Novel defined N7-methylguanosine modification-related lncRNAs for predicting the prognosis of laryngeal squamous cell carcinoma

Objective:Through integrated bioinformatics analysis,the goal of this work was to find new,characterised N7-methylguanosine modification-related long non-coding RNAs(m7G-lncRNAs)that might be used to predict the prognosis of laryngeal squamous cell carcinoma(LSCC).Methods:The clinical data and LSCC gene expression data for the current investigation were initially retrieved from the TCGA database&sanitised.Then,using co-expression analysis of m7G-associated mRNAs&lncRNAs&differential expression analysis(DEA)among LSCC&normal sample categories,we discovered lncRNAs that were connected to m7G.The prognosis prediction model was built for the training category using univariate&multivariate COX regression&LASSO regression analyses,&the model’s efficacy was checked against the test category data.In addition,we conducted DEA of prognostic m7G-lncRNAs among LSCC&normal sample categories&compiled a list of co-expression networks&the structure of prognosis m7G-lncRNAs.To compare the prognoses for individuals with LSCC in the high-&low-risk categories in the prognosis prediction model,survival and risk assessments were also carried out.Finally,we created a nomogram to accurately forecast the outcomes of LSCC patients&created receiver operating characteristic(ROC)curves to assess the prognosis prediction model’s predictive capability.Results:Using co-expression network analysis&differential expression analysis,we discovered 774 m7G-lncRNAs and 551 DEm7G-lncRNAs,respectively.We then constructed a prognosis prediction model for six m7G-lncRNAs(FLG−AS1,RHOA−IT1,AC020913.3,AC027307.2,AC010973.2 and AC010789.1),identified 32 DEPm7G-lncRNAs,analyzed the correlation between 32 DEPm7G-lncRNAs and 13 DEPm7G-mRNAs,and performed survival analyses and risk analyses of the prognosis prediction model to assess the prognostic performance of LSCC patients.By displaying ROC curves and a nomogram,we finally checked the prognosis prediction model's accuracy.Conclusion:By creating novel predictive lncRNA signatures for clinical diagnosis&therapy,our findings will contribute to understanding the pathogenetic process of LSCC.

Expression of Hypoxia Inducible Factor-1α and Its Relationship to Apoptosis and Proliferation in Human Laryngeal Squamous Cell Carcinoma

Summary: To investigate the expression of hypoxia inducible factor-1 alpha (HIF-1α) and its relationship to apoptosis and proliferation in laryngeal squamous cell carcinoma (LSCC), immunohistochemical method was used to detect the expression of HIF-1α and PCNA. Tunnel technique was used to detect in situ cell apoptosis in LSCC. Our results showed that the expression of HIF-1α was related to the clinical stages of cancer and lymph node metastasis (P<0.05). The relationship between HIF-1α and PCNA was statistically significant (P<0.05) and no relationship was found between HIF-1α and apoptosis (P>0.05) It is concluded that HIF-1α plays a role in the carcinogenesis of laryngeal carcinoma and is correlated with proliferation, but bears no relationship with the apoptosis of tumor cells in LSCC.

Expression of Vascular Endothelial Growth Factor and Cyclooxygenase-2 in Laryngeal Squamous Cell Carcinoma and Its significance

n order to study the expressions of vascular endothelial growth factor （VEGF） and cyclooxygenase-2 （COX-2） in human laryngeal squamous cell carcinoma （LSCC） and its significance, the expression of VEGF mRNA and COX-2 mRNA in 62 cases of LSCC and 54 adjacent noncancerous laryngeal tissues and 9 normal human laryngeal mucous tissues was detected by using techniques of semi-quantitative RT-PCR. It was found that the expression level of VEGF and COX-2 mRNA was significantly increased in LSCC as compared with that in the normal human laryngeal mucous tissues （both P〈0. 01）, and the expression level of VEGF and COX-2 mRNA were significantly increased in stage Ⅲ＋ Ⅳtissues of LSCC as compared with the stage Ⅰ ＋ Ⅱ tissues of LSCC （P 〈0.01）. There was a high positive correlation between VEGF and COX-2 expression in LSCC （r= 0. 756,P〈0.01）. These data raise the possibility that VEGF and COX-2 may play key roles in the growth, invasion and metastasis of LSCC.

Neoadjuvant chemotherapy with pingyangmycin can inhibit the amplification and metastasis of laryngeal squamous cell carcinoma

Objective:To evaluate the short-term effects of neoadjuvant chemotherapy with pingyangmycin(PYM)in the treat-ment of laryngeal squamous cell carcinoma.Methods:24 cases of laryngeal squamous cell carcinoma were treated with PYM before the operation,and the surgeries were undergone within one week after neoadjuvant chemotherapy.PCNA,p53,Bcl-2 and CD44v6 were detected in the specimens of tumor,retreated tumor and normal tissue using immunohistochemical methods.Results:Apoptosis could be detected more often in specimens with tumor and retreated tumor after chemotherapy than that before.The expression of PCNA,p53,Bcl-2 and CD44v6 in tumor tissue after neoadjuvant chemotherapy with PYM was weaker than that before the chemotherapy.There was significant difference in the positive ratio of PCNA,p53,Bcl-2 and CD44v6 be-tween retreated tumor and tumor.Conclusion:After neoadjuvant chemotherapy with PYM,a large number of tumor cells died.The amplification and metastasis of tumor were suppressed by neoadjuvant chemotherapy with PYM.

Prognostic Value of Ki67 and VE6F Expression in Laryngeal Squamous Cell Carcinoma

OBJECTIVE To investigate the prognostic value of measuring Ki67 and VEGF expression in laryngeal squamous cell carcinoma (LSCC). METHODS The expression of Ki67 and VEGF in 32 LSCC tissues was studied by immunohistochemical staining. Of these cases, 5 recurred (recurrent group), 3 cases metastasized (metastatic group), 8 cases died (deceased group) and 24 cased survived (survival group) over a 3 year period of follow-up after their operation. RESULTS The expression of Ki67 and VEGF in the deceased group was higher compared to that in the survival group (P<0.01). Overexpression of Ki67 was found in the recurrent group and in the metastatic group (P< 0.05). VEGF expression was higher in the recurrent group than in the non recurrent group (P<0.05). With Cox-regression of multivariate analysis, Ki67 (RR:3.236; P=0.001), the clinical T stage (RR: 1.382; P=0.029) and metastasis in lymph nodes (RR:0.316; P=0.033) were shown to be independent prognostic factors for survival of LSCC patients. CONCLUSION Ki67 and VEGF expression is related to the prognosis of LSCC. Overexpression of the 2 markers indicated poor prognosis of the disease, a finding which might be helpful for the treatment of laryngocar-cinoma.

Prognostic and Predictive Protein Biomarkers in Laryngeal Squamous Cell Carcinoma—A Systematic Review

Background: Despite recent advances in clinical management of laryngeal squamous cell carcinoma (LSCC), the overall 5-year survival continues to be poor. Consequently, biomarkers of treatment response will need to be identified. Proteomic strategies are one way to attempt to identify such biomarkers. Methods: The Medline, Embase and Cochrane Library databases were systematically searched until 1st March 2014 using the terms “larynx”, “squamous cell carcinoma”, “proteomic”, and “biomarker”. Articles which met inclusion criteria were assessed for the type of biomarker investigated, the proteomic technique used, and whether any validation had been performed. Results: Six studies identified biomarkers, including UCRP, ceramides, uPA, MT1-MMP, stratifin, transferrin, albumin, S100 calcium-binding protein A9, stathmin, enolase, PLAU, IGFBP7, MMP14, THBS1, and transthyretin. Transferrin was the only biomarker to appear in more than one study. Conclusions: Our review identified several potential biomarkers of outcome in LSCC. Well designed studies will need to further validate their use in the future.

Changing the paradigm：the potential for targeted therapy in laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma(LSCC) remains a highly morbid and fatal disease. Historically, it has been a model example for organ preservation and treatment stratification paradigms. Unfortunately, survival for LSCC has stagnated over the past few decades. As the era of next-generation sequencing and personalized treatment for cancer approaches, LSCC may be an ideal disease for consideration of further treatment stratification and personalization. Here, we will discuss the important history of LSCC as a model system for organ preservation, unique and potentially targetable genetic signatures of LSCC, and methods for bringing stratified, personalized treatment strategies to the 21^(st) century.

Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by In Situ Hybridization

Objective：Laryngeal squamous cell carcinoma（LSCC） is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol abuse.Human papilloma virus（HPV） is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including LSCC.This retrospective study was to investigate the prevalence of HPV infection in patients with LSCC.Methods：In situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6 mRNA.Results：The positive rate of HPV16 E6 mRNA was 36.36%（36/99） in laryngeal squamous cell carcinoma（LSCC）,whereas only 3 of 50（6%） specimens of the normal laryngeal mucosa as a control group showed positive results（P0.05）.Additionally,there was no corelation between HPV16 and age,gender,clinical stage,nodal status and tumor site（P0.05）.Conclusion：The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types（especially type 16） were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell carcinoma.However,their clinical value remains to be further investigated.

A CORRELATIVE STUDY OF Ki67 AND VASCULAR ENDOTHELIAL GROWTH FACTOR AND THEIR VALUE IN LARYNGEAL SQUAMOUS CELL CARCINOMA

Objective： To study the correlation between Ki67 and vascular endothelial growth factor（VEGF） and the significance of their expression in laryngeal squamous cell carcinoma（LSCC）. Methods： The expressions of Ki67 and VEGF in 40 cases of LSCC and 5 cases of normal laryngeal mucosa were examined by immunohistochemical staining. Results： The expression levels of Ki67 and VEGF in LSCC tissue were higher than in normal laryngeal mucosa （Ki67： P〈0.001, VEGF： P〈0.001）. The two indexes＇ levels in patients of different age or different sex had no significant difference （P〉0.05）. They were higher in LSCC with metastasis of lymph nodes than in patients without metastasis （Ki67： P=0.034, VEGF： P=0.006）. The expressions of the two genes elevated correspondingly along with the development of LSCC T stage （P〈0.05）. In addition, correlation analysis indicated that the expression of Ki67 had a positive correlation with VEGF in LSCC（r=0.823, P〈0.01）. Conclusion： Ki67 and VEGF are objective indexes for the biological behavior of LSCC, and they might be helpful to the diagnosis, treatment and prognosis of laryngocarcinoma.

Prognostic value of body mass index before treatment for laryngeal squamous cell carcinoma

Objective: Patients with head and neck cancer often suffer from malnutrition. This study aims to investigate the influence of body mass index(BMI) on the prognosis of laryngeal squamous cell carcinoma(LSCC).Methods: A total of 473 patients with LSCC initially treated at Sun Yat-sen University Cancer Center between January 2005 and July 2009 were retrospectively reviewed. Survival analysis was performed by the Kaplan-Meier method and Cox regression model.Results: Low BMI before treatment was significantly associated with poor overall survival in patients with LSCC(P<0.001). BMI was an independent prognostic factor for patients with LSCC.Conclusion: Leanness before treatment was associated with poor prognosis in patients with LSCC. Good nutritional status is favorable to improve survival in patients with LSCC.

LSM6 promotes cell proliferation and migration regulated by HMGB1 in laryngeal squamous cell carcinoma

Elevated levels of high mobility group protein B1(HMGB1)play a significant role in the pathogenesis of many diseases,but is particularly important for the formation of malignant tumors.Nonetheless,the function of HMGB1 and the underlying mechanism of laryngeal squamous cell carcinoma(LSCC)remain incompletely understood,causing uncertainty.Here we found immunohistochemistry from 97 LSCC tissues showed HMGB1 was upreg-ulated,which was associated with poor differentiation.HMGB1 knockdown could significantly inhibit wound closure and colony formation.The full-genome gene expression microarray was performed to investigate the mechanism.After knockdown of HMGB1 by siRNA,among the expressed differential genes,10 genes were ran-domly selected for validation.Then,shRNA lentivirus targeting these genes were constructed to explore their role in LSCC by cell proliferation assay.LSM6 downregulation was dramatically promoted by HMGB1 knockdown,resulting in higher expression in LSCC tissues.Furthermore,downregulation of LSM6 could significantly suppress cell proliferation,migration and colony formation.This study indicated that HMGB1 promoted LSCC cell malig-nant phenotypes through regulation of LSM6.We anticipate that HMGB1-LSM6 could be a putative therapeutic target for LSCC.

Relation between the Expression of K-ras in Hep-2 Cells and Development of Laryngeal Carcinoma~*

Objective： To investigate the expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and its significance for establishing a solid foundation for further study of the relationship between human laryngeal squamous cell carcinoma and K-ras gene point mutations. Methods： The expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and human pancreatic carcinoma cell lines （MIAPaCa-2） was detected by using RT-PCR. Results： The expression of K-ras mRNA in Hep-2 and MIAPaCa-2 was strong and positive. Conclusion： The expression of K-ras mRNA in human laryngeal squamous cell carcinoma cell lines （Hep-2） is positive. Development of laryngeal carcinoma might be related to the activation of K-ras gene point mutation.

Effect and mechanism of miR-34a on proliferation, apoptosis and invasion of laryngeal carcinoma cells

Objective: To discuss the effect and mechanism of miR-34 a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells. Methods: The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34 a mimics and miR-34 a NC. The MTT, colony-forming assay, Hoechst staining and Annexin V-PI double staining flow cytometry were employed to detect the effect of miR-34 a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34 a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RTPCR assay to defect the effect of miR-34 a mimics on the expression of survivin and Ki-67 m RNA in laryngeal squamous carcinoma Hep2 cells. Results: Compared with miR-34 a NC group, the cell viability in miR-34 mimics group was significantly decreased(P<0.01), the cell apoptosis rate was significantly increased(P<0.01), the abilities of cell migration and invasion were significantly reduced(P<0.01) and the expression of survivin and Ki-67 m RNA was significantly decreased(P<0.01). Conclusions: The increased expression of miR-34 a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.

PIK3CA mutation in Chinese patients with lung squamous cell carcinoma

Objective： To investigate PIK3CA mutation in Chinese patients with lung squamous cell carcinoma （LSCC） and explore their relationship with clinicopathological profiles. Methods： Tumor samples from 123 cases of LSCC were included in this study. PIK3CA mutations in exon 9 and 20 were screened by pyrosequencing and confirmed by clone sequencing or amplification refractory mutation system （ARMS）. Denaturing performance liquid chromatography （DHPLC） was employed for evaluation of EGFR mutation in exon 19, 21 and KRAS mutation. Results： PIK3CA mutations were found in 3 （2.4%） patients. The mutation type included E545K, E452Q and H1047R. Of these three patients, one coupled with EGFR mutation, and the other two coupled with PIK3CA amplification. All the three patients shared the same clinicopathologic characteristics： male, less than 60 years old, had smoke history, stage III and carried wild-type KRAS. Conclusions： The frequency of PIK3CA mutation is low in Chinese patients with LSCC. The mutational status of PIK3CA is not mutually exclusive to EGFR mutation.

The Prognostic Value of Pathological and Molecular Margins Marked by p53 and eIF4E in Laryngeal Carcinoma

Objective: To study the prognostic value of the pathological margin and molecular margin marked by eIF4E and P53 protein in laryngeal carcinoma. Methods: The prognostic value of pathological and molecular margin was studied in 253 cases and 67 cases respectively, the latter were pathological negative margin chosen from the former. Immunohistochemisty was used to detect the expression of eIF4E and p53 proteins. Results: The rate of pathological, p53 and eIF4E positive margins was 20.2%, 19.4% and 32.8% respectively. The recurrent rate of those with positive margins was higher than that of negative margins, which including pathological margin (70.6% vs 35.1%, P =0.0000), p53 margin (69.2% vs 33.3%, P =0.018) and eIF4E margin (63.6% vs 28.9%, P =0.018); The survival rate of those with negative margins was higher than those with positive margins, including pathological margin (the 5-year cumulative survival rate was 37.52% and 64.37% respectively, P =0.0023), p53 margin (the 5-year cumulative survival rate was 24.62% and 75.69% respectively, P =0.0012) and eIF4E margin (the 5-year cumulative survival rate was 43.31% and 77.52% respectively, P =0.0006). Conclusion: The prognosis of those with both pathological and molecular positive margins was worse than that of the negative margins; Both the eIF4E and p53 were useful markers to pick out the poor prognostic patients from those with pathological negative margin, and the former seemed to be more potential.

Clinical significance of tumor-associated macrophage infiltration in supraglottic laryngeal carcinoma

Tumor-associated macrophages(TAMs) can elicit contrasting effects on tumor progression,depending on different tumor microenvironment.This study aimed to explore the correlation between TAM infiltration and clinicopathologic characteristics,metastasis,and prognosis of supraglottic laryngeal carcinoma.TAMs in intratumoral and peritumoral regions of 84 specimens of supraglottic laryngeal carcinoma tissues were detected by immunohistochemical staining with monoclonal CD68 antibody.The density of peritumoral CD68+ TAMs in recurrence cases(9/11) and in dead cases(17/23) were significantly higher than those in non-recurrence cases(33/73) and in survival cases(25/61),with significant differences(P = 0.024 and 0.007,respectively).The Kaplan-Meier survival analysis showed a significant relationship between the infiltration of both intratumoral and peritumoral CD68 + TAMs and the overall survival of patients.The 5year survival rate was significantly lower in the group with a high density of intratumoral CD68+ TAMs than in the group with a low density(39.6% vs.82.5%,P < 0.05).Similarly,the 5-year survival rate was significantly lower in the group with a high density of peritumoral CD68+ TAMs than in the group with a low density(50.6% vs.73.1%,P < 0.05).Cox regression analysis revealed that T classification,distant metastasis,and intratumoral or peritumoral CD68 + TAMs were independent factors for disease-free survival,whereas T classification and intratumoral CD68 + TAMs were independent factors for overall survival.The results indicate that TAM infiltration in supraglottic laryngeal carcinoma can be used to predict metastasis and prognosis and is an independent factor for prognosis.

Studies on the interrelationship of Chinese laryngeal carcinoma and human papillomavirus

The studies described herein emphasize HPVDNA detection by applying consensus primers and multiple primers of modified polymerase chain reaction (PCR) to 124 cases of fresh tissue samples with different lesions of larynx. The consensus primers used were able to detect 9 types of HPVsDNAs ( HPV 6 , 11 , 16 , 18, 31 , 33,35, 42, and 583 , from which the positive cases were picked out for further identification of their genometypes of HPV-DNA by using multiple primers PCR. The results areas follows : (1)In the group of laryngeal carcinoma,the total positive rate of HPV infection was 49. 1 %(28/57) : 15. 8%(9/57)for HPV18, 12. 3%(7/57)for HPV16,5. 3 %(3/57) dual infection for HPV16 and HPV18 , 3. 5%(2/57) for mixed infection of HPV6/11 and HPV18 , and 12. 3%(7/57)for the other types. (2) In the cervical metastatic lymphnode group, 3 of the 14 cases (21. 4%) of cervical metastatic lymphnode showed positive HPV, among which there was 1 case of HPV16 infection , 1 case of HPV18 infection ,and 1 case of HPV16 and HPV18 dual infection, resulting in a rate at 7. 1% for the respective cases immediately above. (3) In the pecarcinomatous lesion group ,the positive rate of HPV infection tmixed infection of HPV6/11 and HPV18) was 11. 1 %(1/9). (4)In the vocal cord polypus group , the rate of positive reaction (HPV6/11) was 7.1% (1/14). (5) In the nomal laryngeal tissue group , 15 cases of normal laryngeal tissue adjacent to the carcinoma and 15 cases of normal laryngeal tissue opposite to the carcinoma were HPV-DNA negative.The results showed that the occurrence and development of laryngeal carcinoma were closely related to HPV infection. The distribution of genometypes of HPV varied in different lesions of larynx. The carcinogenic action of HPV in laryngeal carcinoma is also discussed in this paper.

Overexpression of interleukin-17 in tumor-associated macrophages is correlated with the differentiation and angiogenesis of laryngeal squamous cell carcinoma

Background Interleukin-l7 （IL-17）, which exerts strong pro-inflammatory effects, has emerged as an important mediator in inflammation-associated cancer. The aim of this study was to clarify the relationship between IL-17 and tumor associated macrophages （TAMs）, and the correlation of the microvessel density in the development of laryngeal squamous cell carcinoma （LSCC）.Methods Histopathological observations and immunohistochemistry staining for IL-17, CD68, and CD34 were performed on 72 specimens （32 cases of LSCC, 20 cases of adjacent tissues of carcinoma as controls, and 20 cases of chronic hypertrophic laryngitis）. Double immunohistochemical staining was done to determine which cells expressed IL-17. Real-time quantitative PCR determined the mRNA expression of IL-17. ELISA was used to detect the expression of the serum level of IL-17 in the three groups.Results The inflammation response had increased in LSCC. Overexpression of IL-17 and CD68 protein were seen in LSCC （P 〈0.01）. The expression of IL-17 was different between well and poorly differentiated LSCC （P 〈0.01）. The IL-17 expressing cells were mainly located in macrophages （CD68＋/IL17＋） as demonstrated by double immunohistochemical staining. IL-17 expression significantly correlated with high microvessel density （CD34＋） in LSCC （P 〈0.05）. Relatively higher mRNA expression levels of IL-17 were seen in LSCC compared to the controls （P 〈0.05）. The serum expression of IL-17 was similar among the three groups （P 〉0.05）.Conclusion IL-17 was expressed by TAMs, and IL-17 may significantly correlate to the differentiation and angiogenesis in the development of LSCC.

Vasculogenic mimicry is a key prognostic factor for laryngeal squamous cell carcinoma： a new pattern of blood supply

Background Recurrence and local lymph node metastasis affected the prognosis of patients with laryngeal cancer. The aim of this study was to analyze clinical and pathological significance of vasculogenic mimicry （VM） in laryngeal squamous cell carcinoma （LSCC） and evaluate its contribution to prognosis. Methods Data of 168 cases of LSCC were reviewed retrospectively to reveal clinical pathology and prognostic significance of VM. CD31 and periodic acid-Schiff double staining was used to identify VM. Results VM in LSCC contributed to lymph node metastasis （P=0.003） and clinical progression. VM correlated to histopathology grade （P=0.001） of LSCC. VM was an adverse prognostic factor for both disease-specific survival （P=0.039） and metastasis-free survival （P=0.042） by univariate survival analyses. And it was an independent prognostic factor for only disease-specific survival （P=0.003） by multivariate survival analyses. Conclusions VM existed in LSCC. LSCC with VM has more Dotential to invasion and metastasis.

Detailed deletion mapping of loss of heterozygosity on 9p13-23 in laryngeal squamous cell carcinoma by microsatellite analysis

Background This study was designed to investigate the hot spots of microsatellite loss of heterozygosity (LOH) on 9p13-23 in laryngeal squamous cell carcinoma and to find out the correlation between the incidence of microsatellite LOH and the clinicopathological parameters Methods Tumor tissues were obtained from paraffin embedded sections with microdissection Genomic DNA was extracted from tumor tissues and peripheral blood lymphocytes with the phenol-chloroform Polymerase chain reaction (PCR) amplification and denaturing gel electrophoresis were carried out in a set of 42 squamous cell carcinoma (SCC) of larynx and corresponding peripheral blood lymphocytes using 13 highly polymorphic microsatellite markers on 9p13-23 The correlation was analyzed between microsatellite LOH at the high frequency on 9p13-23 and clinicopathological parameters in the patients with squamous cell carcinoma of larynx KH*2/5DResults Of the 42 laryngeal cancers, 41 (97 6%) showed LOH in at least one of the microsatellite markers tested on 9p13-23 The most frequently deleted marker was D9S162 in 17 of the 19 (89 5%) informative samples The marker D9S171, which is located on 9p21, had LOH detected in 12 of the 15 informative cases (80 0%) LOH at the D9S1748 marker (closest to the p16 gene locus) was detected in 18 of the 36 informative cases (50 0%) Allelic deletion mapping revealed two minimal regions of LOH encompassing markers D9S161-D9S171 on 9p21 and IFNA-D9S162 on 9p22-23 Multiple LOH (≥4) on 9p21-23 was found more frequently in the patients under 60 years, with supraglottic SCC or cervical lymph node metastasis than those over 60 years, with glottic SCC or without cervical lymph node metastasis ( P <0 01 or 0 01, 0 05, respectively) On the contrary, there was no correlation between T stages or pathologic classification and the frequency of LOH on 9p21-23 in 42 SCC of Larynx Conclusions These findings imply the presence of at least two putative tumor suppressor genes on 9p13-23 in laryngeal SCC Multiple genetic alterations are probably implicated in supraglottic SCC with cervical lymph node metastasis in younger patients