| Objective：Laryngeal cancer is frequently occurring in head and neck cancers. Most laryngealcancers are squamous cell carcinomas. Radiotherapy, chemotherapy, surgery aloneand combination of therapy have been used in the treatment of laryngocarcinoma.However, the survival rate of patients with laryngocarcinoma is low due to its latemetastases and resistance to chemotherapy and radiotherapy post surgery.“Fieldcancerization” is a biological process in which one or groups of cells in specificorgans changes into the pre-cancerous cells under stimulation of the externalcarcinogenic factors. In the chornic carcinogenic factors stimulation, further geneprecancerous occurs to induce malignant tumor, even in peripheral tissues of normalmucosa pathology. Loss of heterozygosity (LOH) reflects the characteristic geneticchanges during tumorigenesis, which is commonly used in study of fieldcancerization. There was less report on field concretization in laryngeal cancers. Inthis study, we attempt to, verify the field cancerization in laryngeal cancer patients byLOH analysis. Through analysis and comparison of gene alternation, possible sitesplay a key role in the process of tumor progression would be investigated. Moreover,the security of sugical margin in current laryngeal cancer treatment was alsoevaluated.Methods：The paraffin blocks from35cases of patients with laryngeal cancer was collected,including five cases of recurrent patients post-surgery. The patients were divided intodifferent groups according to TNM staging, histological type and other circumstances.The paraffin blocks were used to make7μm serial sections. Then HE staining wasperformed, different positions of tumor were distinguished under a microscope.Sugical margins, center area and paracancerous were observed, the appropriatesections were selected to perform laser capture microdissection (LCM). The center ofthe tumor (T), paracancerous (F1-F4, paraneoplastic distance <5mm; F5, adjacentdistance>5mm) and sugical margins (Ma-Md) position microdissection were cut off via LCM, respectively. The DNA was extracted from the cut cells. Then PCRtechniques were used to detect LOH in different region of tumor tissues. Incombination with medical record data, the LOH analysis was compared in eachpartition. To test whether field cancerization was occurred in laryngeal carcinoma, themicrosatellite located on chromosome9p,3p,17p,18q and8p, which were identifiedas potential or known tumor suppressor genes, were studied in this study. Accordingto the difference LOH patterns, to determine the recurrent tumor clone was analyzed.Meanwhile, the differences in adjacent mucosa and surgical margin were alsoanalyzed with above microsatellites.Results：1. There are25cases of laryngeal cancer patients at the cancer center position (T)detected in one or more sites of LOH, the detection rate was83.3%(25/30);2. Nine microsatellites in the center of the tumor detection rate were: D3S1284（31.6%）、D3S1300（44.4%）、D3S1234（55%）、D3S1568（47.1%）、D9S171（44.4%）、D9S1870（42.1%）、TP53（58.8%）、D18S1110（33.3%）and以及D8S518（35.3%）respectively;3. In the five patients with recurrent tumor, LOH patterns were consistent with theprimary tumor;4. It was found that the age of patient, regions of tumor, lymphatic metastasis and Tstage were not related to TP53LOH (P>0.05). Except tumor grade (P=0.021<0.05), the rest microsatellites in LOH detection were also not related to the age ofpatients, regions of tumor, lymphatic metastasis, histological grade and T stage(P>0.05);5. There were some microsatellites loci detected in mucosa of tumor-adjacenttissues (F1-F4, F5), the detection rate of each index is: D3S1284（83.3%）、D3S1300（75%）、D3S1234（72.7%）、D3S1568（75%）、D9S171（87.5%）、D9S1870（75%）、TP53（80%）、 D8S518（33.3%） and D18S1110（33.3%）,respectively;6. Microsatellites loci in the sugical margins (Ma-Md) were also detected with different degrees of LOH, the detection rate of each index are:D3S1284（83.3%）、D3S1300（37.5%）、D3S1234（54.5%）、D3S1568（50%）、D9S171（62.5%）、D9S1870（62.5%）、TP53（70%）、D8S518（16.7%）and D18S1110（16.7%）, respectively;7. There were at least one microsatellite loci LOH in tumor-adjacent mucosa in18patients (60%). In which10patients (No:1,4,6,9,10,12,14,23,26,27)mucosal paraneoplastic manifestations in different regions had the same geneticchanges, six patients (No:11,17,19,20,22,25) adjacent mucosa showed loss ofdifferent loci. Meanwhile, there are also two patients with only one regionoccurred LOH (No:3,8);8. In adjacent mucosa, the D8S518LOH was detected in patients No.17and23,especially the No.17patient, the changing expanded to the sugical margins.D18S1110LOH was detected in patients No.3and20patients in adjacentmucosa, No.20patients8months after surgery recurrence of laryngeal cancer(No.33) occurred;9. The sugical margins of15patients (50%) were detected with at least one locusLOH. In which six patients (NO:4,8,9,10,12,22) mucosal resection marginperformance in different regions had the same genetic changes, one patient (No:3) mucosal resection margin performance for different loci lost. There was onlyone sugical margins region of the remaining eight patients (No:6,11,14,17,19,23,26,27) had been detected with LOH;10. Cancer center (T) and adjacent regions in8patients (No:1,6,10,11,14,19,26,27) showed the same genetic alterations. There were additional genetic changesbetween tumor center (T) and paracancerous mucosa in10patients (No:3,4,8,9,11,12,17,20,23,25). In addition, cancer Center (T) and the sugical marginsperformance had the same genetic changes in6patients (No:3,6,10,11,14,19).However, the genetic changes in mucosal resection margin was unlike cancercenter (T) in the other10patients (No:3,4,8,9,12,17,22,23,26,27);11.12patients (No:3,4,6,8,9,10,11,12,14,17,19,26) surgical margin and adjacentmucosa with the same LOH model, these two regions have a common genetic alterations process. While patients No:3patients margin (Mb), No:22(Mb, Md),No:27(Ma) showed differences in adjacent mucosa LOH patterns;12. The LOH was found in five cancer patients with recurrent tumors. The LOH ofadjacent mucosa was similar to tumor tissues in3patients (No:31,32,34).However, there were genetic changes between cancer center (T) and mucosaadditional in No.33and No.35patients. The sugical margins of4patients withrecurrent tumors (No:32,33,34,35) had been found LOH,and the LOH was lessin sugical margins than cancer center (T) with recurrent tumors. In comparisonwith primary tumor, there was difference of LOH between sugical margins andadjacent mucosa in patients with recurrent tumors. There was less LOH in thesugical margins of No.31patients, and the LOH was also different to primarytumor in No.32,33,34,35patients;13. The LOH of adjacent mucosa (F1-F4, F5) was unrelated with the age of patients,location of tumor, T stage, histological grade (P>0.05). However, the lymphaticwas significantly related (P=0.008<0.05);14. The LOH of sugical margins (Ma-Md) was unrelated with age, location of tumor,T stage, histological grade (P>0.05), but correlated with lymphatic (P=0.003<0.05);15. TP53in adjacent mucosa (F1-F4, F5) was unrelated with age, location of tumor,tumor grade, T stage (P>0.05), but correlated with lymphatic (P=0.049<0.05).The remaining indicators in adjacent mucosa (F1-F4, F5) was unrelated with age,location of tumor, lymph node metastasis, histological grade, T stage (P>0.05).All indexes in the sugical margins (Ma-Md) detected in patients were not relatedto age, location of tumor, lymph node metastasis, histological grade, T stage (P>0.05).Conclusions：1. Microsatellites D3S1300, D3S1234, D3S1568, D9S171, D9S1870, TP53tumorwas detected with over40%, which indicated that in laryngeal cancers, thesespecific sites can be considered for the detection of LOH in laryngeal cancer;2. There was clonality correlation exists between recurrent laryngeal cancer and primary tumor, which could be local recurrence. It suggested that negativemargin determination in histology could be imperfection as surgical resection;3. The relationship of other indicators between LOH and the age of laryngeal cancerpatients, location of tumor, lymph node metastasis, histological grade, T stage werenot certain yet,but TP53associated with pathological grading;4. Laryngeal cancer is multifactorial, multi-stage, multi-step process during itsdevelopment;5. There was field cancerization in most mucosa in laryngeal patients. The area wasdifferent in each patient. Importantly, a significant portion of patients had fieldcancerization in the surgical margins;6. D18S1110and D8S518may play a key role in the development of precancerouscell to tumor cells, further study should be performed;7. The LOH of TP53in adjacent mucosa can be used to assess whether the lymphnode metastasis in laryngeal cancer occurs. |
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