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.

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.

Prediction of response to neoadjuvant chemotherapy by MammaTyper® across breast cancer subtypes: A retrospective cross-sectional study.

BACKGROUND: Neoadjuvant chemotherapy (NACT) is widely used in the treatment of triple-negative and HER2-positive breast cancer (BC), but its use in estrogen receptor (ER) and/or progesterone receptor (PR) positive/HER2-negative BC is questioned because of the low pathologic complete response (pCR) rates. This retrospective study assessed the mRNA-based MammaTyper® assay's capability of predicting pCR with NACT, and ER, PR, Ki67, and HER2 status at immunohistochemical (IHC) through transcriptomics. METHODS: Diagnostic biopsies from 76 BC patients treated at the Cremona Hospital between 2012-2018 were analyzed. Relative mRNA expression levels of ERBB2, ESR1, PGR, and MKI67 were measured using the MammaTyper® kit and integrated into a pCR score. Predicting capability of pCR and standard IHC biomarkers could be assessed with ROC curves in 75 and 76 patients, respectively. RESULTS: Overall, 68.0% patients obtained a MammaTyper® high-score and 32.0% a MammaTyper® low-score. Among high-score patients, 62.7% achieved pCR, compared to 16.7% in the low-score group (p = 0.0003). The binary MammaTyper® score showed good prediction of pCR in the overall cohort (area under curve [AUC] = 0.756) and in HR+/HER2-negative cases (AUC = 0.774). In cases with residual disease, the continuous MammaTyper® score correlated moderately with residual tumor size and decrease in tumor size. MammaTyper® showed substantial agreement with IHC for ESR1/ER and ERBB2/HER2, and moderate agreement for PGR/PR and MKI67/Ki67. CONCLUSION: Overall, MammaTyper® pCR score may serve as a standardized tool for predicting NACT response in HR+/HER2-negative BC, potentially guiding treatment strategies. Additionally, it could provide a more standardized and reproducible assessment of ER, PR, HER2, and Ki67 status.

Alteration of G1/S transition regulators influences recurrences in head and neck squamous carcinomas.

Head-Neck Squamous Cell Carcinoma (HN-SCC) is a clinically challenging disease associated with a high mortality rate. The chemo-radiotherapy treatments that aim to preserve the organ represent the current gold standard therapy for advanced laryngeal disease, reserving surgery only for non-responsive or relapsed cases. Despite these aggressive approaches, local persistent or recurrent disease remains the primary cause of treatment failure but we still do not have known factors and/or markers able to predict the outcome of the disease and in particular the risk of local relapse. Here we address this point on a series of 54 cases of HN-SCC for whom the presence of local relapse was known. Using immunohistochemistry (IHC) analysis to evaluate protein expression and localization in the recurrence free and recurrence positive samples, we studied the expression of key cell cycle regulators including p53, p16, p27, pRB, Cyclin D1, Cyclin D3, and Stathmin. Overall by analyzing seven different cell cycle regulators we can hypothesize that the alteration of G1/S regulation represents a fundamental event in the onset/progression of HN-SCC cancers and that the associate use of Cyclin D1/p16 expression should be considered as a possible biomarker toward the identification of those patients that will probably develop a recurrent disease and thus should benefit of a more aggressive treatment.

Correlation between IgA tissue transglutaminase antibody ratio and histological finding in celiac disease.

OBJECTIVES: Positivity of both immunoglobulin A anti-tissue transglutaminase (TTG) and anti-endomysium antibodies (EMA) has a positive predictive value of nearly 100% for celiac disease (CD). The objective of the present study was to evaluate whether patients of any age, with high pretest probability of CD and high titre of anti-TTG and EMA positivity, have a high probability of intestinal damage and may not require the biopsy for final diagnosis. METHODS: A retrospective analysis of 412 consecutively referred patients, age range 10 months to 72 years, who underwent small-bowel biopsy for suspicion of CD and positivity to both anti-TTG and EMA, was performed at 4 Italian centers. Biopsies were evaluated independently by 2 pathologists using Marsh modified classification; in cases of dissimilar results, a third pathologist examined the biopsy. The final histological finding diagnosis was expressed as the prevalent or highest score assigned by the pathologist board. RESULTS: Three hundred ninety-six patients (96.1%) had histological findings consistent with CD (grade 2 and 3a, 3b, or 3c of modified Marsh classification). An anti-TTG ratio ≥ 7 was able to identify with the 3 assays used (Celikey, anti-TTG immunoglobulin A, EuTTG) all of the patients with significant mucosal damage (Marsh ≥ 2) independent of age and sex; specificity and positive predictive value were 100%. An anti-TTG ratio >20 was more specific (99.8%) for identification of patients with villous atrophy (Marsh 3 a, b, or c). CONCLUSIONS: Patients with positivity of anti-TTG ≥ 7-fold cutoff, confirmed by positivity to EMA, have a high-degree probability of duodenal damage. In selected conditions, a duodenal biopsy may be avoided and a confirmed greatly positive anti-TTG result could be the basis to prescribe a gluten-free diet.

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.

Epithelioid melanoma of the rectum: reappraisal of a deceptive microscopic simulator.

We report 2 cases of rectal melanoma. The patients were a 48-year-old man and an 88-year-old woman. In both cases, the tumor presented as polypoid and necrotic masses, making initial diagnostic assessment difficult because of the regressive tissue changes. Microscopically, tumors were mostly composed of epithelioid, highly atypical cells mimicking poorly differentiated carcinoma or large cell lymphoma. Tumor cells were negative for keratin, leukocyte common antigen, and epithelial membrane antigen and positive for S100 protein and several melanoma markers, although in endoscopic biopsy specimens, the positive reaction was focal due to the limited viable tissue. One patient was treated with abdominoperineal resection. Pathologic examination of the surgical specimen revealed a polypoid mass infiltrating the muscle coat of the rectum. Local lymph nodes contained metastatic deposits. Microscopic examination of the rectal mucosa adjacent to the infiltrating neoplasm revealed colonization of the intestinal crypts by atypical melanocytes. This patient is alive 1 year after surgery with no evidence of recurrent disease. The other case was not deemed amenable to radical surgery because of the patient's advanced age and evidence of diffuse metastatic disease. She was alive 6 months after the initial diagnosis but was lost to follow-up. Data indicate that the diagnosis of melanoma, although feasible in large resection specimens, may be problematic in endoscopic biopsy, especially when only small tissue fragments are available and tumor regression hampers the accuracy of immunohistochemical stains. Documentation of intracryptic atypical melanocytes in nonneoplastic mucosa conceptually corroborates claims that rectal melanoma is related to preinvasive precursors as in cutaneous melanoma.

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.

Sentinel node biopsy in head and neck squamous cell cancer: 5-year follow-up of a European multicenter trial.

BACKGROUND: Sentinel node biopsy (SNB) may represent an alternative to elective neck dissection for the staging of patients with early head and neck squamous cell carcinoma (HNSCC). To date, the technique has been successfully described in a number of small single-institution studies. This report describes the long-term follow-up of a large European multicenter trial evaluating the accuracy of the technique. METHODS: A total of 227 SNB procedures were carried out across 6 centers, of which 134 were performed in clinically T1/2 N0 patients. All patients underwent SNB with preoperative lymphoscintigraphy, intraoperative blue dye, and handheld gamma probe. Sentinel nodes were evaluated with hematoxylin and eosin (H&E) staining, step-serial sectioning (SSS), and immunohistochemistry (IHC). There were 79 patients who underwent SNB as the sole staging tool, while 55 patients underwent SNB-assisted elective neck dissection. RESULTS: Sentinel nodes were successfully identified in 125 of 134 patients (93%), with a lower success rate observed for floor-of-mouth tumors (FoM; 88% vs. 96%, P = 0.138). Also, 42 patients were upstaged (34%); of these, 10 patients harbored only micrometastatic disease. At a minimum follow-up of 5 years, the overall sensitivity of SNB was 91%. The sensitivity and negative predictive values (NPV) were lower for patients with FoM tumors compared with other sites (80% vs. 97% and 88% vs. 98%, respectively, P = 0.034). CONCLUSIONS: Sentinel node biopsy is a reliable and reproducible means of staging the clinically N0 neck for patients with cT1/T2 HNSCC. It can be used as the sole staging tool for the majority of these patients, but cannot currently be recommended for patients with tumors in the floor of the mouth.

Alcohol, tobacco and genetic susceptibility in relation to cancers of the upper aerodigestive tract in northern Italy.

AIMS AND BACKGROUND: Each year in Italy there are approximately 14,000 new cases and 7,000 deaths from cancer of the upper aerodigestive tract, which includes malignant tumors originating from the oral cavity, pharynx, larynx and esophagus. Established etiological factors include tobacco consumption and heavy alcohol drinking. The study of single nucleotide polymorphisms in upper aerodigestive tract cancer etiology may help to identify high-risk subgroups and to better understand the pathways leading to the development of these cancers. METHODS: Italian results on about 500 cases and 500 controls from a large case-control study (ARCAGE) conducted in 10 European countries are presented with the major objectives of updating results on the effects of alcohol and tobacco consumptions in northern Italy, investigating the role of genetic variation with regard to the metabolism of alcohol and carcinogens from tobacco smoke, and evaluating possible interactions of these single nucleotide polymorphisms with these carcinogens. RESULTS: The present study confirmed the importance of tobacco smoking and alcohol drinking as the main risk factors for upper aerodigestive tract cancers, indicating that about 68% of cancers among populations in northern Italy can be attributed to the combination of these risk factors. Significant associations between metabolizing phase I genes (CYP1A1 and CYP2A6), phase II genes (GSTA2) and upper aerodigestive tract cancers were found. A polymorphism of ADH1C has been associated with an increased risk of upper aerodigestive tract cancers, suggesting that the less rapid alcohol metabolizers are more susceptible to upper aerodigestive tract cancer risk. CONCLUSIONS: Our results suggest that the ADH1C allele modifies the carcinogenic dose response for alcohol in the upper aerodigestive tract, giving rise to a gene-environment interaction. The role of genes as possible modifiers of life-style risks seems the most reliable.

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.

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.

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.

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.

Does tumor depth affect nodal upstaging in squamous cell carcinoma of the head and neck?

PURPOSE: The aim of this study was to determine whether tumor depth affects upstaging of the clinically node-negative neck, as determined by sentinel lymph node biopsy with full pathologic evaluation of harvested nodes including step-serial sectioning (SSS) and immunohistochemistry (IHC). PATIENTS AND METHODS: One hundred seventy-two patients with cT1/2 N0 squamous cell carcinoma (SCC) of the oral cavity/oropharynx undergoing primary resection and either sentinel node biopsy (SNB) or SNB-assisted neck dissection as a staging tool for the cN0 neck. Harvested nodes were examined with hematoxylin-eosin staining, SSS, and IHC. Patients upstaged by SSS/IHC were denoted pN1mi. RESULTS: One hundred one of 172 patients were staged pN0, with 71 (41%) patients upstaged. Increasing tumor depth was associated with higher likelihood of upstaging (P < .001). Tumor depth showed a positive correlation with nodal stage according to TNM classification (P < .001). Tumor depth greater than 4 mm appears to be the most appropriate cutoff for risk stratification, although tumors in the oropharynx may require a lower value. CONCLUSION: Tumor depth is an important prognostic factor for patients with SCC of the oral cavity or oropharynx. Tumors greater than 4 mm are associated with greater risk of upstaging; however, this optimum cutoff value may vary between primary tumor sites.

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.

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.