The Glioma-IRE project - Molecular profiling in patients with glioma: steps toward an individualized diagnostic and therapeutic approach.

BACKGROUND: This study aimed to characterize the genetic profile of patients with glioma and discuss the impact of next-generation sequencing in glioma diagnosis and treatment. METHODS: Between 2019 and 2022, we analyzed the genetic profile of 99 patients with glioma through the Oncomine Focus Assay. The assay enables the detection of mutations in 52 driver genes, including single nucleotide variants (SNVs), copy number variants (CNVs), and gene fusions. We also collected and analyzed patients' clinic characteristics and treatment outcomes. RESULTS: Over a period of 35 months, 700 patients with glioma followed by our neuro-oncology unit were screened, and 99 were enrolled in the study; most of the patients were excluded for inadequate non-morphological MRI or lack/inadequacy of the tissue samples. Based on our findings, most patients with glioma present mutations, such as SNVs, CNVs or gene fusions. Our data were similar to those reported by The Cancer Genome Atlas Program in terms of frequency of SNVs and CNVs, while we observed more cases of gene fusions. Median overall survival, progression-free survival, and time to progression were significantly lower for patients with grade VI glioblastoma than those with other gliomas. Only four patients were offered a targeted treatment based on the mutation detected; however, only one received treatment, the others could not receive the selected treatment because of worsening clinical status. CONCLUSION: Routine timely molecular profiling in patients with glioma should be implemented to offer patients an individualized diagnostic approach and provide them with advanced targeted therapy options if available.

Alpha, Beta, and Gamma Human Papillomaviruses in Genital Lichen Sclerosus: A Retrospective Cross-Sectional Study.

BACKGROUND: Lichen sclerosus (LS) is an inflammatory disease mostly arising at the genital level. It is unclear whether human papillomaviruses (HPVs) have an etiological significance in LS, and data on their prevalence in patients with LS are controversial. OBJECTIVES: The authors assessed alpha, beta, and gamma HPV prevalence in patients with genital LS. The association of HPV positivity with demographic and clinical factors was also investigated. METHODS: One hundred thirty-two formalin-fixed, paraffin-embedded LS samples (2016-2020) were retrieved from the archives of a pathology department. Alpha HPVs were genotyped with the INNO-LiPA HPV Genotyping Extra II kit. Beta and gamma HPVs were searched by multiplex Polymerase Chain Reaction. Immunostaining for p16 INK4a was performed on high-risk HPV-positive samples. RESULTS: Patients had a median age of 61 years, were mostly women ( n = 73, 55.3%), and with an early disease stage ( n = 79, 59.8%). Alpha HPVs were detected in 12/132 cases (9.1%). Among the 5 high-risk HPV-positive cases, only 2 displayed a strong and diffuse p16 INK4a staining. Beta genus was the most prevalent (35/132, 26.5%) and HPV5 was the most frequent beta genotype (25/132, 18.9%). There were 3 gamma HPV-positive cases among those with a valid result (3/131, 2.3%). Multiple infections with genotypes belonging to different genera were infrequent (3/131, 2.3%). No significant differences in the prevalence of the individual genera were observed according to sex and disease stage. CONCLUSIONS: Of the 3 HPV genera, beta genus showed the highest prevalence. Further research is needed to clarify whether the presence of beta HPVs in genital LS has a clinical significance.

FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma.

BACKGROUND & AIMS: About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF+ iCCA, responses are partial and/or limited by resistance mechanisms, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical unmet need. Herein, we aimed to generate a murine model of FF-driven iCCA and use this to uncover actionable FF-associated dependencies. METHODS: Four iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by transplantation into immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies. RESULTS: Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and the Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo. CONCLUSIONS: FF-driven iCCA pathogenesis was successfully modeled on a Tp53-/- murine background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-ERK signaling deserves consideration for precision-based approaches against human FF+ iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA.

Oral human papillomavirus infection in HIV-infected and HIV-uninfected MSM: the OHMAR prospective cohort study.

OBJECTIVE: We aimed to assess incidence and clearance of oral human papillomavirus (HPV) infection and the respective risk factors in HIV-infected and uninfected men who have sex with men (MSM). METHODS: Oral rinse and gargles were collected semiannually from 244 MSM (103, 42.2% HIV-infected). HPV-DNA testing was performed with the Linear Array HPV Genotyping test. A Markov model was used for estimation of incidence, clearance and risk factor analysis. RESULTS: Incidence rates for any HPV were 21.2 and 15.0×1000 person-months in HIV-infected and uninfected MSM, respectively. The respective figures for high-risk HPVs were 10.7 and 6.5×1000 person-months. The clearance rate was 4-12 times higher than the respective incidence rate. HIV-infected MSM with >95 lifetime oral sex partners showed increased incidence of any HPV (adjusted HR, aHR: 8.46, 95% CI 1.89 to 37.92). Condomless oral sex appeared the strongest predictor for incident infection by high-risk HPVs in this group (aHR: 13.40, 95% CI 2.55 to 70.53). Those aged >46 years (aHR: 0.30, 95% CI 0.12 to 0.74) and those with nadir CD4+ T count of <200 cells/mm3 (aHR: 0.14, 95% CI 0.03 to 0.75) displayed a significantly reduced clearance of any and high-risk HPVs, respectively. HIV-uninfected MSM aged >46 years had increased risk of acquiring any HPV (aHR: 3.70, 95% CI 1.30 to 10.52) and high-risk HPV (aHR: 5.33, 95% CI 1.06 to 26.68). Any HPV clearance declined in those with more than six recent oral sex partners (aHR: 0.18, 95% CI 0.05 to 0.65). CONCLUSIONS: Acquisition of oral HPV infection in MSM seems to occur rarely, whereas clearance seems to be a frequent event. Oral HPV natural history in these at-risk subjects is differently influenced by age and sex behaviour, depending on HIV status.

Oral testing for high-risk human papillomavirus DNA and E6/E7 messenger RNA in healthy individuals at risk for oral infection.

BACKGROUND: Testing for oral high-risk human papillomavirus (HPV) DNA may be useful for identifying individuals at increased risk for HPV-driven oropharyngeal cancer (OPC). However, positivity for HPV DNA provides no information on the transforming potential of the infection. In contrast, the detection of high-risk HPV E6/E7 messenger RNA (mRNA) may help to identify clinically significant infections because of the indispensable role of E6/E7 viral oncoproteins in the carcinogenic process. METHODS: Oral rinses were collected with a mouthwash from cancer-free individuals at increased risk for oral HPV infection. High-risk HPV DNA and mRNA were evaluated via the testing of the oral rinses with the Linear Array HPV genotyping test and the Aptima HPV assay, respectively. RESULTS: Overall, 310 subjects with no clinical evidence of lesions of the oral cavity and oropharynx were included in the study. Thirty-three (10.6%) harbored high-risk HPV DNA in their oral rinse. These cases, together with 10 random samples negative for high-risk HPV DNA, were tested with the Aptima assay. A valid result was obtained for 41 of the 43 specimens (95.3%). Among the 31 cases that were positive for high-risk HPV DNA and had a valid Aptima result, 4 (12.9%) were positive for HPV mRNA. HPV mRNA was not detected in any of the samples negative for high-risk HPV DNA. CONCLUSIONS: HPV mRNA is detectable in oral rinses of cancer-free subjects. Oral HPV mRNA testing may be useful in the screening and/or early detection of HPV-driven OPC by possibly identifying active and transforming oral infections. The testing of individuals at increased risk for HPV-related OPC via simply and noninvasively collected oral specimens is an attractive option for future screening strategies.

Altered peritumoral microRNA expression predicts head and neck cancer patients with a high risk of recurrence.

Head and neck squamous cell carcinoma is typically characterized by a high incidence of local recurrences. It has been extensively shown that mucosa from head and neck squamous cell carcinoma patients carries both genetic and gene expression alterations, which are mostly attributable to major etiologic agents of head and neck squamous cell carcinoma. We previously identified a signature of microRNAs (miRNAs) whose high expression in tumors is predictive of recurrence. Here, we investigated whether the deregulation of miRNA expression in the tumor-surrounding mucosa is correlated to disease recurrence. Specifically, comparing the miRNA expression in matched tumoral, peritumoral, and normal tissues collected from head and neck squamous cell carcinoma patients, we identified 35 miRNAs that are deregulated in both tumoral and peritumoral tissues as compared with normal matched samples. Four of these composed a miRNA signature that predicts head and neck squamous cell carcinoma local recurrence independently from prognostic clinical variables. The predictive power of the miRNA signature increased when using the expression levels derived from both the peritumoral and the tumoral tissues. The expression signal of the miRNAs composing the predictive signature correlated with the transcriptional levels of genes mostly associated with proliferation. Our results show that expression of miRNAs in tumor-surrounding mucosa may strongly contribute to the identification of head and neck squamous cell carcinoma patients at high risk of local recurrence.

Cytology and human papillomavirus testing on cytobrushing samples from patients with head and neck squamous cell carcinoma.

BACKGROUND: The increasing incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) highlights the need for simple and effective tools to evaluate head and neck lesions and their HPV status. The main objective of the current study was to investigate the association between abnormal cytology and HPV infection, assessed on cytobrushing samples, and histologically confirmed HNSCC. Second, the authors attempted to investigate whether HPV status on cytobrushing samples reflected that of the tumoral tissue. METHODS: A total of 164 samples from HNSCC, nonmalignant lesions, or healthy mucosae of the oral cavity and oropharynx were collected by cytobrushing in PreservCyt solution and evaluated by liquid-based cytology and Linear Array HPV genotyping test. All the findings from the cytologic samples were compared with those from the corresponding histologic samples. RESULTS: Patients with abnormal cytology had a significantly higher risk of having an HNSCC (odds ratio [OR], 9.18; 95% confidence inteval [95% CI], 3.27-26.49). The association was stronger for oral cancer (OR, 10.86; 95% CI, 2.51-51.06) than oropharyngeal cancer (OR, 8.45; 95% CI, 1.62-49.82). HPV positivity in the oropharyngeal cytobrushing was associated with a nearly 5-fold higher risk of having abnormal cytology (OR, 4.57; 95% CI, 1.57-13.57) as well as histologically proven oropharyngeal cancer (OR, 5.09; 95% CI, 1.09-31.61). Comparing the HPV status on cytologic and corresponding histologic samples from patients with HNSCC, we found that 90.4% of the cases were concordant (kappa, 0.796). CONCLUSIONS: Abnormal brushing cytology is strongly associated with a diagnosis of HNSCC, whereas HPV positivity on cytobrushing samples is only associated with oropharyngeal cancer. HPV testing on cytobrushing samples represents a valid option for the assessment of HPV infection in patients with oropharyngeal cancer.

Interobserver agreement in the interpretation of anal cytology.

BACKGROUND: Anal cytology represents a tool for anal cancer screening in high-risk populations. In addition to accuracy, the reproducibility of the interpretation is of key importance. The authors evaluated the agreement of anal cytologic interpretation between two cytopathologists. METHODS: Liquid-based cytologic slides from human immunodeficiency virus (HIV)-negative men who have sex with men (MSM) were evaluated by two readers with at least 10 years of expertise in cervical cytology. Cases with a discordant interpretation were reviewed, and a consensus was reached. Human papillomavirus (HPV) genotyping was performed using a proprietary HPV genotyping test. Unweighted and weighted Cohen kappa and 95% confidence interval (CI) values were calculated. RESULTS: Overall, 713 slides that were adequate for interpretation were evaluated (MSM: median age, 33 years). An HPV test was performed on 620 samples (87.0%). Considering a dichotomous interpretation (negative for intraepithelial lesion or malignancy vs. atypical squamous cells of undetermined significance or worse), the crude agreement between the two readers was 93.3% (kappa = 0.82; 95% CI, 0.77-0.87). Once a consensus for discordant cases was reached, the best agreement was found for the negative for intraepithelial lesion or malignancy category (511 of 528 samples; 96.8%), whereas the atypical squamous cells of undetermined significance category showed the lowest agreement (90 of 117 samples, 76.9%). Considering the individual cytologic categories, overall agreement was 92.1% (kappa = 0.85; 95% CI, 0.81-0.89). The discordant interpretations were not associated with high-risk HPV infection, HPV16 infection, or MSM age. CONCLUSIONS: The results indicating excellent interobserver agreement in this study substantiate the use of anal cytology in the setting of human immunodeficiency virus-negative MSM.

Combined TP53 status in tumor-free resection margins and circulating microRNA profiling predicts the risk of locoregional recurrence in head and neck cancer.

Locoregional recurrences represent a frequently unexpected problem in head and neck squamous cell carcinoma (HNSCC). Relapse often (10-30%) occurs in patients with histologically negative resection margins (RMs), probably due to residual tumor cells or hidden pre-cancerous lesions in normal mucosa, both missed by histopathological examination. Therefore, definition of a 'clean' or tumor-negative RM is controversial, demanding for novel approaches to be accurately explored. Here, we evaluated next generation sequencing (NGS) and digital PCR (dPCR) as tools to profile TP53 mutational status and circulating microRNA expression aiming at scoring the locoregional risk of recurrence by means of molecular analyses. Serial monitoring of these biomarkers allowed identifying patients at high risk, laying the ground for accurate tracking of disease evolution and potential intensification of post-operative treatments. Additionally, our pipeline demonstrated its applicability into the clinical routine, being cost-effective and feasible in terms of patient sampling, holding promise to accurately (re)-stage RMs in the era of precision medicine.

Incidence of abnormal anal cytology in HIV-infected and HIV-uninfected men who have sex with men.

INTRODUCTION: Anal cytology is used in the prevention of anal cancer, which disproportionally affects men who have sex with men (MSM). Data on the incidence of cytologic abnormalities in these individuals are scant. METHODS: MSM with baseline negative anal cytology and at least one further adequate cytology were included. Incidence rate for positive atypical squamous cells of undetermined significance (ASC-US+) was calculated. Kaplan-Meier curves were compared by log-rank test according to HIV status, baseline high-risk human papillomavirus (HPV) (high-risk HPV-negative, HPV16-positive, other high-risk HPV-positive [non-HPV16]) and high-risk HPV persistence (positive from baseline to the first ASC-US+ or last visit for those who remained cytologically negative). Cox univariate and multivariate analyses were performed. RESULTS: A total of 250 MSM were included: 52/153 (34.0%) HIV-uninfected MSM had an ASC-US+ report at follow-up (incidence: 13.1 × 100 person-years; 95% CI, 9.8-17.2); 48/97 (49.5%) HIV-infected MSM developed cytologic abnormalities (incidence: 16.0 × 100 person-years; 95% CI, 11.8-21.2). ASC-US+ incidence in HIV-uninfected and HIV-infected MSM did not differ significantly (p = .32). Kaplan-Meier curves did not differ significantly according to baseline high-risk HPV. Differences were significant between those with and without persistent high-risk HPVs, both among HIV-uninfected (p = .03) and HIV-infected MSM (p = .008). Age (adjusted hazard ratio [aHR], 0.98; 95% CI, 0.96-0.99), high-risk HPV persistence (aHR, 1.57; 95% CI, 1.02-2.39), and condomless receptive anal sex (aHR, 1.99; 95% CI, 1.31-3.03) were predictors for incident ASC-US+. CONCLUSIONS: Despite the limited number of subjects, in our study HIV-uninfected and HIV-infected MSM have a similar ASC-US+ incidence. Occurrence of ASC-US+ was significantly affected by age, high-risk HPV persistence, and condomless receptive anal sex. The assessment of HPV persistence might identify those MSM at higher risk for anal lesions.

HSF-1/miR-145-5p transcriptional axis enhances hyperthermic intraperitoneal chemotherapy efficacy on peritoneal ovarian carcinosis.

Hyperthermic intraperitoneal administration of chemotherapy (HIPEC) increases local drug concentrations and reduces systemic side effects associated with prolonged adjuvant intraperitoneal exposure in patients affected by either peritoneal malignancies or metastatic diseases originating from gastric, colon, kidney, and ovarian primary tumors. Mechanistically, the anticancer effects of HIPEC have been poorly explored. Herein we documented that HIPEC treatment promoted miR-145-5p expression paired with a significant downregulation of its oncogenic target genes c-MYC, EGFR, OCT4, and MUC1 in a pilot cohort of patients with ovarian peritoneal metastatic lesions. RNA sequencing analyses of ovarian peritoneal metastatic nodules from HIPEC treated patients unveils HSF-1 as a transcriptional regulator factor of miR-145-5p expression. Notably, either depletion of HSF-1 expression or chemical inhibition of its transcriptional activity impaired miR-145-5p tumor suppressor activity and the response to cisplatin in ovarian cancer cell lines incubated at 42 °C. In aggregate, our findings highlight a novel transcriptional network involving HSF-1, miR145-5p, MYC, EGFR, MUC1, and OCT4 whose proper activity contributes to HIPEC anticancer efficacy in the treatment of ovarian metastatic peritoneal lesions.

Claspin as a biomarker of human papillomavirus-related high grade lesions of uterine cervix.

BACKGROUND: Claspin is a nuclear protein involved in DNA replication and damage response and is a key mediator for the S-phase checkpoint. Claspin expression is significantly high in several human solid tumors. Furthermore, high levels of claspin have been found in cervical cancer cell lines. Nevertheless, no data are available regarding claspin expression in cervical tissues. METHODS: In order to investigate whether claspin immunoreactivity is related to the lesion severity and High-Risk (HR) HPV infection, we analyzed claspin expression by immunohistochemistry in a series of cervical biopsies which represent the steps occurring during cervical carcinogenesis (normal tissues, Cervical Intraepithelial Neoplasias 1, 2 and 3, Squamous Cell Carcinomas). All patients also had a cervico-vaginal sample for HPV testing, collected immediately before the colposcopy-guided biopsy. The HR-HPV DNA detection was performed by the HR-HPV Hybrid Capture 2 test. HPV genotyping was performed using the Linear Array HPV Genotyping Test. RESULTS: Our results evidenced a constant and significant increase of the rate of claspin positivity from the normal tissues to carcinomas (pχ2(trend) < 0.0001). In fact, the normal tissues displayed either no or faint claspin immunoreactivity, whereas a moderate/high positivity was observed in 16% of the CIN1, 76% of the CIN2, 87.5% of the CIN3 and 93.3% of the cancers. Moreover, we found a statistically significant correlation between claspin expression and HR-HPV infection (pχ2 < 0.0001), irrespective of the genotype. Finally, we demonstrated the feasibility of claspin immunostaining in cervical cytology. CONCLUSIONS: Our findings indicate that in vivo claspin expression is significantly related to HR-HPV infection and lesion grade both in histological and cytological samples. Therefore, the analysis of claspin expression could be clinically relevant in the diagnosis of HPV-related cervical lesions, in particular when applied to cervico-vaginal cytology. Moreover, giving information on the proliferation rate of each lesion, claspin immunostaining may contribute to the evaluation of progression risk, thus being helpful in patient management. Nevertheless, only large prospective studies may clarify the true clinical usefulness of claspin expression in distinguishing lesions with different progression potential.

Anal cytological abnormalities and epidemiological correlates among men who have sex with men at risk for HIV-1 infection.

BACKGROUND: The incidence of anal cancer, a Human Papillomavirus (HPV)-related neoplasia, has been increasing in recent decades, mainly in men who have sex with men (MSM). Cytological changes of the anal epithelium induced by HPV can be detected through an anal pap smear. This study aimed to evaluate the prevalence and epidemiological correlates of anal cytological abnormalities among relatively young MSM at risk for HIV-1 infection, to help clarify whether or not this population deserves further investigation to assess the presence of anal cancer precursor lesions. METHODS: MSM were recruited among attendees of a large STI clinic for a HIV-1 screening program. Anal samples, collected with a Dracon swab in PreservCyt, were used both for liquid-based cytology and HPV testing by the Linear Array HPV Genotyping Test. Data regarding socio-demographic characteristics and sexual behavior were collected in face-to-face interviews. RESULTS: A total of 346 MSM were recruited (median age 32 years). Overall, 72.5% of the individuals had an anal HPV infection, with 56.1% of them being infected by oncogenic HPV genotypes. Anal cytological abnormalities were found in 29.8% of the cases (16.7% ASC-US and 13.1% L-SIL). Presence of ASC-US+ was strongly associated with infection by any HPV type (OR = 4.21, 95% CI: 1.97-9.23), and particularly by HPV 16 and/or 18 (OR = 5.62, 95% CI: 2.33-13.81). A higher proportion of ASC-US+ was found in older MSM, in those with a higher number of lifetime partners and in those with a history of ano-genital warts. However, none of these variables or the others analyzed showed any significant association with abnormal cytological findings. CONCLUSIONS: The presence of anal cytological abnormalities in about one third of the recruited MSM and their strong association with HPV infection, in particular that caused by HPV 16 and/or 18, might provide a further complement to the data that now support the introduction of HPV vaccination among MSM to protect them from the development of HPV-associated diseases. Additional studies are needed to determine whether and how screening for anal cancer precursor lesions should be performed in younger MSM.

p16/Ki-67 dual staining in cervico-vaginal cytology: correlation with histology, Human Papillomavirus detection and genotyping in women undergoing colposcopy.

OBJECTIVES: To evaluate the CINtec PLUS assay (mtm laboratories), a new immunocytochemical method for the simultaneous detection of p16(INK4a) and Ki-67, in liquid-based cervico-vaginal cytology, investigating the association of the dual staining with HPV infection and genotyping as well as cytological and histological abnormalities. METHODS: 140 women with a cervico-vaginal sample obtained immediately before the colposcopy were enrolled. This cytological sample was used for HPV testing with the Linear Array HPV Genotyping Test, the dual staining with the CINtec PLUS kit and the morphology assessment. RESULTS: Cytology results were 38 NILM, 16 ASC-US, 32L-SIL, 54H-SIL or worse. 113 patients also had a colposcopy-guided biopsy, classified as 14 negative, 35 CIN1, 24 CIN2, 37 CIN3, 3 invasive SCC. A strong association between p16/Ki-67 and HR-HPV infection was found (COR=6.86, 95% CI: 1.84-31.14). Importantly, the association between p16/Ki-67 positivity and HPV16 and/or 18 infection was 2-fold stronger compared to that with the infection by other HR-HPV types (COR=9.92, 95% CI: 2.39-47.77 vs COR=4.20, 95% CI: 0.99-20.87). In addition, p16/Ki-67 positivity rate significantly increased with the severity of the cytological and histological abnormalities (p<0.05 in both cases). p16/Ki-67 positivity resulted strongly associated with a CIN2+ diagnosis (COR=10.86 95% CI: 4.16-29.12). CONCLUSIONS: This preliminary study evidenced that p16/Ki-67 immunostaining might have a relevant clinical role, since the dual staining was significantly associated with HR-HPV infection, particularly with HPV 16 and 18, and the increasing grade of the cervical lesions, the positivity for this biomarker being strongly related to the presence of a CIN2+ lesion.

Incidence and clearance of anal high-risk Human Papillomavirus infection and their risk factors in men who have sex with men living with HIV.

HIV-infected men who have sex with men (MSM) display the highest prevalence of anal infection by high-risk Human Papillomaviruses (hrHPVs) and incidence of anal carcinoma. Anal specimens were genotyped by the Linear Array. Incidence and clearance of anal infection by hrHPVs, hrHPVs other than HPV16, low-risk HPVs, and four individual types (6,11,16,18) were estimated using a two-state Markov model. Determinants for incidence and clearance were assessed by logistic regression. Overall, 204 individuals were included (median age 42 years, IQR = 34-49). For hrHPVs, incidence and clearance rates were 36.1 × 1000 person-months (p-m) (95% CI 23.3-56.5) and 15.6 × 1000 p-m (95% CI 10.7-23.3), respectively. HPV16 showed a higher incidence than HPV18 (10.2 vs. 7.2 × 1000 p-m). Its clearance was more than twofold lower than that of HPV18 (30.1 vs. 78.2 × 1000 p-m). MSM receiving cART displayed a 68% to 88% decrease in risk of acquiring hrHPVs, hrHPVs other than HPV16, HPV16, and HPV18 (adjusted Hazard Ratio [aHR] 0.13, 95% CI 0.02-0.67; aHR 0.22, 95% CI 0.06-0.78; aHR 0.32, 95% CI 0.12-0.90; aHR 0.12, 95% CI 0.04-0.31, respectively) than patients not treated. A nadir CD4 + count < 200 cells/mm3 significantly reduced the clearance of hrHPVs other than HPV16 (aHR 0.39, 95% CI 0.17-0.90). cART use reduces the risk of acquiring anal infection by hrHPVs.

Prevalence of 13 polyomaviruses in actinic keratosis and matched healthy skin samples of immunocompetent individuals.

BACKGROUND: Actinic keratosis (AK) is a precursor of cutaneous squamous cell carcinoma (cSCC). UV radiation is the major risk factor for AK, but certain human papillomaviruses (HPVs) of the beta genus are also involved in its development. Differently, the role of polyomaviruses (PyVs) in skin carcinogenesis is still debated. Fiftheen PyVs have been isolated from human tissues so far, including Merkel cell polyomavirus (MCPyV), the aetiological agent of Merkel cell carcinoma. METHODS: The presence of 13 PyVs was assessed in skin samples from AK patients (n = 342). Matched fresh-frozen scrapings from healthy skin (HS) and AK lesions from 242 patients, and formalin-fixed paraffin-embedded AK biopsies from a different cohort of 100 patients were analyzed by multiplex PyVs genotyping assay. RESULTS: The most frequent lesion site was the scalp in men (27.3%), and the cheek area in women (29.0%). Differences between men and women were significant for the scalp, the cheek area and the lips. Almost all the scrapings were PyV-positive (HS: 89.7%, AK: 94.6%; p = 0.04). The three most frequent PyVs were MCPyV, HPyV6 and JCPyV (HS: 87.2%, 58.7%, 6.6%, respectively; AK: 88.8%, 51.2%, 9.9%, respectively). HPyV9, TSPyV, BKPyV, HPyV7, LIPyV and SV40 were detected in < 2% of the scrapings. In most cases, matched HS and AK scrapings were both positive (MCPyV: 78.1%, HPyV6: 41.7%), or both negative for the individual genotypes (for the remaining PyVs). PyV prevalence in AK biopsies was 22.0%. Only MCPyV (21.0%) and HPyV6 (3.0%) were detected in these samples. CONCLUSIONS: PyV prevalence in HS and AK scrapings was high, but detection of PyVs exclusively in AK scrapings was rare. PyV positivity rate in AK biopsies was modest. Further research is need to reach firm conclusions regarding the role of these viruses in AK development.

A Real-World Systematic Analysis of Driver Mutations' Prevalence in Early- and Advanced-Stage NSCLC: Implications for Targeted Therapies in the Adjuvant Setting.

The approval of osimertinib for adjuvant treatment of stage I-II-III EGFR-mutated NSCLC (early stage) represents a paradigm shift, raising the question of whether other genotype-matched therapeutics approved for advanced-stage NSCLC can also provide clinical benefit in the adjuvant setting. However, there is a paucity of real-world data on the prevalence of actionable genomic alterations (GAs) in early-stage NSCLC. We used next-generation sequencing, complemented by immunohistochemistry and fluorescence in situ hybridization, to screen our single-institution cohort of 1961 NSCLC consecutive cases for actionable molecular targets. The prevalence of actionable GAs was comparable in early versus advanced-stage NSCLC, the only exception being KRAS mutations (more frequent in early-stage cases). Consistent with advanced-stage tumors being more aggressive, co-occurrence of TP53 and EGFR GAs as well as copy number gains were less frequent in early-stage tumors. EGFR mutations and high expression of PD-L1 were inversely associated, whereas KRAS mutations and high PD-L1 reactivity showed positive association. Recapitulating advanced-stage tumors, early-stage NSCLC had the highest share of EGFR mutations in lepidic and acinar subtypes. Resected lepidic tumors contained the highest proportion of the KRAS G12C actionable variant. These results, obtained with routine diagnostic technologies in an unselected clinical setting, provide a significant addition of real-world data in early-stage NSCLC.

Diagnostic and prognostic validity of the human papillomavirus E6/E7 mRNA test in cervical cytological samples of HC2-positive patients.

The study aimed to assess the clinical utility in identifying CIN2 or worse (CIN2+), of the Pretect HPV-Proofer test for E6/E7 mRNA detection in Hybrid Capture 2 (HC2)-positive patients, who underwent colposcopy. In particular, the study analyzed the mRNA test performance as the third test in a subgroup of HC2+ patients with less severe than high-grade squamous intraepithelial lesions (HSIL-). We analyzed 464 cervico-vaginal samples by liquid-based cytology (LBC) and PreTect HPV-Proofer. Moreover 231 patients also had a biopsy at baseline and 75, with HSIL-, were followed up within 2 years by LBC, colposcopy, and histology when indicated. The highest sensitivity for CIN2+ belonged to the mRNA compared to LBC, at the HSIL+ threshold (72% vs. 58%), whereas the LBC showed the highest specificity and positive predictive value (PPV) (99 and 93% vs. 73 and 39%, respectively). Focusing on the 408 HSIL- patients, the mRNA positivity was significantly more associated with CIN2+ than CIN2- lesions (p < 0.0001). Moreover, among the 75 HSIL- followed up patients, the mRNA displayed high longitudinal Specificity (89%), even if the sensitivity and the PPV were low (50 and 20%, respectively). The present data suggest that the mRNA test may have a diagnostic and a potentially prognostic role in HC2+/HSIL- patients.

High expression of HLA-E in colorectal carcinoma is associated with a favorable prognosis.

BACKGROUND: Human Leukocyte Antigen (HLA)-E is a non-classical class I HLA molecule that can be stabilized by ligands donated by other classical (HLA-A, -B, -C) and non-classical (HLA-G) family members. HLA-E engages a variety of immune receptors expressed by cytotoxic T lymphocytes (CTLs), Natural killer (NK) cells and NK-CTLs. In view of the opposing outcomes (activation or inhibition) of the different HLA-E receptors, the preferred role (if any) of HLA-E expressed in vivo on tumor cells remains to be established. METHODS: Taking advantage of MEM-E/02, a recently characterized antibody to denatured HLA-E molecules, HLA-E expression was assessed by immunohistochemistry on an archival collection (formalin-fixed paraffin-embedded) of 149 colorectal primary carcinoma lesions paired with their morphologically normal mucosae. Lymphoid infiltrates were assessed for the expression of the HLA-E-specific, inhibitory, non-rearranging receptor NKG2A. RESULTS: High HLA-E expression did not significantly correlate with the expression of classical HLA-B and HLA-C molecules, but it did correlate with high expression of its preferential ligand donor HLA-A. In addition, it correlated with lymphoid cell infiltrates expressing the inhibitory NKG2A receptor, and was an independent predictor of good prognosis, particularly in a subset of patients whose tumors express HLA-A levels resembling those of their paired normal counterparts (HLA-A). Thus, combination phenotypes (HLA-Elo-int/HLA-AE and HLA-Ehi/HLA-AE) of classical and non-classical class I HLA molecules mark two graded levels of good prognosis. CONCLUSIONS: These results suggest that HLA-E favors activating immune responses to colorectal carcinoma. They also provide evidence in humans that tumor cells entertain extensive negotiation with the immune system until a compromise between recognition and escape is reached. It is implied that this process occurs stepwise, as predicted by the widely accepted 'immunoediting' model.

Comparative evaluation of different DNA extraction methods for HPV genotyping by linear array and INNO-LiPA.

In order to investigate the influence of DNA extraction on two PCR-based HPV genotyping tests (Linear Array, Roche and INNO-LiPA Extra, Innogenetics), three different procedures were used to purify DNA from 28 cervico-vaginal samples tested previously by the Hybrid Capture 2: the AmpliLute Liquid Media Extraction kit (Roche), the QIAamp DNA Blood mini kit (QIAGEN), and the NucliSENS EasyMAG automated platform (bioMérieux). All HC2-positive samples were found positive by both assays, independently of the extract used. Type-specific concordance (i.e., identical HPV type-specific profile in all the extracts of the same sample) was observed in 55% and 75% of the cases testing samples by the Linear Array and the INNO-LiPA, respectively. Using the DNA extracted with the two manual methods the results were concordant in 75% of the cases both for the Linear Array and the INNO-LiPA. When comparing the Linear Array results obtained on either of the two manual extracts with those obtained following automated extraction, 65% of the samples showed type-specific concordance in both cases. The INNO-LiPA results were concordant in 80% of the cases comparing the AmpliLute versus the automated extract, while concordant results were observed in 90% of the cases when comparing the QIAGEN versus the automated extract. In conclusion, the Linear Array and INNO-LiPA results are affected by the method of DNA extraction. Consequently, different HPV type-specific profiles may be observed using different extracts of the same sample. The use of consistent protocols for DNA purification is a priority to guarantee intra-assay reproducibility over time.