithelial ovarian cancer; however, there are a few cytogenetic studies that have contributed to the clinical management. It is probably because tumors are difficult to culture and the resultant metaphase has complex numerical and structural chromosomal anomalies. Cytogenetics entered the molecular era with the introduction of in situ hybridization, a procedure that allows researchers to localise the positions of specific DNA sequences on chromosomes. Since the first in situ hybridization experiment in 1969(3),many variations of the procedure have been developed and its sensitivity has increased enormously.Today,most in situ hybridization procedures use fluorescent probes to detect DNA Changes associated with neoplastic progression occur at specific nucleotide sites within DNA that can be localized by sequencing. Certain aspects of these changes can also be recognized at the cytogenetic level by methods that detect chromosomal changes like deletions, translocations, inversions and gene amplifications. To date, no characteristic markers have been found for ovarian cancers. FISH, though expensive reduces the time in detecting chromosomal abnormalities in benign as well as malignant tumors. The current study aims to

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identify the pattern of involvement of chromosome 12, 17 and x in ovarian tumors by using respective FISH probes.

Materials and methods The current one year study was carried out on 20 cases of ovarian tumors:5 benign epithelial (three serous cyst adenomas and two mucinous cyst adenomas),ten malignant epithelial(seven papillary serous adenocarcinomas , two mucinous adenocarcinomas ,and one clear cell carcinoma) ,three mature cystic teratomas and two granulosa cell tumors. The aim of the study was to analyse the numerical abnormalities involving the chromosome 12, 17 and X by using FISH technique, in various types of histologically diagnosed ovarian tumors and to identify the marker chromosomes, if any. These three chromosomes were selected on the basis of review of previous cytogenetic studies carried out on ovarian tumors, which show that involvement of these chromosomes occurs very frequently. The selected patients were confirmed to have ovarian tumors on the basis of general physical examination, systemic and local examination radiological workup and CA 125 levels. Tissue was collected in the hypotonic solution and first minced mechanically into cell suspension. Cell suspension was exposed consecutively to 0.04%Versene, Earle’s solution for 30 minutes at 37 C.Cells were treated with hypotonic solution,0.05M KCL(1:4)for 15 minutes at 37C.The material was fixed in methanol and acetic acid (3:1)and slides were made by air drying method. For conventional cytogenetic study, slides were prepared from cultured lymphocytes of patients with confirmed malignant tumors. The prepared slides were subjected to GTG- banding and these slides were scanned under AX101 metaphase imaging system. FISH using Alphoid DNA probes (centromere enumeration probes) were used and the site of hybridization of probes with cellular DNA observed under fluorescence microscope for fluorescent signals. The probes used were: CEP 12 spectrum green probe for chromosome 12, which gives green signal; CEP17

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spectrum orange probe for chromosome 17, which gives orange signal; and CEP X spectrum orange probe for chromosome, which gives orange signal. The protocol for FISH was followed according to Diane et al with slight modifications. For visualizing the hybridization, 10 microlitres of DAPI counter stain was applied to the target area and covered with a cover slip. The slide was then incubated at RT for 10 minutes and then viewed under a fluorescent microscope using a suitable filter set for FISH analysis, at least20 interphase cells were studied in each case.

Results In benign epithelial ovarian tomors,monosomy of chromosome 12 was observed in 20% of the cells; while nullisomy was seen in 70% of the cells.80% of the cells showed monosomy for chromosome 17.For chromosome X, no significant change was seen as all the cells were normal showing two orange signals(Table1 &2). In papillary adenocarcinomas, monosomy 12 was seen in 100% of the cells. Monosomy 17 was seen in 50% of the cells and nullisomy X was seen in 100% of the cells. In mucinous adenocarcinomas, nullisomy 12 was observed in 90% of the cells. Both monosomy17 and trisomy 17 was observed in 50% each of the cells.85% of the cells showed monosomy X .In clear cell carcinoma, nullisomy 12 was seen in all the cells.Nullisomy17 was observed in 75% of the cells and 25% cells showed trisomy 17.Nullisomy X was seen in 85% of the cells. In mature cystic teratomas, all the cells observed showed two signals for three chromosomes 12, 17 and X. In granulosa cell tumors, trisomy 12 was observed in 70% of the cells while no numerical change was seen for chromosome 17 and X.

Discussion

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While early detection permits treatment success, fewer than 25% ovarian cancers are currently detected at stage 1.Approaches to ovarian cancer screening include TVS, Serum 125 and markers but these include limitations in sensitivity and specificity. Gene expressions and proteomic arrays were used to identify markers that can be used in combination with clinical picture for early detection (4). Cytogenetics provides important insights into the molecular pathogenesis of human cancers. Previous studies of ovarian carcinoma indicated the presence of multiple complex clonal chromosome abnormalities. Specific and recurrent chromosome abnormalities may occur in regions of the genome that are involved in the conversion of normal cells to those with tumorigenic potential. FISH remains one of the few techniques capable of detecting these multiple, simultaneously occurring genetic abnormalities. Besides being used as an important tool for gene mapping, FISH is also applied to ascertain the presence, absence copy number or location of a particular chromosome locus/gene in cancer cells. The FISH analysis can be applied not only to chromosomes (metaphase based FISH), but to the interphase nuclei (interphase-based FISH), as well as to cells from tissues embedded in paraffin touch preparations or smears. FISH analysis of epithelial ovarian tumors for chromosome 12 showed a loss of signal for chromosome 12 in the form of monosomy or nullisomy. Benign tumors showed monosomy or nullisomy while in case of malignant epithelial tumors, majority of the cases showed nullisomy i.e,complete loss of chromosome 12.Numerical aberrations of chromosome 12 in epithelial ovarian tumors are highly controversial; both trisomy 12 and monosomy 12 have been observed. Trisomy 12 has been reported both in benign and malignant epithelial tumors by a team of workers (5, 6, 7). In our study, as well as in an earlier study(8), loss of chromosome 12 was seen in majority of tumor samples and this suggests that frequent loss of chromosome 12 may indicate that this chromosome is involved in the tumorigenesis of epithelial ovarian tumors. In epithelial ovarian tumors, reports of numerical aberrations of chromosome 17 have been predominantly restricted to losses in carcinoma (9, 10, and 11). In the present study, loss of chromosome 17 was seen in both benign and malignant epithelial ovarian tumors; while in malignant epithelial tumors, gain of

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chromosome 17 was also observed which confirms the existing view that trisomy 17 is specific for invasive disease(12). Loss of a single X chromosome is the most frequent aberration present in malignant epithelial ovarian tumor (13). On FISH analysis of epithelial ovarian tumors for chromosome X, monosomy X was seen in some malignant tumors, while in some, nullisomy X was observed. In benign epithelial tumors, no loss or gain of chromosome X was seen. Cytogenetic analysis on mature cystic teratomas has shown that the majority of cases have a 46XX normal karyotype (14,15).Our observations were in accordance with these cytogenetic findings ,i.e. no abnormality regarding the number of chromosome 12 , 17 and X , was observed in any of the three cases on FISH analysis. Trisomy 12 has been reported as a feature of ovarian granulosa cell tumors, which suggests that this may be a non random cytogenetic aberration. Trisomy 12 was observed in all cases of granulosa cell tumors, which is in accordance with the observations made by workers earlier (16, 17). No abnormality regarding the number of chromosome 17 and X was observed in any of the two cases on FISH analysis. In our study, we observed a preferential loss of chromosomes 12 and 17 in both benign and malignant epithelial ovarian tumors; whereas gain of chromosome 1was seen in granulosa cell tumors. This suggests the role of tumor suppressor genes /oncogenes present on these chromosomes and cell cycle regulators governed by these genes in the pathogenesis and /or progression of ovarian tumors. These chromosomes may serve as target areas of the genome for further studies so as to exactly localise the primary genetic insult responsible for tumor development.

Conclusions

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On FISH analysis, aberrations of chromosome 12, 17, and X were found in epithelial ovarian tumors. In granulosa cell tumors, trisomy 12 was seen and no aberrations of chromosome 17 and X were observed. Whereas in germ cell tumors, no involvement of chromosome 12, 17 and X was seen. Since, the size of the study was small and limited by time and financial constraints, no definite marker chromosome can be suggested. However, on comparing our observations with the previous literature and nature of cytogenetic aberrations observed, it can be stated that chromosome 12 can be used as a marker chromosome for epithelial and sex cord stromal tumors. Chromosome 17 and X can also be used as a marker in epithelial ovarian tumors. There is no doubt that chromosomes 12, 17 and X and tumor suppressor genes/oncogenes present on these chromosomes are involved in the origin and /or progression of ovarian tumors. Further research is necessary on larger study samples which will one day lead us to the detection of genes involved in the initiation and progression of ovarian tumors; which will pave way to diagnose the women prone to develop ovarian tumors by specific test at generic level and will help in preventing the initiation and progression of ovarian tumor by gene therapy in susceptible women and will also help in better treatment of patients suffering from ovarian tumors. Further genetic research is thus required to identify the genes whose function is altered and the exact role played by these aberrations in the tumorigenesis of ovarian tumors.

Bibiliography

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