RET Fusion Testing in Patients With NSCLC: The RETING Study.

Introduction: RET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. Methods: This situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion-positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. Results: The most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). Conclusions: In-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.

Cervical Lymph Node Metastasis From Recurrent Glioblastoma Detected by MRI and 2-[18F]FDG PET/CT.

ABSTRACT: A 53-year-old man diagnosed with recurrent glioblastoma after multimodal treatment on a follow-up brain MRI. Because of a palpable mass in the neck, the patient underwent a whole-body 2-[18F]FDG PET/CT, which revealed hypermetabolic laterocervical confluent lymphadenopathies. A nodal cervical biopsy indicated distant metastases from glioblastoma. Recent studies have confirmed dural meningeal lymphatics, as part of the glymphatic system, which provide clearance of interstitial solutes from the brain parenchyma into cervical lymphatics. Nodal cervical metastases from glioblastoma, using this pathway, are extremely rare and have been almost unthinkable some years ago.

Detecting anal human papillomavirus infection in men who have sex with men living with HIV: implications of assay variability.

BACKGROUND: Incidence of anal cancer (AC) caused by persistent human papillomavirus (HPV) infection has risen in the last years in men who have sex with men (MSM) living with HIV. There is consensus that this population should be screened for anal precancerous lesions, but the role of HPV DNA testing in AC screening programmes is still under debate. OBJECTIVES: This study employed two molecular test to detect anal HPV DNA and compared assay performance and prognostic value for the diagnosis of histology proven high-grade intraepithelial anal lesions. METHODS: MSM living with HIV attended their regular check-up visits consisting of detection of anal HPV infection, anal cytology, digital anorectal examination and high resolution anoscopy. HPV DNA was detected using Hybrid Capture 2 High-Risk test (HC2, total assay) and LINEAR ARRAY HPV Genotyping Test (LA, type-specific assay) RESULTS: Among 274 participant, prevalence of HPV DNA was 48.5% by HC2 and 89.4% by LA. HPV16 (30.6%) and HPV6 (19.6%) were the most common genotypes identified. Prevalence of multiple HPV infections was 56.2%. Agreement between HPV DNA assays was 75.2% (κ=0.51; 95% CI 0.42 to 0.60). Total HPV detection demonstrated high sensitivity (90%; 95% CI 68.3 to 98.8) and moderate specificity (58.4%; 95% CI 50.2 to 66.3), while type-specific HPV16/18 genotyping provided an increase in specificity and showed the highest area under the curve (0.81; 95% CI 0.74 to 0.89) and Youden's index (0.63). CONCLUSIONS: Both methodologies identified a high prevalence of anal HPV infection and multiple HPV infections in MSM living with HIV, showing a moderate overall agreement between them. Either total HPV detection or type-specific HPV16/18 detection together with a threshold ≥atypical squamous cells of undetermined significance for abnormal cytology showed an acceptable diagnostic accuracy.

An Integrated Approach for the Early Detection of Endometrial and Ovarian Cancers (Screenwide Study): Rationale, Study Design and Pilot Study.

Screenwide is a case-control study (2017−2021) including women with incident endometrial and ovarian cancers (EC and OC), BRCA1/2 and MMR pathogenic variant carriers, and age-matched controls from three centers in Spain. Participants completed a personal interview on their sociodemographic factors, occupational exposure, medication, lifestyle, and medical history. We collected biological specimens, including blood samples, self-collected vaginal specimens, cervical pap-brush samples, uterine specimens, and, when available, tumor samples. The planned analyses included evaluation of the potential risk factors for EC/OC; evaluation of molecular biomarkers in minimally invasive samples; evaluation of the cost-effectiveness of molecular tests; and the generation of predictive scores to integrate different epidemiologic, clinical, and molecular factors. Overall, 182 EC, 69 OC, 98 BRCA pathogenic variant carriers, 104 MMR pathogenic variant carriers, and 385 controls were enrolled. The overall participation rate was 85.7%. The pilot study using 61 samples from nine EC cases and four controls showed that genetic variants at the variant allele fraction > 5% found in tumors (n = 61 variants across the nine tumors) were detected in paired endometrial aspirates, clinician-collected cervical samples, and vaginal self-samples with detection rates of 90% (55/61), 79% (48/61), and 72% (44/61) by duplex sequencing, respectively. Among the controls, only one somatic mutation was detected in a cervical sample. We enrolled more than 800 women to evaluate new early detection strategies. The preliminary data suggest that our methodological approach could be useful for the early detection of gynecological cancers.

Sensitivity of cervical cytology in endometrial cancer detection in a tertiary hospital in Spain.

INTRODUCTION: Cervical cytology is a well-stablished cervical cancer screening method. However, due to the anatomical continuity of the genital tract, it can also detect signs of endometrial disease. Our aim was to estimate the sensitivity of cervical cytology in endometrial cancer detection and prognosis in a large population over a 30-year period in a large academic tertiary hospital in Spain. METHODOLOGY: We performed a search for women diagnosed with endometrial cancer from 1990 to 2020, who were surgically treated and had a previous cervical cytology result. Information Technologies Department databases from Bellvitge University Hospital and the Screenwide case-control study's database were used. Cervical cytology results were classified as abnormal when squamous lesions, glandular atypia or malignant cells were identified. RESULTS: Overall, we evaluated 371 women with endometrial cancer and a documented cervical cytology performed within 3 years previous to surgical treatment. Overall, the sensitivity of cervical cytology for endometrial cancer detection was 25.6%. Several clinico-pathological characteristics, such as non-endometrioid histology and a higher stage, were correlated with higher sensitivity. DISCUSSION: We observed a low sensitivity of cervical cytology to effectively diagnose endometrial cancer. However, recent technological advances using genomics and epigenomics may offer a promising perspective to detect endometrial cancer with high sensitivity in these cervical specimens.

Comparison of two sample collection devices for anal cytology in HIV-positive men who have sex with men: Cytology brush and Dacron swab.

OBJECTIVE: HIV-positive men who have sex with men (MSM) are a vulnerable group for anal cancer (AC), a cancer with a well-described precursor lesion, which can be detected early in screening programs using anal liquid-based cytology (aLBC). We aim to compare two aLBC sample collection devices: cytology brush (CB) and Dacron swab (DS). METHODS: Retrospective analysis of two consecutive study periods, the first using CB and the second DS. Participants underwent an aLBC, a human papillomavirus (HPV) DNA test and a high-resolution anoscopy (HRA), and a biopsy was performed for suspicious lesions. The sensitivity and specificity of aLBC, area under the receiver operating characteristic (ROC) curve (AUC), and concordance between cytology and HRA were assessed using Cohen's kappa coefficient. RESULTS: A total of 239 participants were enrolled (CB group, 120; DS group, 119). aLBC was benign in 46% of samples, and high-grade squamous intraepithelial lesion (HSIL) was detected in 11.7%. Prevalence of biopsy-proven HSIL was 15.3%. No differences in cytological and histological results were observed between the groups. aLBC-HRA concordance was weak for benign results (CB group, k = 0.309; DS group, k = 0.350) as well as for HSIL (k = 0.321 and 0.387, respectively). Sensitivity and specificity were 100% and 51.4%, respectively, in the CB group and 88% and 54.3% in the DS group (AUC = 0.711 and 0.759, respectively, P-value = .514). Representation of the transformation zone (TZ) was adequate in 83.3% of samples in the CB group and 50.4% in the DS group (P-value <.001). CONCLUSION: Our data suggest that both devices had similar accuracy to detect anal HSIL, although samples collected with CB are more likely to have an adequate TZ representation, the presence of which could be an indicator of sample quality.

Sensitivity of cervico-vaginal cytology in endometrial carcinoma: A systematic review and meta-analysis.

Cervico-vaginal cytology is primarily a cervical cancer screening test. The anatomical continuity of the uterine cavity with the cervix makes the Papanicolaou (Pap) test accessible to evaluate signs of disease shed from the endometrium. Our aim was to determine the sensitivity of routine Pap test in endometrial carcinoma detection and its relationship with clinico-pathologic factors. We performed a systematic review of studies reporting Pap test results prior to diagnosis of or surgery for endometrial carcinoma between 1990 and 2018 in PubMed or Web of Science. Two independent reviewers extracted data and assessed study quality using an adapted Newcastle-Ottawa Quality Assessment Scale and Quality Assessment of Diagnostic Accuracy Studies tool. We identified 45 studies including a total of 6599 women with endometrial cancer. Abnormal Pap test results prior to diagnosis of or surgery for endometrial carcinoma were observed in 45% (95% CI, 40%-50%) of study participants. This percentage was significantly higher among those of non-endometrioid histology compared with endometrioid subtypes (77% [95% CI, 66%-87%] vs 44% [95% CI, 34%-53%], respectively; P heterogeneity <.001). Several clinico-pathologic factors were related to a higher percentage of abnormal Pap test results, including high-stage, myometrial invasion >50%, high histological grade, positive peritoneal cytology, presence of lymph node metastasis, cervical involvement, and lymphovascular invasion (P heterogeneity <.05 for all variables). Routine cervical cytology can detect endometrial cancer in almost half of patients, whereas sensitivity is higher among individuals with non-endometrioid histology or more advanced cancers. This review summarizes the current clinical and prognostic value of cervical cytology in endometrial carcinoma. Recent technological developments using molecular biomarkers may improve accuracy for early cancer detection.

The Natural-Based Antitumor Compound T21 Decreases Survivin Levels through Potent STAT3 Inhibition in Lung Cancer Models.

Lung cancer is the leading cause of cancer-related deaths worldwide; hence novel treatments for this malignancy are eagerly needed. Since natural-based compounds represent a rich source of novel chemical entities in drug discovery, we have focused our attention on tambjamines, natural compounds isolated from marine invertebrates that have shown diverse pharmacological activities. Based on these structures, we have recently identified the novel indole-based tambjamine analog 21 (T21) as a promising antitumor agent, which modulates the expression of apoptotic proteins such as survivin. This antiapoptotic protein plays an important role in carcinogenesis and chemoresistance. In this work, we have elucidated the molecular mechanism by which the anticancer compound T21 exerts survivin inhibition and have validated this protein as a therapeutic target in different lung cancer models. T21 was able to reduce survivin protein levels in vitro by repressing its gene expression through the blockade of Janus kinase/Signal Transducer and Activator of Transcription-3 (JAK/STAT3)/survivin signaling pathway. Interestingly, this occurred even when the pathway was overstimulated with its ligand interleukin 6 (IL-6), which is frequently overexpressed in lung cancer patients who show poor clinical outcomes. Altogether, these results show T21 as a potent anticancer compound that effectively decreases survivin levels through STAT3 inhibition in lung cancer, appearing as a promising therapeutic drug for cancer treatment.

MET-Oncogenic and JAK2-Inactivating Alterations Are Independent Factors That Affect Regulation of PD-L1 Expression in Lung Cancer.

Purpose: The blockade of immune checkpoints such as PD-L1 and PD-1 is being exploited therapeutically in several types of malignancies. Here, we aimed to understand the contribution of the genetics of lung cancer to the ability of tumor cells to escape immunosurveillance checkpoints.Experimental Design: More than 150 primary non-small cell lung cancers, including pulmonary sarcomatoid carcinomas, were tested for levels of the HLA-I complex, PD-L1, tumor-infiltrating CD8+ lymphocytes, and alterations in main lung cancer genes. Correlations were validated in cancer cell lines using appropriate treatments to activate or inhibit selected pathways. We also performed RNA sequencing to assess changes in gene expression after these treatments.Results:MET-oncogenic activation tended to associate with positive PD-L1 immunostaining, whereas STK11 mutations were correlated with negative immunostaining. In MET-altered cancer cells, MET triggered a transcriptional increase of PD-L1 that was independent of the IFNγ-mediated JAK/STAT pathway. The activation of MET also upregulated other immunosuppressive genes (PDCD1LG2 and SOCS1) and transcripts involved in angiogenesis (VEGFA and NRP1) and in cell proliferation. We also report recurrent inactivating mutations in JAK2 that co-occur with alterations in MET and STK11, which prevented the induction of immunoresponse-related genes following treatment with IFNγ.Conclusions: We show that MET activation promotes the expression of several negative checkpoint regulators of the immunoresponse, including PD-L1. In addition, we report inactivation of JAK2 in lung cancer cells that prevented the response to IFNγ. These alterations are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors. Clin Cancer Res; 24(18); 4579-87. ©2018 AACR.

Predictive and Prognostic Brain Metastases Assessment in Luminal Breast Cancer Patients: FN14 and GRP94 from Diagnosis to Prophylaxis.

FN14 has been implicated in many intracellular signaling pathways, and GRP94 is a well-known endoplasmic reticulum protein regulated by glucose. Recently, both have been associated with metastasis progression in breast cancer patients. We studied the usefulness of FN14 and GRP94 expression to stratify breast cancer patients according their risk of brain metastasis (BrM) progression. We analyzed FN14 and GRP94 by immunohistochemistry in a retrospective multicenter study using tissue microarrays from 208 patients with breast carcinomas, of whom 52 had developed BrM. Clinical and pathological characteristics and biomarkers expression in Luminal and non-Luminal patients were analyzed using a multivariate logistic regression model adjusted for covariates, and brain metastasis-free survival (BrMFS) was estimated using the Kaplan-Meier method and the Cox proportional hazards model. FN14 expression was associated with BrM progression mainly in Luminal breast cancer patients with a sensitivity (53.85%) and specificity (89.60%) similar to Her2 expression (46.15 and 89.84%, respectively). Moreover, the likelihood to develop BrM in FN14-positive Luminal carcinomas increased 36.70-fold (3.65-368.25, p = 0.002). Furthermore, the worst prognostic factor for BrMFS in patients with Luminal carcinomas was FN14 overexpression (HR = 8.25; 95% CI: 2.77-24.61; p = 0.00015). In these patients, GRP94 overexpression also increased the risk of BrM (HR = 3.58; 95% CI: 0.98-13.11; p = 0.054-Wald test). Therefore, FN14 expression in Luminal breast carcinomas is a predictive/prognostic biomarker of BrM, which combined with GRP94 predicts BrM progression in non-Luminal tumors 4.04-fold (1.19-8.22, p = 0.025), suggesting that both biomarkers are useful to stratify BrM risk at early diagnosis. We propose a new follow-up protocol for the early prevention of clinical BrM of breast cancer patients with BrM risk.

Visceral leishmaniasis with mediastinal lymphadenopathy diagnosed with endoscopic ultrasound-guided fine needle aspiration.

A 44-year-old man with a HIV infection and levels of CD4 < 100/mm3, under antiretroviral therapy and with a previous medical history of visceral leishmaniasis (VL), was admitted to hospital with progressive muscular weakness and paraesthesia in both legs for three months. Imaging procedures were performed, showing a leptomeningeal thickening and enlargement of hilar and mediastinal lymph nodes.

Visceral leishmaniasis with mediastinal lymphadenopathy diagnosed by endoscopic ultrasound-guided fine needle aspiration

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Combined Bronchial Artery Embolization and Endobronchial Resection for Bronchial Carcinoid: A Safety and Feasibility Pilot Study.

BACKGROUND: There is growing evidence to support bronchoscopic resection of well-circumscribed typical carcinoids. However, massive bleeding and risk of recurrence can potentially complicate this approach. OBJECTIVES: The aim of this study was to assess the safety and feasibility of endobronchial resection of carcinoids preceded by bronchial artery embolization. METHODS: Five patients with centrally located typical carcinoids were recruited, 4 with a curative intent and 1 for palliation of a carcinoid with mediastinal invasion. All patients underwent selective embolization of the feeding bronchial artery 24-48 h prior to endobronchial resection, which was performed with a rigid bronchoscope and neodymium:yttrium-aluminium-perovskite laser. RESULTS: Minimal bleeding was noted during tumour resection. After a median (range) follow-up of 20 (14-48) months, only the case with segmental extension of the tumour had local recurrence, which was treated successfully using cryotherapy (with negative endobronchial biopsies since), and no cases of metastatic spread occurred. One patient, in whom the histopathological diagnosis was changed from typical to atypical carcinoid following resection, went on to have a surgical bilobectomy 3 months later. Extensive fibrosis was noted at the site of original tumour resection with no evidence of residual disease. CONCLUSIONS: Bronchial artery embolization prior to endobronchial resection of centrally located carcinoids is feasible and safe. The reduction in bleeding may facilitate and simplify the procedure. The possible application of this combined therapy to the management of atypical carcinoids warrants the design of a larger prospective clinical trial.

FN14 and GRP94 expression are prognostic/predictive biomarkers of brain metastasis outcome that open up new therapeutic strategies.

Brain metastasis is a devastating problem in patients with breast, lung and melanoma tumors. GRP94 and FN14 are predictive biomarkers over-expressed in primary breast carcinomas that metastasized in brain. To further validate these brain metastasis biomarkers, we performed a multicenter study including 318 patients with breast carcinomas. Among these patients, there were 138 patients with metastasis, of whom 84 had brain metastasis. The likelihood of developing brain metastasis increased by 5.24-fold (95%CI 2.83-9.71) and 2.55- (95%CI 1.52-4.3) in the presence of FN14 and GRP94, respectively. Moreover, FN14 was more sensitive than ErbB2 (38.27 vs. 24.68) with similar specificity (89.43 vs. 89.55) to predict brain metastasis and had identical prognostic value than triple negative patients (p < 0.0001). Furthermore, we used GRP94 and FN14 pathways and GUILD, a network-based disease-gene prioritization program, to pinpoint the genes likely to be therapeutic targets, which resulted in FN14 as the main modulator and thalidomide as the best scored drug. The treatment of mice with brain metastasis improves survival decreasing reactive astrocytes and angiogenesis, and down-regulate FN14 and its ligand TWEAK. In conclusion our results indicate that FN14 and GRP94 are prediction/prognosis markers which open up new possibilities for preventing/treating brain metastasis.

Ecto-nucleotidases activities in the contents of ovarian endometriomas: potential biomarkers of endometriosis.

Endometriosis, defined as the growth of endometrial tissue outside the uterus, is a common gynecologic condition affecting millions of women worldwide. It is an inflammatory, estrogen-dependent complex disorder, with broad symptomatic variability, pelvic pain, and infertility being the main characteristics. Ovarian endometriomas are frequently developed in women with endometriosis. Late diagnosis is one of the main problems of endometriosis; thus, it is important to identify biomarkers for early diagnosis. The aim of the present work is to evaluate the ecto-nucleotidases activities in the contents of endometriomas. These enzymes, through the regulation of extracellular ATP and adenosine levels, are key enzymes in inflammatory processes, and their expression has been previously characterized in human endometrium. To achieve our objective, the echo-guided aspirated fluids of endometriomas were analyzed by evaluating the ecto-nucleotidases activities and compared with simple cysts. Our results show that enzyme activities are quantifiable in the ovarian cysts aspirates and that endometriomas show significantly higher ecto-nucleotidases activities than simple cysts (5.5-fold increase for ATPase and 20-fold for ADPase), thus being possible candidates for new endometriosis biomarkers. Moreover, we demonstrate the presence of ecto-nucleotidases bearing exosomes in these fluids. These results add up to the knowledge of the physiopathologic mechanisms underlying endometriosis and, open up a promising new field of study.

Protecting the underscreened women in developed countries: the value of HPV test.

BACKGROUND: Poor attendance to cervical cancer (CC) screening is a major risk factor for CC. Efforts to capture underscreened women are considerable and once women agree to participate, the provision of longitudinal validity of the screening test is of paramount relevance. We evaluate the addition of high risk HPV test (HPV) to cervical cytology as a primary screening test among underscreened women in the longitudinal prediction of intraepithelial lesions grade 2 or worse (CIN2+). METHODS: Women were included in the study if they were older than 39 years and with no evidence of cervical cytology in the previous five years within the Public Primary Health Care System in Catalonia (Spain). 1,832 underscreened women from eight public primary health areas were identified during 2007-2008 and followed-up for over three years to estimate longitudinal detection of CIN2+. Accuracy of each screening test and the combination of both to detect CIN2+ was estimated. The risk of developing CIN2+ lesions according to histology data by cytology and HPV test results at baseline was estimated using the Kaplan-Meier method. RESULTS: At baseline, 6.7% of participants were HPV positive, 2.2% had an abnormal cytology and 1.3% had both tests positive. At the end of follow-up, 18 out of 767 (2.3%) underscreened women had a CIN2+, two of which were invasive CC. The three-year longitudinal sensitivity and specificity estimates to detect CIN2+ were 90.5% and 93.0% for HPV test and 38.2% and 97.8% for cytology. The negative predictive value was >99.0% for each test. No additional gains in validity parameters of HPV test were observed when adding cytology as co-test. The referral to colposcopy was higher for HPV but generated 53% higher detection of CIN2+ compared to cytology. CONCLUSIONS: Underscreened women had high burden of cervical disease. Primary HPV screening followed by cytology triage could be the optimal strategy to identify CIN2+ leading to longer and safe screen intervals.