Low-Density Lipoprotein Receptor Is a Key Driver of Aggressiveness in Thyroid Tumor Cells.

We previously described the role of low-density lipoprotein (LDL) in aggressiveness in papillary thyroid cancer (PTC). Moreover, the MAPK signaling pathway in the presence of BRAF V600E mutation is associated with more aggressive PTC. Although the link between MAPK cascade and LDL receptor (LDLR) expression has been previously described, it is unknown whether LDL can potentiate the adverse effects of PTC through it. We aimed to investigate whether the presence of LDL might accelerate the oncogenic processes through MAPK pathway in presence or absence of BRAF V600E in two thyroid cell lines: TPC1 and BCPAP (wild-type and BRAF V600E, respectively). LDLR, PI3K-AKT and RAS/RAF/MAPK (MEK)/ERK were analyzed via Western blot; cell proliferation was measured via MTT assay, cell migration was studied through wound-healing assay and LDL uptake was analyzed by fluorometric and confocal analysis. TPC1 demonstrated a time-specific downregulation of the LDLR, while BCPAP resulted in a receptor deregulation after LDL exposition. LDL uptake was increased in BCPAP over-time, as well as cell proliferation (20% higher) in comparison to TPC1. Both cell lines differed in migration pattern with a wound closure of 83.5 ± 9.7% after LDL coculture in TPC1, while a loss in the adhesion capacity was detected in BCPAP. The siRNA knockdown of LDLR in LDL-treated BCPAP cells resulted in a p-ERK expression downregulation and cell proliferation modulation, demonstrating a link between LDLR and MAPK pathway. The modulation of BRAF-V600E using vemurafenib-impaired LDLR expression decreased cellular proliferation. Our results suggest that LDLR regulation is cell line-specific, regulating the RAS/RAF/MAPK (MEK)/ERK pathway in the LDL-signaling cascade and where BRAF V600E can play a critical role. In conclusion, targeting LDLR and this downstream signaling cascade, could be a new therapeutic strategy for PTC with more aggressive behavior, especially in those harboring BRAF V600E.

LDL, HDL and endocrine-related cancer: From pathogenic mechanisms to therapies.

Cholesterol is essential for a variety of functions in endocrine-related cells, including hormone and steroid production. We have reviewed the progress to date in research on the role of the main cholesterol-containing lipoproteins; low-density lipoprotein (LDL) and high-density lipoprotein (HDL), and their impact on intracellular cholesterol homeostasis and carcinogenic pathways in endocrine-related cancers. Neither LDL-cholesterol (LDL-C) nor HDL-cholesterol (HDL-C) was consistently associated with endocrine-related cancer risk. However, preclinical studies showed that LDL receptor plays a critical role in endocrine-related tumor cells, mainly by enhancing circulating LDL-C uptake and modulating tumorigenic signaling pathways. Although scavenger receptor type BI-mediated uptake of HDL could enhance cell proliferation in breast, prostate, and ovarian cancer, these effects may be counteracted by the antioxidant and anti-inflammatory properties of HDL. Moreover, 27-hydroxycholesterol a metabolite of cholesterol promotes tumorigenic processes in breast and epithelial thyroid cancer. Furthermore, statins have been reported to reduce the incidence of breast, prostate, pancreatic, and ovarian cancer in large clinical trials, in part because of their ability to lower cholesterol synthesis. Overall, cholesterol homeostasis deregulation in endocrine-related cancers offers new therapeutic opportunities, but more mechanistic studies are needed to translate the preclinical findings into clinical therapies.

Cholesterol and 27-hydroxycholesterol promote thyroid carcinoma aggressiveness.

Cholesterol mediates its proliferative and metastatic effects via the metabolite 27-hydroxycholesterol (27-HC), at least in breast and endometrial cancer. We determined the serum lipoprotein profile, intratumoral cholesterol and 27-HC levels in a cohort of patients with well-differentiated papillary thyroid carcinoma (PTC; low/intermediate and high risk), advanced thyroid cancers (poorly differentiated, PDTC and anaplastic thyroid carcinoma, ATC) and benign thyroid tumors, as well as the expression of genes involved in cholesterol metabolism. We investigated the gene expression profile, cellular proliferation, and migration in Nthy-ori 3.1 and CAL-62 cell lines loaded with human low-density lipoprotein (LDL). Patients with more aggressive tumors (high-risk PTC and PDTC/ATC) showed a decrease in blood LDL cholesterol and apolipoprotein B. These changes were associated with an increase in the expression of the thyroid's LDL receptor, whereas 3-hydroxy-3-methylglutaryl-CoA reductase and 25-hydroxycholesterol 7-alpha-hydroxylase were downregulated, with an intratumoral increase of the 27-HC metabolite. Furthermore, LDL promoted proliferation in both the Nthy-ori 3.1 and CAL-62 thyroid cellular models, but only in ATC cells was its cellular migration increased significantly. We conclude that cholesterol and intratumoral accumulation of 27-HC promote the aggressive behavior process of PTC. Targeting cholesterol metabolism could be a new therapeutic strategy in thyroid tumors with poor prognosis.

[2018 Consensus statement by the Spanish Society of Pathology and the Spanish Society of Medical Oncology on the diagnosis and treatment of cancer of unknown primary]. / Consenso 2018 de la Sociedad Española de Anatomía Patológica y la Sociedad Española de Oncología Médica sobre el diagnóstico y tratamiento del cáncer de origen desconocido.

Cancer of unknown primary is defined as a heterogeneous group of tumours that present with metastasis, and in which attempts to identify the original site have failed. They differ from other primary tumours in their biological features and how they spread, which means they can be considered a separate entity. There are several hypotheses regarding their origin, but the most plausible explanation for their aggressiveness and chemoresistance seems to involve chromosomal instability. Depending on the type of study done, cancer of unknown primary can account for 2-9% of all cancer patients, mostly 60-75 years old. This article reviews the main clinical, pathological and molecular studies conducted to analyse and determine the origin of cancer of unknown primary. The main strategies for patient management and treatment, by both clinicians and pathologists, are also addressed.

Airway and vascular maturation stimulated by tracheal occlusion do not correlate in the rabbit model of diaphragmatic hernia.

BACKGROUND: In animal models of congenital diaphragmatic hernia (CDH), tracheal occlusion (TO) has induced maturation of both airway spaces and vascular structures. Airway and vascular response to TO are assumed to occur in parallel. This study aims to describe and measure the relationship between airway and vascular maturation induced by TO. METHODS: A rabbit model of CDH on gestational day (GD) 23 and TO on GD 28 (term = GD 31) has been used. Two study groups have been defined: DH (diaphragmatic hernia) and TO (DH treated with TO). Animals were collected on GD 30 and blood flow data of the pulmonary artery (pulsatility index (PI) and fractional moving blood volume) were ultrasonographically measured. Lung morphometry consisted of measurements of radial alveolar count (RAC) and arterial muscular thickness. RESULTS: Animals in the DH group (n = 9) had the worst hemodynamic parameters; their lungs were hypoplastic and had the thickest arterial muscular layer. Animals in the TO group (n = 10) had all these effects reversed. There were no correlations among hemodynamic, airway, and vascular parameters, except for RAC and PI (r = -0.528, P = 0.043). CONCLUSION: Airway and vascular maturation after TO appear to be uncorrelated effects. TO could trigger several pathways that separately regulate airway and vascular responses.

Human papillomavirus as a favorable prognostic biomarker in squamous cell carcinomas of the vagina.

OBJECTIVE: Recent evidence has confirmed two independent pathways in the development of vaginal squamous cell carcinoma (VaSCC): one related to and the other independent of human papillomavirus (HPV). The aim of our study was to evaluate whether HPV status has prognostic significance in this neoplasm. METHODS: All confirmed primary VaSCCs diagnosed and treated from 1995 to 2009 in two institutions were retrospectively evaluated (n=57). HPV infection was detected by PCR using SPF-10 primers and typed with the INNO-LIPA HPV assay and p16(INK4a) expression by immunohistochemistry. Disease-free and overall survival (DFS and OS) were analyzed by Kaplan-Meier analysis with the log-rank test and a multivariate Cox proportional hazard's model. RESULTS: HR-HPV DNA was detected in 70.2% patients. HPV16 was the most prevalent genotype (67.5% of cases). p16(INK4a) was positive in 97.5% HPV-positive and 17.6% HPV-negative tumors (p<.001). FIGO stage was associated with DFS (p=.042) and OS (p=.008). HPV-positive tumors showed better DFS (p=.042) and OS (p=.035) than HPV-negative tumors. Multivariate analysis confirmed better DFS and OS of HPV-positive patients independent of age and stage. This reduced risk of progression and mortality in HPV-positive patients was limited to women with FIGO stages I and II tumors (HR=0.26; 95% CI 0.10-0.69; p=0.006). CONCLUSIONS: HPV-positive early stage (FIGO I and II) VaSCCs have a better prognosis than early HPV-negative tumors. HPV detection and/or p16(INK4a) immunostaining can be easily implemented in routine pathology and should be considered as valuable prognostic biomarkers in the study of patients with VaSCC.

Does human papillomavirus infection imply a different prognosis in vulvar squamous cell carcinoma?

BACKGROUND: Two independent pathways in the development of vulvar squamous cell carcinoma (VSCC) have been described, one related to and the other independent of high-risk human papillomavirus (HR-HPV). The aim of our study was to evaluate whether the HPV status has a prognostic significance or can predict response to radiotherapy. METHODS: All VSCC diagnosed from 1995 to 2009 were retrospectively evaluated (n=98). HPV infection was detected by amplification of HPV DNA by PCR using SPF-10 primers and typed by the INNO-LIPA HPV research assay. p16(INK4a) expression was determined by immunohistochemistry. Disease-free and overall survival (DFS and OS) were estimated by Kaplan-Meier analysis with the log-rank test and a multivariate Cox proportional hazard's model. RESULTS: HR-HPV DNA was detected in 19.4% of patients. HPV16 was the most prevalent genotype (73.7% of cases). p16(INK4a) stained 100% HPV-positive and 1.3% HPV-negative tumors (p<.001). No differences were found between HPV-positive and -negative tumors in terms of either DFS (39.8% vs. 49.8% at 5 years; p=.831), or OS (67.2% vs. 71.4% at 5 years; p=.791). No differences in survival were observed between HPV-positive and -negative patients requiring radiotherapy (hazard ratio [HR] 1.04, 95% confidence interval [CI] .45 to 2.41). FIGO stages III-IV (p=.002), lymph node metastasis (p=.030), size ≥ 20 mm (p=.023), invasion depth (p=.020) and ulceration (p=.032) were associated with increased mortality but in multivariated only lymph node metastasis retained the association (HR 13.28, 95% CI 1.19 to 148.61). CONCLUSIONS: HPV-positive and -negative VSCCs have a similar prognosis. Radiotherapy does not increase survival in HPV-positive women.

Primary squamous cell carcinoma of the vagina: human papillomavirus detection, p16(INK4A) overexpression and clinicopathological correlations.

AIM: To determine the role of human papillomavirus (HPV) in the pathogenesis of primary squamous cell carcinoma of the vagina (SCCVa), and to evaluate its clinicopathological significance. METHODS AND RESULTS: All cases of SCCVa diagnosed over a 15-year period from two hospitals in Barcelona, Spain (n=32) were retrieved. Patients with a history of carcinoma of the cervix diagnosed <5 years before were excluded. HPV was detected and typed by polymerase chain reaction (PCR) using SPF10 primers. Immunohistochemistry was performed for p16 and p53. HPV was detected in 25 cases (78.1%). HPV16 was the most prevalent type. Patients with HPV-positive tumours were associated frequently with a history of carcinoma or intraepithelial neoplasia of the cervix or vulva diagnosed more than 5 years before (56% versus 0%; P=0.01). HPV-positive tumours were more frequently of non-keratinizing, basaloid or warty type than HPV-negative neoplasms (84% versus 14.3%; P<0.001), and showed diffuse positive immunoreactivity for p16(INK4a) (96%, versus 14.3%; P<0.001). The sensitivity and specificity of p16 to identify HPV-positive tumours were 96% and 85.7%, respectively. CONCLUSIONS: A high number of SCCVs are related to HPV infection and may be identified by immunohistochemistry for p16. HPV-positive tumours tend to affect women with history of cervical neoplasia.

Colposcopy prediction of progression in human papillomavirus infections with minor cervical lesions.

OBJECTIVES: To evaluate the risk of progression to cervical intraepithelial neoplasia (CIN) grade 2 or 3 in women with positive human papillomavirus (HPV) testing and low-grade (low-grade squamous intraepithelial lesions), borderline (atypical squamous cells of undetermined significance), or no cervical lesions, and to determine the accuracy of initial colposcopy to predict progression. METHODS: Women with HPV infection and low-grade squamous intraepithelial lesions, atypical squamous cells, or normal cytology were recruited and grouped according to cytologic or histologic diagnosis. Exclusion criteria were histologic CIN 2 or 3, previous cervical cancer and HPV infection, cervical disease, or treatment for CIN 2 or 3 in the past 3 years. Four-hundred sixty-five women were included and monitored by cytology, Hybrid Capture-2 test, and colposcopy every 6 months. Colposcopy results were described as normal, with minor or major changes, and lesion size was recorded in quadrants. RESULTS: Forty-three women (9.3%) had progression to CIN 2 or 3. No significant differences were found in rate of progression between women with low-grade squamous intraepithelial lesions, atypical squamous cells, or negative results (8.2%, 13.4%, and 9.8%, respectively; P=.679). Neither colposcopy pattern (P=.284) nor lesion size (P=.170) at recruitment provided any information on the risk of progression. History of cervical lesion and worsening of the colposcopy pattern during follow-up were associated with progression (P<.001). CONCLUSION: Initial colposcopy findings do not provide relevant information on the risk of progression in HPV-positive women with minor or no cervical lesions. These women have a similar risk of progression and should benefit from the same follow-up strategies.

HPV-negative vulvar intraepithelial neoplasia (VIN) with basaloid histologic pattern: an unrecognized variant of simplex (differentiated) VIN.

Vulvar intraepithelial neoplasia (VIN) is classified into 2 clinicopathologic subtypes, classic, related to human papillomavirus (HPV) infection and affecting relatively young women, and simplex (differentiated), negative for HPV and affecting elderly women. Histologically, classic VIN may be basaloid and characterized by a replacement of the whole epidermis by a homogeneous population of small, "undifferentiated" keratinocytes, which are diffusely positive for p16(INK4a) and negative for p53. Simplex VIN is characterized by atypia of the basal layer with high degree of cellular differentiation and shows negative staining for p16(INK4a) and frequent positivity for p53. Simplex VIN is frequently associated with squamous cell hyperplasia and lichen sclerosus. From a series of 110 invasive squamous cell carcinomas of the vulva negative for HPV by highly sensitive polymerase chain reaction, 51 had VIN lesions located at least 1 cm away from the tumor. In 4 (7.8%) cases, the VIN had basaloid histologic features. All cases showed obvious architectural disorganization with a homogeneous population of basaloid, undifferentiated keratinocytes with scanty cytoplasm replacing the whole epidermis. Immunohistochemically, all cases were negative for p16(INK4a) and strongly positive for p53 with suprabasilar extension of positive cells. All patients were postmenopausal (median age 61.0 y; range, 45-76). Squamous cell hyperplasia was identified in 1 case and lichen sclerosus in 1 case. The invasive squamous cell carcinoma was of keratinizing type in 3 cases and basaloid in 1 case. In conclusion, simplex, HPV-negative VIN may occasionally have basaloid morphology. Immunostaining for p16(INK4a) and p53 protein may be helpful in the identification of these lesions and the differential diagnosis with classic, HPV-positive basaloid VIN.

Value of p16(INK4a) as a marker of progression/regression in cervical intraepithelial neoplasia grade 1.

OBJECTIVE: The objective of this study was to evaluate the usefulness of p16(INK4a) staining to classify cervical intraepithelial neoplasia grade 1 according to its progression/regression risk. STUDY DESIGN: Patients with a histologic diagnosis of cervical intraepithelial neoplasia grade 1 were prospectively recruited (n = 138). Simultaneous detection of high-risk human papillomaviruses and p16(INK4a) evaluation were performed. Follow-up was conducted every 6 months by cytology and colposcopy and annually by high-risk human papillomavirus testing, for at least 12 months (mean, 29.0). Progression was defined as a histologic diagnosis of cervical intraepithelial neoplasia grades 2-3, regression as a negative cytology and high-risk human papillomaviruses, and persistence as a cytologic result of low-grade squamous intraepithelial lesions and/or a positive test for high-risk human papillomaviruses. RESULTS: Progression was observed in 14 women (10.1%), 66 (47.6%) regressed, and 58 (42.0%) had a persistent disease. p16(INK4a) was positive in 77 (55.8%) initial biopsy specimens. Progression to cervical intraepithelial neoplasia grades 2-3 was identified in 14 of 77 (18.2%) women with positive and none of 61 (0.00%) women with negative p16(INK4a) immunostaining (P < .001). CONCLUSION: p16(INK4a) negative cervical intraepithelial neoplasia grade 1 lesions rarely progress and may benefit from a less intensive follow-up.