B-cell infiltration is associated with survival outcomes following programmed cell death protein 1 inhibition in head and neck squamous cell carcinoma.

BACKGROUND: Programmed cell death protein 1 (PD-1) axis blockade has become the mainstay in the treatment of recurrent and/or metastatic (R/M) head and neck squamous cell cancer (HNSCC). Programmed death-ligand 1 (PD-L1) is the only approved biomarker for patient selection; however, response rate is limited even among high expressors. Our primary objective was to investigate the association of immune cell-related biomarkers in the tumor and tumor microenvironment with PD-1 checkpoint inhibitors' outcomes in patients with R/M HNSCC. PATIENTS AND METHODS: NCT03652142 was a prospective study in nivolumab-treated platinum-refractory R/M HNSCC, aiming to evaluate biomarkers of response to treatment. Tumor biopsies and blood samples were collected from 60 patients at baseline, post-treatment, and at progression. Immune cells in the tumor and stromal compartments were quantified by immunofluorescence using a five-protein panel (CD3, CD8, CD20, FoxP3, cytokeratin). Tertiary lymphoid structures (TLSs), PD-L1 expression, and peripheral blood immune cell composition were also evaluated for associations with outcome. Our findings were validated by gene set enrichment analysis (GSEA) messenger RNA in situ expression data from the same patients, for B-cell- and TLS-associated genes. RESULTS: High pre-treatment density of stromal B cells was associated with prolonged progression-free survival (PFS) (P = 0.011). This result was validated by GSEA, as stromal enrichment with B-cell-associated genes showed association with response to nivolumab. PD-L1 positivity combined with high B-cell counts in stroma defined a subgroup with significantly longer PFS and overall survival (P = 0.013 and P = 0.0028, respectively). CONCLUSIONS: Increased B cells in pre-treatment HNSCC biopsy samples correlate with prolonged benefit from PD-1-based immunotherapy and could further enhance the predictive value of PD-L1 expression.

A phase 2, randomized, double-blind, placebo- controlled study of chemo-immunotherapy combination using motolimod with pegylated liposomal doxorubicin in recurrent or persistent ovarian cancer: a Gynecologic Oncology Group partners study.

BACKGROUND: A phase 2, randomized, placebo-controlled trial was conducted in women with recurrent epithelial ovarian carcinoma to evaluate the efficacy and safety of motolimod-a Toll-like receptor 8 (TLR8) agonist that stimulates robust innate immune responses-combined with pegylated liposomal doxorubicin (PLD), a chemotherapeutic that induces immunogenic cell death. PATIENTS AND METHODS: Women with ovarian, fallopian tube, or primary peritoneal carcinoma were randomized 1 : 1 to receive PLD in combination with blinded motolimod or placebo. Randomization was stratified by platinum-free interval (≤6 versus >6-12 months) and Gynecologic Oncology Group (GOG) performance status (0 versus 1). Treatment cycles were repeated every 28 days until disease progression. RESULTS: The addition of motolimod to PLD did not significantly improve overall survival (OS; log rank one-sided P = 0.923, HR = 1.22) or progression-free survival (PFS; log rank one-sided P = 0.943, HR = 1.21). The combination was well tolerated, with no synergistic or unexpected serious toxicity. Most patients experienced adverse events of fatigue, anemia, nausea, decreased white blood cells, and constipation. In pre-specified subgroup analyses, motolimod-treated patients who experienced injection site reactions (ISR) had a lower risk of death compared with those who did not experience ISR. Additionally, pre-treatment in vitro responses of immune biomarkers to TLR8 stimulation predicted OS outcomes in patients receiving motolimod on study. Immune score (tumor infiltrating lymphocytes; TIL), TLR8 single-nucleotide polymorphisms, mutational status in BRCA and other DNA repair genes, and autoantibody biomarkers did not correlate with OS or PFS. CONCLUSIONS: The addition of motolimod to PLD did not improve clinical outcomes compared with placebo. However, subset analyses identified statistically significant differences in the OS of motolimod-treated patients on the basis of ISR and in vitro immune responses. Collectively, these data may provide important clues for identifying patients for treatment with immunomodulatory agents in novel combinations and/or delivery approaches. TRIAL REGISTRATION: Clinicaltrials.gov, NCT 01666444.

Identification of melanoma cells and lymphocyte subpopulations in lymph node metastases by FTIR imaging histopathology.

While early stages of melanoma are usually cured by surgery, metastatic melanomas are difficult to treat because the widely available options have low response rates. Careful and precise diagnosis and staging are essential to determine patient's risk and to select appropriate treatments. Fortunately, the recent progress in immunotherapy is very encouraging. In this context, it is important to characterize the intratumoral infiltration of immune cells in each patient, which is however not done routinely due to the lack of standardized methods. In this study, we used Fourier transform infrared (FTIR) imaging combined with multivariate statistical analyses to investigate non-metastatic and metastatic lymph nodes from melanoma patients. Our results show that the different cell types have different infrared spectral features allowing automated identification of these cell types. High recognition rates were obtained using a supervised partial least square discriminant analysis (PLS-DA) model. Melanoma cells were recognized with 87.1% sensitivity and 85.7% specificity, showing that FTIR spectroscopy has similar detection power as immunohistochemistry. Besides, FTIR imaging could also distinguish lymphocyte subpopulations (B and T cells). Finally, we investigated the changes in lymphocytes due to the presence of metastases. Interestingly, specific features of spectra of lymphocytes present in metastatic or tumor-free lymph nodes could be evidenced by PCA. A PLS-DA model was capable of predicting whether lymphocytes originated from invaded or non-invaded lymph nodes. These data demonstrate that FTIR imaging is capable to distinguish known and also novel biological features in human tissues, with potential practical relevance for histopathological diagnosis and biomarker assessment.

Hepatic tuberculoma mimicking hepatocellular carcinoma in an immunocompetent host.

Hepatic tuberculosis as a part of disseminated tuberculosis is seen in 50-80% of cases. Isolated hepatic tuberculosis is very uncommon even in countries with high prevalence of tuberculosis. It can occur as a primary case or due to reactivation of an old tubercular focus. We report a case of a 59-year-old Caucasian woman who presented with persistent right upper quadrant pain and a hepatic lesion on an abdominal CT. She had a history of pulmonary tuberculosis 15 years ago with localised lung tuberculosis treated with lobectomy and antituberculous drugs.

Usual interstitial pneumonia associated with cytomegalovirus infection after percutaneous transluminal coronary angioplasty.

An unusual case of cytomegalovirus (CMV) pneumonia in a diabetic patient is presented. The diagnosis was based on typical histopathological findings including intranuclear inclusion bodies combined with molecular identification of CMV in tissue specimens. The possibility of CMV reactivation associated with a previous cardiac procedure, which led to the development of usual interstitial pneumonia, is discussed. Clinicians should be aware of CMV-associated severe bilateral pneumonia developing after cardiac procedures even in non-transplant patients. The correct diagnosis depends on clinical awareness in the appropriate setting along with proof of viral infection.

Detection of human telomerase reverse transcriptase mRNA in urine of patients with bladder cancer: evaluation of an emerging tumor marker.

OBJECTIVES: To assess the value of the telomerase enzyme as a bladder cancer detection marker, we investigated the expression of the catalytic subunit of the complex (human telomerase reverse transcriptase [hTERT]) in the urine of patients with malignant or benign urinary lesions, as well as of healthy individuals, and compared the results with urine cytology. METHODS: Spontaneously voided samples were obtained from two groups of subjects: group 1, 146 previously untreated patients with a histologic diagnosis of transitional cell carcinoma or other urothelial neoplasm; and group 2, 128 control individuals, either healthy or with a nonmalignant bladder disease. Total RNA extracts from sedimented urothelial cells were analyzed by a reverse transcriptase-polymerase chain reaction assay for the presence of a 146-bp hTERT transcript. Urine samples were also examined by standard cytology. RESULTS: Expression of hTERT was detected in 134 (92%) of 146 patients with bladder cancer, and only 64 (44%) yielded a positive result by cytology (P <0.001). The sensitivity advantage of the former technique became particularly evident in the detection of low-grade transitional cell carcinoma (93% versus 28%, P <0.001). Accordingly, the negative predictive value of the molecular assay was markedly greater than the one calculated for cytologic screening (91% versus 60%). On the other hand, both methods were at least 96% specific, with their positive predictive indexes exceeding 94%. CONCLUSIONS: Our findings suggest that the assessment of hTERT expression in urine sediments represents a reliable tool for the detection of primary urothelial neoplasms, equally specific, yet far more sensitive, than conventional cytology.

Expression of HLA-DR is reduced in tumor infiltrating immune cells (TIICs) and regional lymph nodes of non-small-cell lung carcinomas. A putative mechanism of tumor-induced immunosuppression?

BACKGROUND: Deregulation of MHC class II molecules consists of a favorable mechanism of tumor evasion from immune surveillance. Among these molecules, HLA-DR antigens are the predominant ones in cancer. In the present study we sought to investigate the ability of tumor infiltrating immune cells (TIICs) to express HLA-DR antigen in the primary tumor site and reactive regional lymph nodes (LNs) in non small cell lung cancer (NSCLC). MATERIALS AND METHODS: Material consisting of 60 NSCLCs with corresponding regional LNs was studied by immunohistochemistry for human leukocyte antigen D-region related (HLA-DR) expression. Control reactive LNs, regional to several different malignant and non-malignant disorders, were also included in the study. RESULTS: Primary tumor site investigation revealed positive HLA-DR cancer cells in 22% of cases, whereas TIICs rarely expressed HLA-DR antigens. The lack of HLA-DR expression in TIICs was gradually attenuated as the distance from the primary tumor site decreased. Regional LN investigation showed that all follicles (paracapsular and deep cortical ones) were HLA-DR-negative in 60% of the LNs; in the remaining 40%, the paracapsular follicles remained negative, while all deep cortical ones were positive. Interestingly, LNs possessing only HLA-DR-negative follicles were more proximal to the primary tumor site compared to those that had only the paracapsular follicles negative. All control reactive LNs, regional to several distinct malignant and non-malignant disorders, were found to be HLA-DR-positive. CONCLUSION: The impairment of HLA-DR expression, detected both in neoplastic and by-stander immune cells, may justify the immunosuppression observed in NSCLC. This phenomenon may be due to a putative soluble factor in the tumor environment secreted by cancer cells.

Deregulated expression of c-mos in non-small cell lung carcinomas: relationship with p53 status, genomic instability, and tumor kinetics.

Little is known about the status of the mitogen-activating protein kinase pathways in lung cancer. One of the key molecules taking part in these pathways is the product of the c-mos proto-oncogene, which plays an important role in oocyte maturation. In vitro investigations in somatic cells have shown that c-mos expression has opposing effects on the cell cycle, which suggests that this proto-oncogene may represent an important determinant of aberrant cell function (genomic instability and altered kinetics). A recent study suggests that these effects may be p53 dependent. In view of the apparent link between c-mos and p53, we investigated in a series of 56 non-small cell lung carcinomas: a) the status of c-mos; b) its relationship to genomic instability (aneuploidy) and two kinetic parameters of the tumors, proliferation and apoptotic indexes (AI); and c) its association with p53 alterations and their concomitant relationship with the above parameters. We found c-mos overexpression in 27% of the tumors. Expression was higher in stages II/III (34%) than in stage I (17%; P = 0.018). Complete concordance was observed between c-mos overexpression and elevated c-mos mRNA levels. Because c-mos gene amplification was not detected, its deregulated expression may be attributable to increased transcription. Of the c-mos positive [c-mos(P)] cases, 77% were associated with aneuploidy. Sequencing showed two silent mutations and one missense (R-->L) at codon 22, located in a region critical for c-mos stability. In contrast to the findings of some in vitro studies, c-mos(P) tumors had a lower mean AI score than the c-mos negative [c-mos(N)] tumors had, implying that induction of apoptosis may have been defective. Indeed, 86% of the tumors overexpressing c-mos showed p53 alterations. The carcinomas with concomitant alterations of c-mos and p53 [c-mos(P)/p53 positive] had significantly lower AI values (P < 0.001) and were more frequently associated with aneuploidy (P = 0.015) than the c-mos(N)/p53 negative tumors but not the c-mos(N)/p53 positive tumors, which suggests that p53 status is the main determinant of ploidy status and apoptosis in our series. This finding also strengthens the concept that wild-type p53 plays a "safeguard" role in preventing oncogene-mediated activation.

Association of allelic loss at the FHIT locus and p53 alterations with tumour kinetics and chromosomal instability in non-small cell lung carcinomas (NSCLCs).

The FHIT gene, located at the FRA3B fragile site of chromosome 3p14.2, encodes a 16.8 kD homologue of the yeast enzyme diadenosine tetraphosphate (Ap(4)A) hydrolase. Frequent allelic losses at this region in various malignancies, including non-small cell lung carcinomas (NSCLCs), imply that FHIT may represent a tumour suppressor gene (TSG). Increasing evidence suggests that multiple TSG impairment has a synergistic effect on tumour growth. The present study of 67 NSCLCs investigated the allelic imbalance (AIm) within the FHIT locus and its relationship with p53 abnormalities, kinetic parameters [proliferative activity or proliferation index (PI) and apoptotic index (AI)], and ploidy status of the carcinomas. Allelic imbalance at FHIT was observed in 35 out of 55 informative (heterozygous: H) cases (64%). Similar frequencies of loss of heterozygosity (LOH) were noticed among squamous cell lung carcinomas and adenocarcinomas. The high percentage of AIm in stage I tumours (71%) is indicative of its relatively early involvement in NSCL carcinogenesis. No association was found between LOH at FHIT, kinetic parameters, and ploidy status of the tumours. Concurrent loss at FHIT and p53 overexpression [FHIT(LOH)/p53(P)] was the most frequent pattern and was observed in 39% of the informative cases. The latter pattern was not associated with smoking, supporting the hypothesis that in patients with a history of tobacco exposure, FHIT allelic loss may not be a consequence of p53 checkpoint defects, but the outcome of tobacco-induced mutagenesis. Statistically significant differences in the presence of FHIT(LOH)/p53(P) and FHIT(LOH)/p53(N) patterns were noted at the proliferative and apoptotic level, whereas ploidy was similar amongst all groups, implying that wild-type (wt) p53 may play a safeguard role against altered FHIT function. However, the possibility of a masking effect from wt p53 cannot be excluded, since the FHIT(LOH)/p53(P) profile demonstrated a higher growth index (GI=PI/AI mean value ratio) than FHIT(H)/p53(P) (32 vs. 8), although this was not significant. Further studies are needed in order to elucidate the role of FHIT and its relationships with other cell-cycle regulatory molecules involved in NSCL carcinogenesis.

Molecular aspects of multiple myeloma.

Multiple myeloma (MM) is a B-cell neoplasm characterized by bone marrow infiltration with malignant plasma cells, which synthesize and secrete monoclonal immunoglobulin (Ig) fragments. Despite the considerable progress in the understanding of MM biology, the molecular basis of the disease remains elusive. The initial transformation is thought to occur in a postgerminal center B-lineage cell, carrying a somatically hypermutated Ig heavy chain (IGH) gene. This plasmablastic precursor cell colonizes the bone marrow, propagates clonally and differentiates into a slowly proliferating myeloma cell population, all under the influence of specific cell adhesion molecules and cytokines. Production of interleukin-6 by stromal cells, osteoblasts and, in some cases, neoplastic cells is an essential element of myeloma cell growth, with the cytokine stimulus being delivered intracellularly via the Jack-STAT and ras signaling pathways. While karyotypic changes have been identified in up to 50% of MM patients, recent molecular cytogenetic techniques have revealed chromosomal abnormalities in the vast majority of examined cases. Translocations mostly involve illegal switch rearrangements of the IGH locus with various partner genes (CCND1, FGFR3, c-maf). Such events have been assigned a critical role in MM development. Mutations in coding and regulatory regions, as well as aberrant expression patterns of several oncogenes (c-myc, ras) and tumor suppressor genes (p16, p15) have been reported. Key regulators of programmed cell death (BCL-2, Fas), tumor expansion (metalloproteinases) and drug responsiveness (topoisomerase II alpha) have also been implicated in the pathogenesis of this hematologic malignancy. A tumorigenic role for human herpesvirus 8 (HHV8) was postulated recently, following the detection of viral sequences in bone marrow dendritic cells of MM patients. However, since several research groups were unable to confirm this observation, the role of HHV8 remains unclear. Translation of the advances in MM molecular biology into novel therapeutic strategies is essential in order to improve disease prognosis.

c-mos immunoreactivity is an indicator of good prognosis in lung cancer.

AIMS: Reports concerning the expression of cytoplasmic components of the mitogen-activating protein kinase (MAPK) pathway in lung cancer are limited. One of the molecules participating in this pathway is the product of the c-mos proto-oncogene. In vitro investigations, in somatic cells, have shown that c-mos expression has opposing effects on cell cycle progression suggesting that it may represent an important determinant of aberrant cell function. In this study we analysed, by immunohistochemical means, its status in a series of lung carcinomas and correlated the findings with clinicopathological parameters and survival of the patients. METHODS AND RESULTS: Sixty cases of lung carcinomas were included in the study. These comprised 52 non-small (NSCLCs) and eight small cell lung carcinomas (SCLCs). Sections from the carcinomas were immunostained with the polyclonal anti-c-mos antibody P-19. Specificity was tested by using the appropriate control peptide and control cell lines. Expression was observed in 63% of the cases, with NSCLCs showing higher reactivity (67%) than SCLCs (37.5%). Staining was observed mainly to the cytoplasm and membranes of the cancerous cells, but some nuclei reacted as well. An intratumour heterogeneous immunoreactivity was noticed. The most interesting and unexpected finding was that c-mos positive staining was associated with better recurrence-free survival in our series, regardless of histological type (P = 0.035). Furthermore, favourable disease-related and recurrence-free survival was observed in the SqC group with c-mos immunoreactivity (P < 0. 001). CONCLUSIONS: c-mos proto-oncogene is expressed in a significant proportion of lung carcinomas and may play a role in its development. The fact that its expression is associated with a relatively good prognosis may be indicative of a negative impact on tumour growth.

Altered expression of the cell cycle regulatory molecules pRb, p53 and MDM2 exert a synergetic effect on tumor growth and chromosomal instability in non-small cell lung carcinomas (NSCLCs).

BACKGROUND: Recent in vitro studies provide evidence that the cell cycle molecules pRb, p53 and MDM2 form a tightly regulated protein network. In this study, we examined the relationship of this protein network in a series of non-small cell lung carcinomas (NSCLCs), with the kinetic parameters, including proliferative activity or proliferation index (PI) and apoptotic index (AI), and ploidy status of the tumors. MATERIAL AND METHODS: A total of 87 NSCLCs were examined using immunohistochemical and molecular methods in order to estimate the status of the pRb-p53-MDM2 network. The kinetic parameters and the ploidy status of the tumors were assessed by in situ assays. The possible associations between alterations of the network, kinetic parameters and ploidy status of the carcinomas were assessed with a series of statistical methods. RESULTS: Aberrant expression of pRb (Ab) and overexpression of p53 (P) and MDM2 (P) proteins were observed in 39%, 57%, and 68% of the carcinomas, respectively. The comprehensive analysis revealed that concurrent alterations in all three cell cycle regulatory molecules were the most frequent pattern, pRb(Ab)/p53(P)/MDM2(P); this "full abnormal" phenotype represented approximately 27% of the cases. This immunoprofile obtained the highest PI/AI value; whereas, the "normal" phenotype was the lowest one (p = 0.004). Furthermore, the pattern pRb(Ab)/p53(P)/MDM2(P) acquired the highest PI (p < 0.001) and lowest AI (p < 0.001) scores. Interestingly, the groups of carcinomas with impaired expression of one or two molecules attained PI/AI ratio values clustered in a narrow range placed in the middle of the scores exhibited by the "normal" and "full abnormal" phenotypes. These tumors had significantly lower AI, but similar PI values, compared with those noticed in the normal pattern. In addition, it was observed that the pRb(Ab)/p53(P)/MDM2(P) phenotype was also significantly associated with aneuploidy (p = 0.002) and a tendency was observed when the expression of two components was altered (p = 0.055). CONCLUSIONS: Our findings suggest that simultaneous deregulation of all members of the pRb-p53-MDM2 network confers an additive effect on tumor growth. The apoptotic pathway seems to be more susceptible to its defects than the cell proliferation machinery. The findings of the ploidy analysis, which are in parallel with those regarding the proliferative activity and the apoptotic rate study, further support the concept that these molecules constitute a tightly regulated network participating in cell cycle control and chromosomal stability.

"High risk" HPV types are frequently detected in potentially malignant and malignant oral lesions, but not in normal oral mucosa.

Studies on the involvement of the human papillomavirus (HPV) in initiation and progression of oral neoplasia have generated conflicting results. The observed discrepancy is attributable mainly to the varying sensitivity of the applied methodologies and to epidemiologic factors of the examined patient groups. To evaluate the role of HPV in oral carcinogenesis, we analyzed 53 potentially neoplastic and neoplastic oral lesions consisting of 29 cases of hyperplasia, 5 cases of dysplasia, and 19 cases of squamous cell carcinomas, as well as 16 oral specimens derived from healthy individuals. A highly sensitive nested polymerase chain reaction (PCR) assay was used, along with type-specific PCR, restriction fragment length polymorphism analysis, dot blotting, and nonisotopic in situ hybridization. Nested PCR revealed the presence of HPV DNA in 48 of the 53 (91%) pathologic samples analyzed, whereas none (0%) of the normal specimens was found to be infected. Positivity for HPV was independent of histology and the smoking habits of the analyzed group of patients. At least one "high risk" type, such as HPV 16, 18, and 33, was detected by type-specific PCR in 47 (98%) infected specimens, whereas only 1 (2%) squamous cell carcinoma was solely infected by a "low risk" type (HPV 6). HPV 16 was the prevailing viral type, being present in 71% of infected cases. Single HPV 16 and HPV 18 infections were confirmed by restriction fragment length polymorphism. HPV 58 was detected by dot blotting in three hyperplastic lesions. HPV positivity and genotyping were further confirmed, and the physical status of this virus was evaluated by nonisotopic in situ hybridization. Diffuse and punctate signals, indicative of the episomal and integrative pattern of HPV infection, were observed for low- and high-risk types, respectively. Our findings are suggestive of an early involvement of high-risk HPV types in oral carcinogenesis.

Expression of HLA-DR, costimulatory molecules B7-1, B7-2, intercellular adhesion molecule-1 (ICAM-1) and Fas ligand (FasL) on gastric epithelial cells in Helicobacter pylori gastritis; influence of H. pylori eradication.

There is evidence that Helicobacter pylori infection up-regulates the expression of HLA class II molecules by gastric epithelial cells (GEC). In this study we evaluated whether GEC are capable of expression of costimulatory molecules in H. pylori gastritis. The expression of FasL by GEC, before and after eradication of H. pylori, was also studied. Thirty patients (23 men) aged 27-81 years (53.67 +/- 13.99 years (mean +/- s.d.)) with dyspepsia were studied. Upper gastrointestinal endoscopy was performed and six biopsies were obtained (antrum, n = 3; corpus, n = 3) for Campylobacter-Like Organisms (CLO) test and histology; 23 (16 men) were H. pylori+ and seven (all men) were H. pylori- by both methods and served as controls. Helicobacter pylori eradication therapy was given to H. pylori+ patients and all patients were re-endoscoped after 116 +/- 9 days. Formalin-fixed paraffin-embedded tissue sections were stained by the ABC immunoalkaline phosphatase method. In H. pylori gastritis HLA-DR was expressed and correlated with disease activity (P < 0.01). No HLA-DR was observed in controls. In H. pylori-eradicated patients significant decrease of HLA-DR was found (antrum, P < 0. 001). ICAM-1 was expressed by GEC in 80% of H. pylori+ patients; ICAM-1 expression did not correlate with gastritis parameters and decreased significantly after eradication (antrum, P < 0.01). B7-1 and B7-2 were expressed on H. pylori+ samples and their expression decreased after eradication, albeit not significantly. Weak epithelial expression of both B7 molecules was observed in all the controls. FasL was steadily expressed by GEC in both H. pylori+ and H. pylori- patients and remained almost unchanged after eradication. These findings suggest that GEC may acquire antigen-presenting cell properties in H. pylori infection through de novo expression of HLA-DR and costimulatory molecules. This seems to be attenuated after eradication and resolution of mucosal inflammation. The same cells exhibit the capacity to control the inflammatory process, probably by inducing apoptotic cell death to Fas-bearing infiltrating lymphocytes.

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.