Role of accelerated aging in limb muscle wasting of patients with COPD.

Purpose: Skeletal muscle wasting is an independent predictor of health-related quality of life and survival in patients with COPD, but the complexity of molecular mechanisms associated with this process has not been fully elucidated. We aimed to determine whether an impaired ability to repair DNA damage contributes to muscle wasting and the accelerated aging phenotype in patients with COPD. Patients and methods: The levels of phosphorylated H2AX (γH2AX), a molecule that promotes DNA repair, were assessed in vastus lateralis biopsies from 10 COPD patients with low fat-free mass index (FFMI; COPDL), 10 with preserved FFMI and 10 age- and gender-matched healthy controls. A panel of selected markers for cellular aging processes (CDKN2A/p16ink4a, SIRT1, SIRT6, and telomere length) were also assessed. Markers of oxidative stress and cell damage and a panel of pro-inflammatory and anti-inflammatory cytokines were evaluated. Markers of muscle regeneration and apoptosis were also measured. Results: We observed a decrease in γH2AX expression in COPDL, which occurred in association with a tendency to increase in CDKN2A/p16ink4a, and a significant decrease in SIRT1 and SIRT6 protein levels. Cellular damage and muscle inflammatory markers were also increased in COPDL. Conclusion: These data are in keeping with an accelerated aging phenotype as a result of impaired DNA repair and dysregulation of cellular homeostasis in the muscle of COPDL. These data indicate cellular degeneration via stress-induced premature senescence and associated inflammatory responses abetted by the senescence-associated secretory phenotype and reflect an increased expression of markers of oxidative stress and inflammation.

An electrochemiluminescence biosensor for detection of CdkN2A/p16 anti-oncogene based on functional electrospun nanofibers and core-shell luminescent composite nanoparticles.

An electrochemiluminescence (ECL) biosensor based on functional electrospun nanofibers for hybridization detection of specific CdkN2A/p16 anti-oncogene at trace level via binding luminescent composite nanoparticles for signal amplification has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped polycaprolactam 6 (PA6) electrospun nanofibers (PA6-MWCNTs) was prepared via electrospinning, which served as the nanosized backbones for silica nanoparticles (SiO2) electrodeposition. The functional electrospun nanofibers (PA6-MWCNTs-SiO2) used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the sensitivity of hybridization. The sandwich construction of ssDNA1-CdkN2A/p16 anti-oncogene -tri(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)32+)/silver nanoparticles (AgNPs) doped gold (Au) core-shell luminescent composite nanoparticles (RuAg@AuNPs)-labeled ssDNA2 (RuAg@Au-ssDNA2) was fabricated through a hybridization reaction. It was observed that high amount of doped Ru(bpy)32+ in RuAg@AuNPs successfully amplify the recognition signal by adding tripropylamine (TPrA). The change of ECL intensity was found to have a linear relationship in respect to the logarithm of the CdkN2A/p16 anti-oncogene concentrations in the wide range of 1.0 × 10-15~1.0 × 10-12 M, with a detection limit of 0.5 fM (S/N = 3) which is comparable or better than that in reported anti-oncogene assays. Excellent sensitivity and selectivity make the developed biosensor a promising tool for the detection of tumor biomarkers.

p16INK4a : A surrogate marker of high-risk human papillomavirus infection in squamous cell carcinoma of the nasal vestibule.

BACKGROUND: The purpose of this study was to analyze the role of p16INK4a and the prevalence of human papillomavirus (HPV) in squamous cell carcinoma (SCC) of the nasal vestibule. METHODS: Patients diagnosed from 1995 to 2014 were included in this study. Assessment of p16INK4a and HPV-DNA polymerase chain reaction (PCR) was performed and analyzed with respect to baseline, clinicopathological, and outcome parameters. The p16INK4a positivity was defined as unequivocal nuclear and cytoplasmic staining of ≥70% of the cells, whereas 50%-69% was considered to be a "borderline" result. RESULTS: There were 46 patients with SCCs of the nasal vestibule, of whom 31 (67.4%) were available for p16INK4a and 30 (65.2%) for analysis of HPV. Expression of p16INK4a was present in 19.4% and showed coincidence with high-risk HPV (P < .001). Neither p16INK4a nor HPV-DNA had significant impact on outcome. CONCLUSION: Significant immunoreactivity for p16INK4a was present in about one-fifth of the samples and figured as a surrogate marker of high-risk HPV infection. There was no influence on outcome.

Abnormal p53 and p16 staining patterns distinguish uterine leiomyosarcoma from inflammatory myofibroblastic tumour.

AIMS: Uterine myxoid leiomyosarcoma may show relatively bland histological appearances, despite its aggressive behaviour. Distinguishing uterine leiomyosarcoma from the more indolent inflammatory myofibroblastic tumour (IMT), which is amenable to targeted therapies, can be challenging. A significant subset of leiomyosarcomas harbour TP53 and/or CDKN2A genomic alterations. Here, we examined the diagnostic value of p53 and p16 immunohistochemistry in the distinction of uterine conventional and myxoid leiomyosarcoma from IMT, in correlation with targeted sequencing of TP53 and CDKN2A. METHODS AND RESULTS: We performed p53 and p16 immunohistochemistry in 49 tumours, including 23 uterine leiomyosarcomas (12 myxoid, 11 conventional) and 26 IMT (12 uterine, 14 extrauterine). TP53 and CDKN2A coding regions were sequenced in 20 cases (four myxoid, 11 conventional uterine leiomyosarcomas; four uterine, one extrauterine IMT). Abnormal p53 staining patterns (strong/diffuse or null) were observed in six of 12 (50%) myxoid and six of 11 (55%) conventional leiomyosarcomas but none of the IMT (P < 0.0001), correlating with TP53 mutation/deletion (P = 0.0001). P16 loss was detected in five of 10 (50%) myxoid and two of 11 (18%) conventional leiomyosarcomas, but none of the IMT (P = 0.0005), correlating with CDKN2A deletion (P = 0.014). Strong/diffuse p16 staining in six of 21 (29%) leiomyosarcomas and three of 26 (12%) IMT did not correlate with CDKN2A alterations. CONCLUSIONS: Abnormal p53 staining and p16 loss are observed frequently in uterine leiomyosarcomas, with 100% specificity and 70% sensitivity against IMT, and correlating with genomic alterations. Conversely, IMT shows normal p53 and p16 staining, highlighting the use of these markers in the differential diagnosis of uterine mesenchymal neoplasms.

Malignant tenosynovial giant cell tumor with CDKN2A/B genomic alteration: a histological, immunohistochemical, and molecular study.

Diffuse-type tenosynovial giant cell tumor (D-T TSGCT) is regarded as a benign but locally aggressive neoplasm with significant recurrent potential. We report a case of malignant D-T TSGCT with pleural metastases arising in the left knee in a 57-year-old man. The tumor demonstrated atypical features, including a solid infiltrative pattern with spindling of the tumor cells, nuclear pleomorphism with prominent nucleoli, and markedly increased mitotic activity (>20 mitoses/10 high-power fields). The immunoprofile demonstrated clusterin+, D2-40+, CD68+, p63+, MDM2+, and p16+ tumor. The next-generation sequencing-based assay demonstrated loss of the CDKN2A/B gene. Pleural metastases with identical histologic and immunohistochemical features were identified 2 years later after primary tumor resection. To the best of our knowledge, this is the first reported case of D-T TSGCT with CDKN2A/B genomic alteration, MDM2 expression, and p16 loss. Clinicians and pathologists should be aware of the morphologic variability and the metastatic propensity of this entity.

Loss of p16 expression and copy number changes of CDKN2A in a spectrum of spitzoid melanocytic lesions.

Spitzoid melanocytic lesions, including Spitz nevi (benign), spitzoid melanoma (malignant), and borderline atypical Spitz tumors (ASTs), frequently present challenges for accurate diagnosis and prognosis. Evaluation for loss of the tumor suppressor p16, encoded by CDKN2A gene on chromosome 9p21.3, has been proposed to be useful for evaluation of spitzoid melanocytic lesions. However, reports on the utility of p16 immunohistochemistry for spitzoid lesions have been conflicting, and few studies have directly compared p16 immunohistochemistry with fluorescence in situ hybridization (FISH) for CDKN2A genomic status. We analyzed a spectrum of benign (n=24), borderline (n=27), and malignant (n=19) spitzoid lesions for p16 protein expression by immunohistochemistry and CDKN2A copy number by FISH. Immunohistochemistry was evaluated by 2 scoring methods: H score and 2-tiered score (positive or negative for p16 loss). By immunohistochemistry, loss of p16 expression was not observed in Spitz nevi (0/24) but was seen in ASTs (7/27; 26%) and spitzoid melanomas (3/19; 16%). By H score, p16 expression was significantly higher in Spitz nevi relative to ASTs or spitzoid melanomas. Similarly, copy number aberrations of CDKN2A by FISH were absent in Spitz nevi but were found in 2 (9.5%) of 21 ASTs and 4 (33%) of 12 spitzoid melanomas. Our findings from this large cohort suggest that p16 aberrations are highly specific for borderline and malignant spitzoid neoplasms relative to Spitz nevi. Similar to ASTs, p16 loss in spitzoid melanomas may occur in the presence or absence of genomic CDKN2A loss.

The overexpression of p16 is not a surrogate marker for high-risk human papilloma virus genotypes and predicts clinical outcomes for vulvar cancer.

BACKGROUND: We aimed to evaluate the correlation between p16(ink4a)-overexpression and high risk (hr)HPV-DNA in vulvar squamous cell carcinoma (vSCC) tumors as well as the impact of both biomarkers on the prognosis of vSCC patients. METHODS: PCR-detection of (hr)HPV-DNA and immunohistochemical staining for p16(ink4a) were conducted in 85 vSCC tumors. Survival analyses included the Kaplan-Meier method, log-rank test and Cox proportional hazards model. RESULTS: p16(ink4a)-overexpression and (hr)HPV-DNA were detected in 35 and 37 of the 85 tumors, respectively. Among the 35 p16(ink4a)-positive tumors, 10 lacked (hr)HPV-DNA (29 %). Among the 50 p16(ink4a)-negative tumors, (hr)HPV-DNA was detected in 12 cases (24 %). The median follow-up was 89.20 months (range 1.7-189.5 months). P16(ink4a)-overexpression, but not (hr)HPV-DNA positivity of the primary tumor, was correlated with prolonged overall survival (OS) (p = 0.009). P16(ink4a)-overexpression predicted a better response to radiotherapy (p < 0.001). Univariate analysis has demonstrated that age (p = 0.025), tumor grade (p = 0.001), lymph node metastasis (p < 0.001), FIGO stage (p < 0.001), p16(ink4a)-overexpression (p = 0.022), and adjuvant RTX (p < 0.001) were prognostic factors for OS. Multivariate analysis has demonstrated that lymph node metastasis (HR 1-2.74, 95 % CI 1.50-5.02, p = 0.019), tumor grade (HR 1-2.80, 95 % CI 1.33-5.90, p = 0.007) and p16(ink4a)-overexpression (HR 1-2.11, 95 % CI 1.13-3.95, p = 0.001) are independent prognostic factors. CONCLUSION: The discovered overlap suggests the use of p16(ink4a) in combination with HPV-DNA detection as an ancillary test for future research and clinical studies in vSCC. The prognostic and predictive value of p16(ink4a)-overexpression should be tested in larger cohort studies.

Immunohistochemical evaluation of p16 expression in cutaneous histiocytic, fibrohistiocytic and undifferentiated lesions.

BACKGROUND: Expression of p16 is frequently evaluated in melanocytic lesions. Expression of p16 in cutaneous histiocytic, fibrohistiocytic and undifferentiated lesions has not been well characterized. METHODS: We evaluated p16 expression in a cohort of histiocytic (reticulohistiocytoma, Langerhans cell histiocytosis, xanthogranuloma, Rosai Dorfman disease and xanthoma), fibrohistiocytic (dermatofibroma, epithelioid fibrous histiocytoma and dermatofibrosarcoma protuberans) and undifferentiated (atypical fibroxanthoma and pleomorphic undifferentiated sarcoma) lesions. A group of melanocytic lesions (Spitz nevus, ordinary nevus, spitzoid melanoma and non-spitzoid melanoma) were also evaluated as reference. Each case was scored by the proportion of p16-positive cells and by staining intensity. RESULTS: Immunoreactivity for p16 was found in almost all histiocytic (28/30, 93%) and fibrohistiocytic (22/24, 92%) lesions. About half of the undifferentiated lesions also exhibited p16 staining (9/17, 53%). Most of the melanocytic cases examined in this study expressed p16. A wide range of staining intensity and proportion of p16-positive cells was observed in most groups. CONCLUSION: Expression of p16 is common, albeit variable in proportion and intensity, amongst a wide variety of cutaneous histiocytic, fibrohistiocytic and undifferentiated lesions. Further studies are required to determine if p16 expression is useful in distinguishing benign from malignant neoplasms of these types.

CDKN2A and BAP1 germline mutations predispose to melanoma and mesothelioma.

BAP1 germline mutations predispose to a cancer predisposition syndrome that includes mesothelioma, cutaneous melanoma, uveal melanoma and other cancers. This co-occurrence suggests that these tumors share a common carcinogenic pathway. To evaluate this hypothesis, we studied 40 Italian families with mesothelioma and/or melanoma. The probands were sequenced for BAP1 and for the most common melanoma predisposition genes (i.e. CDKN2A, CDK4, TERT, MITF and POT1) to investigate if these genes may also confer susceptibility to mesothelioma. In two out of six families with both mesothelioma and melanoma we identified either a germline nonsense mutation (c.1153C > T, p.Arg385*) in BAP1 or a recurrent pathogenic germline mutation (c.301G > T, p.Gly101Trp) in CDKN2A. Our study suggests that CDKN2A, in addition to BAP1, could be involved in the melanoma and mesothelioma susceptibility, leading to the rare familial cancer syndromes. It also suggests that these tumors share key steps that drive carcinogenesis and that other genes may be involved in inherited predisposition to malignant mesothelioma and melanoma.