Aberrant P16 Expression Research Articles

Uterine Inflammatory Myofibroblastic Tumors: p16 as a Surrogate for CDKN2A Deletion and Predictor of Aggressive Behavior.

Uterine inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal neoplasms of uncertain malignant potential. Aside from the recently described risk stratification score, which has not been validated by other studies, and rare reports of aberrant p16 expression in malignant tumors, there are no criteria to reliably predict behavior. Herein, we evaluated the clinicopathologic features and p16 expression patterns in 30 IMTs, with genomic profiling performed in a subset (13 malignant, 3 benign). Fifteen patients had malignant IMTs, defined by extrauterine disease at diagnosis (n=5) or recurrence (n=10; median: 24 mo). Patients ranged from 8 to 65 (median: 51) years and tumors from 6 to 22 (median: 12.5) cm. In primary tumors (n=13), infiltrative borders were noted in 10, moderate/severe cytologic atypia in 9, tumor cell necrosis in 7, and lymphovascular invasion in 6, while mitoses ranged from 0 to 21 (median: 7) per 10 high-power fields. In contrast, 15 patients with benign IMTs ranged from 28 to 65 (median: 44) years, with follow-up of 18 to 114 (median: 41) months. Tumors ranged from 1.9 to 8.5 (median: 5.5) cm, 2 demonstrated infiltrative borders, and 1 had moderate cytologic atypia. No other high-risk histologic features were observed. Application of the previously described clinicopathologic risk stratification score in all primary IMTs with complete data (n=18) classified 8 as high-risk (all malignant), 8 as intermediate-risk (3 malignant, 5 benign), and 2 as low-risk (benign). p16 was aberrant in all malignant IMTs, with <1% expression noted in 10, overexpression (>90%) in 4, and subclonal loss in 1; all benign tumors had patchy staining (20% to 80%; median 50%). Molecular analysis detected CDKN2A deletions in 8 of 9 tumors with <1% p16 expression, while the other harbored a TERT promoter mutation. TERT promoter mutations were also identified in 2 of 3 IMTs with p16 overexpression. Neither of these alterations was detected in the 3 sequenced benign IMTs. Thus, we recommend performing p16 on all uterine IMTs, which, combined with the risk stratification score, is a promising and cost-effective tool for predicting CDKN2A status and outcome in these patients. It may be particularly useful for tumors with incomplete information for risk stratification (ie, morcellated tumors) and for further stratifying intermediate-risk IMTs when sequencing is unavailable.

P16 and P21 are involved in the pathogenesis of endometrial thinning: A cross-sectional study.

P16 plays a role in the negative regulation of cell proliferation, regulating cell apoptosis to control the growth of tumor cells. P21 is a nuclear protein that suppresses DNA synthesis and inhibits cell division. This study aimed to examine the expression and roles of P16 and P21 in endometrial thinning. Thirty cases of endometrial biopsy diagnosed as endometrial thinning were assessed by p16 and p21 immunohistochemistry from March 2014 to August 2020 in Huazhong University of Science and Technology Union Shenzhen Hospital. Another thirty cases of normal endometrium in the same period were assessed as controls. The specimens underwent histological analysis, and P16 and P21 were assessed by immunohistochemistry. There were no statistically significant differences in age, menstrual cycle, BMI, sex hormone levels, gravidity and parity between the two groups (all P > .05). In the endometrial thinning group, P16 was expressed in the endometrial adenoid nucleus, cytolymph and interstitial cell nucleus. In the normal group, P16 was mainly expressed in the endometrial adenoid nucleus, with some P16 signals detected in the endometrial interstitial nucleus. P21 expression was mainly detected in the endometrial adenoid nucleus. P16 and P21 amounts in endometrial thinning cases were significantly lower than those of the normal endometrial group. There was no correlation between p16 and p21 amounts. This study revealed aberrant expression of P16 and P21 in the endometrium might be due to a compensatory effect of the thin endometrium to increase cell proliferation and suppress cell apoptosis. However, the pathological roles of P16 and P21 in endometrial thinning and the contribution of cell senescence deserve further investigation.

Diagnostic Accuracy of p16 Expression in Differentiating Cervical Intraepithelial Neoplasia from Invasive Squamous Cell Carcinoma: A Descriptive Analysis at a Tertiary Care Hospital of Bangladesh

Background: Cervical cancer is one of the most common cancers that affect women worldwide. It is well established thathigh-risk human papillomavirus (HR-HPV) is the prime risk factor for the development of cervical cancer. Early and accuratediagnosis of cervical neoplasia is of utmost significance for prolongation of patient survival. A panel of immunomarkers hasbeen developed and tested to overcome the limitations to histopathological diagnosis. Among them p16 is one of the commonlyused immunomarkers now-a-days. In recent years, routine haematoxylin and eosin (H &amp; E) stain coupled with p16immunohistochemistry (IHC) have a major impact in the field of uterine cervical pathology and cancer screening. Objectives: The present study was aimed to determine the p16 scores and its association with histological types, and grades ofISCC including diagnostic accuracy in differentiating ISCC from CIN lesions. Materials and Methods: This descriptive type of cross-sectional study was conducted in the Department of Pathology,Rajshahi Medical College (RMC), Rajshahi, in collaboration with the Department of Pathology, Bangabandhu Sheikh MujibMedical University (BSMMU), Dhaka, during a period of two years from January 2019 to December 2020 by using routine H&amp; E staining and p16 IHC analysis to evaluate the expression of p16 in samples of cervical biopsies from 51 patients withhistopathologically confirmed diagnosed cases of CIN and ISCC, considering the percentage of p16 positive cells and thereaction intensity. Results: This study revealed that the aberrant expression of p16 increased from LSIL to ISCC, thus emphasizing its usefulnessas an adjunct marker for predicting risk of developing cervical cancer in the test patients. Most of the cases of ISCC in this studyexpressed high level of p16, while only one patient with LSIL failed to express the marker. Notably, 90% of LSIL cases werelow to moderate p16 positive, 100% of HSIL cases were moderate to high p16 positive, while most of the ISCC cases (81%) hadhigh p16 expression. Among p16 positive cases, this is an attempt to verify direct association between lesion severity andreaction intensity. The frequency of positive cells and the reaction intensity were statistically significant (P&lt;0.001) whencompared among different histologic types. Most importantly, the present results clearly demonstrated that p16 IHC wascapable of differentiating ISCC from CIN cases. Conclusion: In addition to routine H &amp; E staining and histopathological diagnosis of cervical lesions, p16 immunomarkercould be used for risk assessment of histologically detected lesions and for distinguishing between ISCC and CIN specimenswhich, in turn, could help predict the progression of cervical lesions and thus monitor the screening of cervical cancer in thecommunity.

Dormant tumor cells interact with memory CD8+ T cells in RET transgenic mouse melanoma model.

Melanoma is an aggressive form of skin-cancer. Melanoma cells are characterized by their plasticity, resulting in therapy resistance. Using RET transgenic mouse melanoma model, we characterized dormant tumor cells accumulated in the bone marrow (BM) and investigated their interaction with effector memory CD8+ T cells. We found that cells expressing melanoma-associated antigen tyrosinase related protein (TRP)-2 and stemness marker CD133 represented less than 1.5% of all melanoma cells in primary skin lesions and metastatic lymph nodes. The majority of these cells were negative for the proliferation marker Ki67. In the BM, CD133+TRP-2+ melanoma cells displayed an aberrant expression of p16, p27, Ki67 and PCNA proteins, suggesting their dormant phenotype. Moreover, these cells were characterized by an elevated expression of various molecules characterized stemness, metastatic, angiogenic and immunosuppressive properties such as CD271, CD34, HIF-1α, CXCR3, CXCR4, VEGR2, PD-L1, CTLA-4, CD39 and CCR4 as compared to their CD133- counterparts. Disseminated BM dormant TRP-2+ tumor cells were found to be co-localized with memory CD8+ T cells. Our data suggest that these dormant melanoma cells in the BM could play an important role in the maintenance of memory T cells in the BM.

Histologic and immunohistochemical differences between hereditary and sporadic diffuse gastric carcinoma

We aimed to identify histopathologic features unique in hereditary diffuse gastric cancer (HDGC) by comparing with its sporadic counterpart (SDGC). 11 patients with confirmed CDH1 mutation who were found to have HDGC in a prophylactic total gastrectomy were collected. Median age of HDGC patients was 39 years (range 24-57). All HDGC cases had intramucosal signet ring cell carcinoma. Twenty-three invasive tumor foci from 7 patients with HDGC were available for ancillary studies, and we evaluated each focus separately. Almost all foci (20/23) showed two distinct tumor cell populations, namely, large signet ring cells and small signet ring cells. The large cells were located just beneath the surface epithelium and were positive for mucicarmine and pCEA, while the small cells were found in the deeper lamina propria and were mostly negative for mucicarmine and pCEA. A subset of small cells (6 foci from two resected stomachs) showed poorly differentiated morphology with p16 positivity. All other tumor cells with well-differentiated signet ring cell morphology were negative for p16. In contrast, 18 of 20 SDGCs were positive for p16. In addition, all HDGCs were negative for CDX2, while 19 of 20 SDGCs were positive. We propose that there are three distinct tumor cell populations in HDGC: well-differentiated large cells, well-differentiated small cells, and poorly differentiated small cells, and that the latter group with aberrant p16 expression may represents a more aggressive phenotype. The absence of CDX2 in HDGC suggests that it may develop along a carcinogenetic pathway different from that of SDGC.

Increased phosphorylation on residue S795 of the retinoblastoma protein in esophageal adenocarcinoma.

Due to its increasing incidence and relatively poor prognosis, esophageal adenocarcinoma (EAC) is becoming a significant health problem. Elucidating the mechanisms underlying EAC development is of great importance to improve upon current conventional treatment strategies. Insight into phosphorylation has proven to be useful for the development of diagnostic and molecular treatment strategies in cancer. A pathway largely dependent on phosphorylation and frequently deregulated in cancer is the cell cycle regulating p16-retinoblastoma (Rb) pathway. We investigated kinase activity, specifically phosphorylation within the p16-Rb pathway, in EAC. A high-throughput peptide tyrosine kinase array containing short peptides representing 100 proteins with known phosphorylation sites, was used to assess phosphorylation activity in EAC. Also, specific phosphorylation changes of the cell cycle protein Rb and its upstream regulator P16 were validated through immunoblotting in EAC and normal esophageal cells and tissues. Phosphorylation activity was higher in EAC tissues as compared to normal squamous esophageal tissues. A majority of the proteins significantly higher phosphorylated in EAC were found to be involved in cell structure maintenance and immunity. Validation of Rb phosphorylation in EAC biopsy specimens and cell lines showed hyper phosphorylation of Rb associated with aberrant P16 expression in the cancer tissues. The specific Rb (S795) residue was significantly higher phosphorylated in EAC compared to normal esophageal tissue (Wilcoxon paired rank test, p=0.004). Investigation of Rb (S795) phosphorylation may indicate targets for intervention and give more molecular insight in EAC.

From laryngeal epithelial precursor lesions to squamous carcinoma of the larynx: the role of cell cycle proteins and β-catenin

Novel markers to accurately predict the risk of malignant transformation in laryngeal epithelial precursor lesions (EPL) are needed. We tried to identify some molecular alterations occurring in laryngeal tumorigenesis. In this study, 60 paraffin-embedded EPL and 17 metachronous invasive carcinomas were immunostained for markers associated with proliferation (Ki67), cell cycle control (p53, p21, p16, p27, cyclin D1), and cell adhesion and invasion (laminin and β-catenin). Aberrant expression of p16 and p53 and positivity at cytoplasm for β-catenin and cyclin D1 were detected significantly in EPL with progression to invasive laryngeal carcinoma. All cases with basal and suprabasal reactivity of p53 showed β-catenin overexpression. We found that β-catenin protein expression increased significantly with the grade of dysplasia. This is one of the studies with the largest number of laryngeal EPL and invasive carcinoma studied sequentially. Our data confirm the role of some cell cycle regulatory proteins in the development of laryngeal carcinoma. Cytoplasmic retention of β-catenin in EPL seems to be related with more aggressive biological behavior. Combined increased p53 and cytoplasmic β-catenin protein expression could be biologically important in laryngeal tumorigenesis. Further research is required to clarify the involvement of β-catenin in the mechanism associated with malignant transformation in laryngeal tissues.

Promoter histone H3 lysine 9 di-methylation is associated with DNA methylation and aberrant expression of p16 in gastric cancer cells

In the course of gastric cancer development, gene silencing by DNA hypermethylation is an important mechanism. While DNA methylation often co-exists with histone modifications to regulate gene expression, the function of histone modifications in gene silencing in gastric cancer has not been evaluated in detail. p16, a well-known tumor suppressor gene, is frequently silenced in DNA hypermethylation manner in gastric cancer. Accordingly, we chose p16 to clarify whether there is a correlation among histone H3 lysine 9 (H3-K9) di-methylation, H3-K9 acetylation, DNA methylation and p16 expression in human gastric cancer. Three gastric cancer cells, MKN-45, SGC-7901 and BGC-823, were treated with 5-aza-2'-deoxycytidine (5-Aza-dC) and/or trichostatin A (TSA). We investigated p16 promoter DNA methylation status, p16 mRNA levels, regional and global levels of di-methyl-H3-K9 and acetyl-H3-K9 in four groups: i) 5-Aza-dC, ii) TSA, iii) the combination of 5-Aza-dC and TSA and iv) control group with no treatments. p16 silencing is characterized by DNA hypermethylation, H3-K9 hypoacetylation and H3-K9 hypermethylation at the promoter region. Treatment with TSA, increased H3-K9 acetylation at the hypermethylated promoter, but did not affect H3-K9 di-methylation or p16 expression. By contrast, treatment with 5-Aza-dC, reduced H3-K9 di-methylation, increased H3-K9 acetylation at the hypermethylated promoter and reactivated the expression of p16. Combined treatment restored the expression of p16 synergistically. In addition, 5-Aza-dC and the combined treatment did not result in global alteration of H3-K9 di-methylation. These results suggest that H3-K9 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence p16 in gastric cancer. The decreased H3-K9 di-methylation correlates with DNA demethylation and reactivation of p16. H3-K9 di-methylation as well as DNA methylation related to p16 silencing is limited to the promoter region. In addition to its effect on DNA methylation, 5-Aza-dC can act at histone modification levels to reactivate p16 expression in region-specific and DNA methylation-dependent manner.

Prognostic prediction of the immunohistochemical expression of p53 and p16 in resected non-small cell lung cancer.

p53 and p16(INK4) are the common and important tumor suppressor genes. Aberrant expression of p53 or p16 protein has been reported in various malignancies including lung cancer. Our aim was to investigate the association of p53 and p16 expression in resected non-small cell lung carcinoma (NSCLC) and evaluated their correlation with clinocopathologic features and survival. p16 and p53 expression were detected by immunohistochemical analysis of 90 paraffin specimens of resected NSCLC, including 35 squamous cell carcinoma, 47 adenocarcinoma, and eight large cell carcinoma, between stages I and IV. The immunohistochemical study was performed using the labeled streptavidine-biotin method with anti-p53 and anti-p16 monoclonal antibodies. Fifty-two (57.8%) and 36 (40%) of 90 patients revealed aberrant immunostaining for p53 (p53+) and p16 (p16+), respectively. While 19 cases (21.1%) showed abnormal immunoreactivity for both p16 and p53. (p53+/p16+). There was no correlation of p53 or p16 expression with the clinicopathologic features. The Kaplan-Meier survival analysis demonstrated that patients with p16+, p53+, late stages, and nodal or distal metastasis had poor survival status (P = 0.006, 0.013, <0.001, <0.001 and 0.018, respectively). Further analysis demonstrated that p53 status was a significant prognostic factor in stage I NSCLCs (P < 0.001), and p16 status in stage I and II NSCLCs (P < 0.001, P = 0.003, respectively). Furthermore, patients whose tumors were both p53 and p16 aberrant expression had worse outcome compared with those whose tumors were both normal expression of p53 and p16 (5-year survival rate: 5 vs. 76%, P < 0.001). In Cox's regression model, the aberrant expression of p16, p53, advanced stages and combined aberrant expression of p53/p16 survived for a significant shorter period. The results indicated that aberrant expression of p16 and p53 are significant and independent, predictable prognostic factors for resected NSCLC, especially in early stage of NSCLCs. The worst prognosis was seen in patients whose tumors had both aberrant expression of p53 and p16. Further prospective trials may be aimed at confirming and validating these results.

Aberrant expression of p16 and RB protein in eccrine porocarcinoma.

Eccrine porocarcinoma is a rare malignant tumor of the sweat gland and very little is known about its etiology and molecular basis. To investigate the expression of p16 and retinoblastoma (RB) protein and their relationship, an immunohistochemical method was performed on nine eccrine porocarcinomas and five eccrine poromas. Furthermore, one case of eccrine porocarcinoma was analyzed for p16 gene mutation. A striking inverse correlation between p16 and RB expression was noted in all of the eccrine porocarcinomas and poromas. Strong immunoreactivity for p16 protein was observed in both nuclei and cytoplasm of the tumor cells in eight out of nine cases of eccrine porocarcinomas, while RB expression was negative in these cases. Conversely, one case of eccrine porocarcinoma did not show immunoreactivity for p16 protein, whereas RB protein was positive in the scattered nuclei. On the other hand, immunostaining of p16 was negative in all cases of five poromas, whereas RB-positive nuclei were sparse. No p16 gene mutation was detected in the investigated eccrine porocarcinoma case. These results indicate that detectable p16 protein and loss of RB protein are common occurrences in eccrine porocarcinoma lesions. Moreover, overexpression of p16 protein may be an additional, simple and useful diagnostic marker for eccrine porocarcinoma on routine laboratory screening.

Malignant melanoma.

The rapidly developing fields of melanoma research are revolutionizing the current concepts on melanoma etiology and pathogenesis and are introducing newer diagnostic techniques and potential therapeutic approaches. To present the most current concepts on the etiology and pathogenesis of melanoma and to introduce the recent diagnostic techniques and the potential therapeutic approaches. Data sources were reports on melanoma published in the English language literature and observations made using specimens available at Harvard University, Johns Hopkins Medical Center, Albany Medical College, Loyola University Medical Center, and University of Tennessee Health Science Center. Studies on melanoma containing chromosomal or genetic evaluation were selected for further analysis. Current clinical and pathologic categories with the reported genetic abnormalities were related to the latest information on pigment biology. The data extracted were used to develop a conceptual framework on the pathogenesis of melanoma; the generated model was then evaluated and used to suggest potential therapeutic approaches. (1) Melanoma is not genetically homogeneous, and the existing differences between the pathologic categories, particularly in areas such as type of growth phase (radial vs vertical growth), total vertical dimension, ulceration of primary tumor, and metastatic process, have profound prognostic and therapeutic implications. (2) Chromosomal aberrations and gene mutations are found in sporadic and familial melanomas; among the most important are those affecting the 9p21, which contains the p16 locus, a site known to be critical for normal progression of the cell cycle. Aberrant p16 expression is associated with more aggressive behavior. (3) Melanoma cells possess a remarkable repertoire of biosynthetic capacities represented by the production of hormones, growth factors, and their receptors that may sustain and accelerate tumor development and progression. For example, expression of the tumoral products alpha-melanocyte-stimulating hormone and adrenocorticotropic hormone is regulated in vitro by ultraviolet light, a known carcinogen. (4) Melanomas differ from other tumors in their intrinsic capability to express melanogenic enzymes with the corresponding structural proteins to actually synthesize melanin. Melanogenesis-related proteins are rapidly entering the clinical arena, being used not only as diagnostic markers, but also as potential targets for melanoma therapy.

Malignant melanoma: An update

○ Context.-The rapidly developing fields of melanoma research are revolutionizing the current concepts on melanoma etiology and pathogenesis and are introducing newer diagnostic techniques and potential therapeutic approaches. Objectives.-To present the most current concepts on the etiology and pathogenesis of melanoma and to introduce the recent diagnostic techniques and the potential therapeutic approaches. Methods.-Data sources were reports on melanoma published in the English language literature and observations made using specimens available at Harvard University, Johns Hopkins Medical Center, Albany Medical College, Loyola University Medical Center, and University of Tennessee Health Science Center. Results.-Studies on melanoma containing chromosomal or genetic evaluation were selected for further analysis. Current clinical and pathologic categories with the reported genetic abnormalities were related to the latest information on pigment biology. The data extracted were used to develop a conceptual framework on the pathogenesis of melanoma; the generated model was then evaluated and used to suggest potential therapeutic approaches. Conclusions.-(1) Melanoma is not genetically homogenous, and the existing differences between the pathologic categories, particularly in areas such as type of growth phase (radial vs vertical growth), total vertical dimension, ulceration of primary tumor, and metastatic process, have profound prognostic and therapeutic implications. (2) Chromosomal aberrations and gene mutations are found in sporadic and familial melanomas; among the most important are those affecting the 9p21, which contains the p16 locus, a site known to be critical for normal progression of the cell cycle. Aberrant p16 expression is associated with more aggressive behavior. (3) Melanoma cells possess a remarkable repertoire of biosynthetic capacities represented by the production of hormones, growth factors, and their receptors that may sustain and accelerate tumor development and progression. For example, expression of the tumoral products α-melanocyte-stimulating hormone and adrenocorticotropic hormone is regulated in vitro by ultraviolet light, a known carcinogen. (4) Melanomas differ from other tumors in their intrinsic capability to express melanogenic enzymes with the corresponding structural proteins to actually synthesize melanin. Melanogenesis-related proteins are rapidly entering the clinical arena, being used not only as diagnostic markers, but also as potential targets for melanoma therapy.

Prognostic Implications of Aberrations in p16/pRb Pathway in Urothelial Bladder Carcinomas: A Multivariate Analysis Including p53 Expression and Proliferation Markers

Objective: To assess the prognostic value of the expression of two negative regulators of the cell cycle, namely CDKN2/INK4a gene product (p16) and retinoblastoma gene product (pRb), in urinary bladder cancer in relation to clinicopathological parameters, proliferative fraction and p53 protein accumulation. Methods: Paraffin sections from 139 patients with urothelial carcinomas were stained immunohistochemically with antibodies to p16 (F12), pRb (PMG3–245), p53 (DO1), PCNA (PC10) and Ki–67 (MIB–1). Results: Diminished p16 and pRb expression occurred in 29 and 74% of cases, respectively, being associated with advanced stage but not with histological grade, papillary status or proliferation rate. In most cases (53%) with some fault in the p16/pRb pathway, only one gene was affected. A double–negative p16/pRb phenotype was comparatively uncommon (25%) and was usually seen in T3–T4 tumours. In survival analysis (either univariate or multivariate) aberrant p16 expression was an adverse prognostic parameter only in T3–T4 tumours. In contrast, the abnormal p16/pRb and p53/p16 phenotypes were linked to a diminished overall and disease–free survival (univariate analysis); p53/p16 abnormal expression was also found to be an independent predictor of reduced survival in muscle–invasive tumours, while proliferation markers were the only parameters with independent significance in superficial (Ta–T1) tumours. Conclusion: Our results suggest that lack of p16 immunoexpression, when combined with p53 accumulation, plays an important role in determining the clinical outcome in muscle–invasive urothelial carcinomas.

Expression of p16(INK4A) and alterations of the 9p21-23 chromosome region in non-small-cell lung carcinomas: relationship with tumor growth parameters and ploidy status.

The 9p21-23 chromosome region harbors a number of known and putative tumor-suppressor genes (TSGs). The best characterized gene in this area is p16(INK4A) (CDKN2A). Alterations of its product have been observed in various malignancies, including non-small-cell lung carcinomas (NSCLCs). We earlier investigated the mechanisms underlying p16(INK4A) inactivation. In the present study, we examined, in a series of 87 NSCLCs, its relationship with the kinetic parameters [proliferation index (PI) and apoptotic index (Al)] and the ploidy status of the tumors. In addition, we extended our previous LOH analysis of the 9p21-23 region by examining flanking areas of p16(INK4A). Aberrant p16 expression was observed in 41.4% of the carcinomas. A significant association was found with increased PI (p = 0.037), but not with apoptosis. Aneuploid tumors were more frequently correlated with abnormal p16 staining (p = 0. 05). A high frequency of allelic imbalance (Alm) was noticed at the D9S161 (51.3%) and D9S157 (64.5%) loci, which lie approximately 4cM centromeric and 7cM telomeric, respectively, to CDKN2A. Abnormal p16(INK4A) expression was strongly correlated with Alm at D9S161 (p = 0.004). Allelic losses at D9S157 occurred more frequently in early stages (p = 0.018) and were significantly associated with deletions at D9S161 (p = 0.035). We conclude that, in a sub-set of NSCLCs, (i) abnormal p16 expression contributes to tumor growth mainly by increasing the proliferative activity in the initial stages of carcinogenesis; (ii) the association with aneuploidy merely reflects the impact of aberrant p16 on proliferative activity; and (iii) other putative TSGs possibly reside within the 9p21-23 region that possibly co-operate in certain cases with CDKN2A in the development of NSCLCs.