LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer.

BACKGROUND AND PURPOSE: Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation. RESULTS: In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival-OS: 28.1% for LINE-1 methylation < 35% vs. 69.1% for ≥ 55%; p < 0.0001). When LINE-1 methylation was dichotomized as < 55% versus ≥ 55%, interaction with HPV16 emerged: compared with hypermethylated HPV16-positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR 4.83, 95% CI 2.24-10.38) and progression (HR 4.54, 95% CI 2.18-9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53 ≥ 50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53 ≥ 50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine-phosphate-guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences. CONCLUSIONS: Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations and lead to altered gene expression in OPSCC.

Multicentre study on resection margins in carcinoma of the oral cavity, oro-hypopharynx and larynx.

Objective: The prognostic significance of the resection margins is still subject of conflicting opinions. The purpose of this paper is to report the results of a study on the margins in carcinoma of the oral cavity, oro-hypopharynx and larynx. Methods: A multicentre prospective study was carried out between 2015 and 2018 with the participation of 10 Italian reference hospitals. The primary objective was to evaluate local control in patients with well-defined clinical characteristics and comprehensive histopathological information. Results: During the study period, 455 patients were enrolled; the minimum follow-up was 2 years. Previous treatment, grading and fresh specimen examination were identified as risk factors for local control in multivariate analysis. On the basis of these results, it seems possible to delineate "risk profiles" for different oncological outcomes. Discussion: The prognostic significance of the margins is reduced, and other risk factors emerge, which require diversified treatment and follow-up. Conclusions: Multidisciplinary treatment with adjuvant therapy, if indicated, reduces the prognostic importance of margins. Collaboration with a pathologist is an additional favourable prognostic factor and quality indicator.An appendix with literature review is present in the online version.

miR-9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC.

Radiotherapy (RT) plus the anti-EGFR monoclonal antibody Cetuximab (CTX) is an effective combination therapy for a subset of head and neck squamous cell carcinoma (HNSCC) patients. However, predictive markers of efficacy are missing, resulting in many patients treated with disappointing results and unnecessary toxicities. Here, we report that activation of EGFR upregulates miR-9 expression, which sustains the aggressiveness of HNSCC cells and protects from RT-induced cell death. Mechanistically, by targeting KLF5, miR-9 regulates the expression of the transcription factor Sp1 that, in turn, stimulates tumor growth and confers resistance to RT+CTX in vitro and in vivo. Intriguingly, high miR-9 levels have no effect on the sensitivity of HNSCC cells to cisplatin. In primary HNSCC, miR-9 expression correlated with Sp1 mRNA levels and high miR-9 expression predicted poor prognosis in patients treated with RT+CTX. Overall, we have discovered a new signaling axis linking EGFR activation to Sp1 expression that dictates the response to combination treatments in HNSCC. We propose that miR-9 may represent a valuable biomarker to select which HNSCC patients might benefit from RT+CTX therapy.

CDKN1B mutation and copy number variation are associated with tumor aggressiveness in luminal breast cancer.

The CDKN1B gene, encoding for the CDK inhibitor p27kip1 , is mutated in defined human cancer subtypes, including breast, prostate carcinomas and small intestine neuroendocrine tumors. Lessons learned from small intestine neuroendocrine tumors suggest that CDKN1B mutations could be subclonal, raising the question of whether a deeper sequencing approach could lead to the identification of higher numbers of patients with mutations. Here, we addressed this question and analyzed human cancer biopsies from breast (n = 396), ovarian (n = 110) and head and neck squamous carcinoma (n = 202) patients, using an ultra-deep sequencing approach. Notwithstanding this effort, the mutation rate of CDKN1B remained substantially aligned with values from the literature, showing that essentially only hormone receptor-positive breast cancer displayed CDKN1B mutations in a relevant number of cases (3%). However, the analysis of copy number variation showed that another fraction of luminal breast cancer displayed loss (8%) or gain (6%) of the CDKN1B gene, further reinforcing the idea that the function of p27kip1 is important in this type of tumor. Intriguingly, an enrichment for CDKN1B alterations was found in samples from premenopausal luminal breast cancer patients (n = 227, 4%) and in circulating cell-free DNA from metastatic luminal breast cancer patients (n = 59, 8.5%), suggesting that CDKN1B alterations could correlate with tumor aggressiveness and/or occur later during disease progression. Notably, many of the identified somatic mutations resulted in p27kip1 protein truncation, leading to loss of most of the protein or of its C-terminal domain. Using a gene-editing approach in a luminal breast cancer cell line, MCF-7, we observed that the expression of p27kip1 truncating mutants that lose the C-terminal domains failed to rescue most of the phenotypes induced by CDKN1B gene knockout, indicating that the functions retained by the C-terminal portion are critical for its role as an oncosuppressor, at least in luminal breast cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

A Novel Kindred with Familial Gastrointestinal Stromal Tumors Caused by a Rare KIT Germline Mutation (N655K): Clinico-Pathological Presentation and TKI Sensitivity.

Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, are characterized by activating mutations in KIT or PDGFRA genes. The vast majority of GISTs are sporadic, but rare hereditary forms have been reported, often featuring multifocality and younger age of onset. We here report the identification of a novel kindred affected by familial GIST caused by a KIT germline mutation in exon 13 (N655K). No family affected by hereditary GIST due to this KIT variant has been reported in literature so far. We were able to track the mutation in three members of the family (proband, mother, and second-degree cousin), all affected by multiple GISTs. Due to its rarity, the N655K variant is poorly characterized. We conducted in vitro drug sensitivity assays that indicated that most tyrosine kinase inhibitors (TKIs) currently included in the therapeutic armamentarium for GISTs have a limited inhibitory activity toward this mutation. However, when compared to a classical imatinib-resistant KIT mutation (T670I), N655K was slightly more sensitive to imatinib, and encouraging responses were observed with last-generation TKIs.

Diagnostic Accuracy of Pleural Fluid Cytology, Carcinoembryonic Antigen and C-Reactive Protein Together in Patients With Pulmonary Metastases and Malignant Pleural Effusion.

BACKGROUND/AIM: Pleural effusion (PE) has a heterogeneous aetiology, and differential diagnosis between benign and malignant disease may require invasive procedures in up to 60% of cases. The sensitivity of pleural cytology is limited, and several strategies have been tested to reduce the need of invasive diagnostic approaches. The aim of this study was to evaluate the usefulness of pleural fluid cytology, compared to, and combined with, carcinoembryonic antigen (CEA), C reactive protein (CRP), and lactate dehydrogenase (LDH) assay of pleural fluid (PF) in patients with a history of cancer, exudative non-purulent PE, and suspicion of malignant PE on imaging studies. PATIENTS AND METHODS: The medical records of 40 patients with pulmonary metastases and malignant PE, and 57 controls with benign exudative PE were reviewed. All the patients underwent pleural cytology and CEA, CRP, and LDH assay before VATS-guided biopsy. RESULTS: The sensitivity and specificity were 55.0% and 98.2% (cytology), 35.0% and 98.2% (CEA), 92.5% and 71.9% (CRP), 70.0% and 54.4% (LDH). The multivariate analysis excluded LDH, and the final AUC (cytology+CEA+CRP) was 0.894. CONCLUSION: In all patients with a history of cancer and PE of uncertain origin, the combination of PF cytology plus pleural CEA and CRP assay together should be suggested to recognize malignant plural effusion (MPE), minimising the use of unnecessary invasive investigations.

Diagnosis of Malignant Pleural Effusion Using CT Scan and Pleural-Fluid Cytology Together. A Preliminary Case-Control Study.

BACKGROUND/AIM: The purposes of this study were to evaluate the usefulness of chest computed tomographic (CT) scan plus pleural fluid cytology (PFC) together in patients with malignant pleural effusion (PE), and to compare the results of these diagnostic tools in patients with malignant PE due to non-small-cell lung cancer and pulmonary metastases from other malignancies. PATIENTS AND METHODS: The medical records of 185 patients with PE, who underwent chest CT, PFC and video-assisted thoracoscopy (VATS) thoracentesis followed by VATS-guided biopsy for diagnostic purpose, were reviewed. At the final diagnosis, 123 (66.5%) patients had malignant PE (cases), and 62 (33.5%) had benign PE (controls). RESULTS: Overall, the sensitivity, specificity, and accuracy of CT and PFC were 65.0% vs. 67.5% 98.4% vs. 98.4%, and 76.2% vs. 77.8%, respectively. The combination of CT plus PFC significantly improved sensitivity (86.2%, p=0.003) and accuracy (90.8%, p=0.02). CONCLUSION: CT and PFC used together may lead to approximately 100% specificity and >90% sensitivity in distinguishing between benign and malignant PE.

Interleukin-2 chronotherapy for metastatic renal cell carcinoma: Results of a phase I-II study.

BACKGROUND: Interleukin-2 (IL-2) was the cornerstone treatment for metastatic renal cell carcinoma (RCC) until the advent of tyrosine kinase inhibitors, but it still has therapeutic value. As a single bolus of IL-2 causes toxicity, there is interest in administration regimens with better tolerability and efficacy. Chronotherapy is the administration of therapy according to the circadian rhythm's influence on the immune and hormonal systems. This phase I-II trial evaluated the safety of IL-2 chronotherapy in metastatic RCC patients and determined the maximum tolerated dose. The secondary objective was to identify prognostic factors for survival. METHODS: Three chronomodulation schedules (5:00-13:00, 13:00-21:00, and 21:00-5:00) were tested. Each schedule was an 8-h IL-2 infusion, with a Gaussian distribution of drug concentration peaking at 4 h. To identify the maximum tolerated dose, the dose for different patients was escalated from 2 MIU/m2 (level I) to 18.6 MIU/m2 (level VI). RESULTS: Thirty patients were enrolled and completed treatment. Two patients were treated at 5:00-13:00, 15 at 13:00-21:00, and 13 at 21:00-5:00. Nine cases of grade 3 toxicity occurred in 7 patients at the highest dose (18.6 MIU/m2); no grade 4 toxicity occurred. The maximum tolerated dose was 14.0 MUI/m2. Patients were followed for a median of 16 months (range, 2-107). One patient was lost to follow-up, 3 patients were alive at last contact, and 26 patients died. Six patients achieved long-term survival (≥48 months). There was one complete response, four partial responses, 11 cases of stable disease and 14 of progressive disease. The response rate was 16% (5/30) and disease-control rate was 53% (16/30). Median progression-free survival was 4.5 months, and median overall survival was 14.5 months. Kaplan-Meier analyses revealed significant associations between overall survival and ECOG performance score (0 vs. 1-2), MSKCC score (0-2 vs. ≥ 3), IMDC risk score (0-2 vs. ≥ 3), IL-2 dose level (IV-VI vs. I-III), and prolactin (increase vs. no increase), and but not for chronotherapy schedule. CONCLUSION: IL-2 chronotherapy appears to be safe, moderately toxic and active in metastatic RCC. It may represent a new modality of IL-2 administration for these patients.

Esophageal ultrasound with ultrasound bronchoscope (EUS-B) guided left adrenal biopsy: Case report with review of literature.

A 45-year old man, chronic smoker with a pancoast tumor due to squamous cell carcinoma has been described. The initial diagnosis of lung carcinoma was based on a scant tissue so the exact cell typing was not possible. The initial treatment consisted of platinum based chemotherapy and radiotherapy. He developed a left adrenal lesion on treatment. There was a possibility of metachronous primary. Also, a large tissue sample was required for tumor markers. The lung mass was difficult to access and was static on treatment. A left adrenal biopsy was considered to be more appropriate. A novel approach for left adrenal lesion with esophageal ultrasound using ultrasound bronchoscope (EUS-B) was successfully performed. This article is aimed at describing the use of EUS-B for transdiaphragmatic structures.

Correction to: Unusual presentation of recurrent papillary thyroid microcarcinoma with neck muscles and skin dissemination.

The original version of this article unfortunately contained a mistake. The title of this article was rendered incorrectly; the correct title is as follows: Unusual presentation of recurrent papillary thyroid microcarcinoma with neck muscles and skin dissemination.

Teaching Digital Pathology: The International School of Digital Pathology and Proposed Syllabus.

Digital pathology is an interdisciplinary field where competency in pathology, laboratory techniques, informatics, computer science, information systems, engineering, and even biology converge. This implies that teaching students about digital pathology requires coverage, expertise, and hands-on experience in all these disciplines. With this in mind, a syllabus was developed for a digital pathology summer school aimed at professionals in the aforementioned fields, as well as trainees and doctoral students. The aim of this communication is to share the context, rationale, and syllabus for this school of digital pathology.

Prognostic significance of LINE-1 hypomethylation in oropharyngeal squamous cell carcinoma.

BACKGROUND: Inclusion of new biomarkers to improve a personalized treatment approach for oropharyngeal squamous cell carcinoma (OPSCC) is urgently needed. Hypomethylation of the Long interspersed nucleotide element-1 (LINE-1) repetitive elements, a widely accepted surrogate of overall genomic DNA methylation content, was found to be associated with a poor prognosis in several cancers. At present, no studies have investigated the influence of LINE-1 methylation levels on OPSCC relapse. The main goal of this study was the evaluation of the prognostic value of LINE-1 methylation status in predicting early tumor relapse in locally advanced OPSCC. METHODS: We retrospectively reviewed a cohort of 77 patients with stage III-IVB OPSCC. Methylation of LINE-1 repetitive sequences was evaluated by real-time quantitative methylation-specific PCR in formalin-fixed paraffin-embedded tissues. The prognostic relevance of LINE-1 methylation was assessed by comparing patients who relapsed within 2 years from the end of treatment (cases) with those who did not (controls). Results were validated in an independent cohort of 33 patients with OPSCC. RESULTS: With respect to early OPSCC relapse, the mean LINE-1 methylation level was significantly lower in relapsed cases than in control group (p < 0.01). Interestingly, LINE-1 methylation was lower in relapsed cases than in controls in both HPV16-negative and HPV16-positive OPSCC patients, even if statistical significance was reached only for the former group (p = 0.01). LINE-1 methylation levels were also significantly reduced in relapsed cases with respect to the controls in OPSCC current smokers (p = 0.02). Consistently, in HPV16-negative current smokers, OPSCC relapse was significantly associated with decreased levels of LINE-1 methylation (p = 0.02). Using logistic regression model, we found that patients with hypomethylated LINE-1 were associated with a 3.5 higher risk of early relapse than hypermethylated ones (OR = 3.51; 95% CI 1.03-12.00). Adjustment for potential confounders did not substantially change the risk magnitude. Results from the validation cohort confirmed the lower LINE-1 methylation in patients who early relapsed compared to relapse-free patients. CONCLUSIONS: LINE-1 hypomethylation is associated with higher risk of early relapse in stage III-IVB OPSCC. Further validation in a prospective study is needed for its application in daily clinical practice.

An Integrated Approach Identifies Mediators of Local Recurrence in Head and Neck Squamous Carcinoma.

Purpose: Head and neck squamous cell carcinomas (HNSCCs) cause more than 300,000 deaths worldwide each year. Locoregional and distant recurrences represent worse prognostic events and accepted surrogate markers of patients' overall survival. No valid biomarker and salvage therapy exist to identify and treat patients at high-risk of recurrence. We aimed to verify if selected miRNAs could be used as biomarkers of recurrence in HNSCC.Experimental Design: A NanoString array was used to identify miRNAs associated with locoregional recurrence in 44 patients with HNSCC. Bioinformatic approaches validated the signature and identified potential miRNA targets. Validation experiments were performed using an independent cohort of primary HNSCC samples and a panel of HNSCC cell lines. In vivo experiments validated the in vitro results.Results: Our data identified a four-miRNA signature that classified HNSCC patients at high- or low-risk of recurrence. These miRNAs collectively impinge on the epithelial-mesenchymal transition process. In silico and wet lab approaches showed that miR-9, expressed at high levels in recurrent HNSCC, targets SASH1 and KRT13, whereas miR-1, miR-133, and miR-150, expressed at low levels in recurrent HNSCC, collectively target SP1 and TGFß pathways. A six-gene signature comprising these targets identified patients at high risk of recurrences, as well. Combined pharmacological inhibition of SP1 and TGFß pathways induced HNSCC cell death and, when timely administered, prevented recurrence formation in a preclinical model of HNSCC recurrence.Conclusions: By integrating different experimental approaches and competences, we identified critical mediators of recurrence formation in HNSCC that may merit to be considered for future clinical development. Clin Cancer Res; 23(14); 3769-80. ©2017 AACR.

In stage II/III lymph node-positive breast cancer patients less than 55 years of age, keratin 8 expression in lymph node metastases but not in the primary tumour is an indicator of better survival.

Axillary lymph node status is one of the most important prognostic variables for breast cancer (BC). To investigate and understand the clinical, histopathological and biological factors that affect prognosis in node-positive young breast cancer patients, we compared the phenotype of 100 primary tumours with their corresponding loco-regional lymph node (LN) metastases using conventional immunohistochemistry (IHC) markers currently in use for molecular classification of breast cancer. By comparing the expression of ER, PR, HER-2, Ki67, K8, K5/6 and vimentin, we found that expression of HER-2, Ki67, K8 and vimentin is frequently lost in lymph node metastases. Between the primary tumour and corresponding lymph node metastases, expression of keratins K8 and K5/6 significantly changed. Expression of K8 in lymph node metastases, but not in primary tumours, segregates patients in two sub-groups with different outcomes. Survival of patients with K8-positive LN metastases at 5 years in comparison with patients with K8-negative LN metastases was 75 vs 48 %, at 10 years 62 vs 22 % and at 20 years 53 vs 14 % (p < 0.001). K8 immunostaining of tissue from the lymph node metastasis allows defining a sub-group of lymph node-positive BC patients with a highly unfavourable outcome, for whom therapeutic options might have to be reconsidered.

Are breast cancer molecular classes predictive of survival in patients with long follow-up?

In this study we investigate the clinical outcomes of 305 breast cancer (BC) patients, aged 55 years or younger, with long follow-up and according to intrinsic subtypes. The cohort included 151 lymph node negative (LN-) and 154 lymph node positive (LN+) patients. Luminal A tumors were mainly LN-, well differentiated, and of stage I; among them AR was an indicator of good prognosis. Luminal B and HER2 positive nonluminal cancers showed higher tumor grade and nodal metastases as well as higher proliferation status and stage. Among luminal tumors, those PR positive and vimentin negative showed a longer survival. HER2-positive nonluminal and TN patients showed a poorer outcome, with BC-specific death mostly occurring within 5 and 10 years. Only luminal tumor patients underwent BC death over 10 years. When patients were divided in to LN- and LN+ no differences in survival were observed in the luminal subgroups. LN- patients have good survival even after 20 years of follow-up (about 75%), while for LN+ patients survival at 20 years (around 40%) was comparable to HER2-positive nonluminal and TN groups. In conclusion, in our experience ER-positive breast tumors are better divided by classical clinical stage than molecular classification, and they need longer clinical follow-up especially in cases with lymph node involvement.

Detection of HPV E7 oncoviral protein in cervical lesions by a new antibody.

The availability of a new E7 mAb-based immunohistochemistry (IHC) detection assay, Cervimax, allowed for the first time reliable testing of the E7 protein marker in formalin-fixed and paraffin-embedded tissues from cervical lesions. E7-specific IHC staining patterns were compared with those patterns of cervical cancer biomarkers, including the viral capsid protein L1 and the surrogate biomarkers p16INK4A, p53, hTERT, ubiquitin, and Ki67. The use of a tissue microarray of 138 cervical tissue cores from different pathologic stages allowed for a first profiling of the various markers in comparison with E7. Cervimax staining patterns closely overlap with those from p16INK4A and human telomerase reverse transcriptase (hTERT) in IHC staining for high-grade cervical intraepithelial neoplasia and squamous cell carcinoma. In squamous cell carcinoma, E7 immunostaining matched better to hTERT and ubiquitin profiles. On the contrary, the pattern of E7 and L1 were different in all the squamous lesions. The nuclear staining of E7 significantly discriminates between low-grade cervical intraepithelial neoplasia and high-grade cervical intraepithelial neoplasia in the basal, parabasal, and superficial layers. The results obtained in the presented pilot study suggest E7 as a valid candidate biomarker for all the stages of the malignant progression of cervical cancer; however, more extensive studies are needed to confirm the causal effect of the oncoprotein E7 in the diagnosis of human papillomavirus-induced diseases. These results also suggest that the diagnostic interpretation of cervical lesions could be increased by the combination of E7 and L1 staining in the evaluation of risk of progression, because related to different phases of viral integration.