Favorable survival outcomes in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer sequentially treated with a tyrosine kinase inhibitor and osimertinib in a real-world setting.

PURPOSE: EGFR tyrosine kinase inhibitor (TKI) therapy in EGFR-mutated lung cancer is limited by acquired resistance. In half of the patients treated with first/second-generation (1st/2nd gen) TKI, resistance is associated with EGFR p.T790M mutation. Sequential treatment with osimertinib is highly active in such patients. Currently, there is no approved targeted second-line option for patients receiving first-line osimertinib, which thus may not be the best choice for all patients. The present study aimed to evaluate the feasibility and efficacy of a sequential TKI treatment with 1st/2nd gen TKI, followed by osimertinib in a real-world setting. METHODS: Patients with EGFR-mutated lung cancer treated at two major comprehensive cancer centers were retrospectively analyzed by the Kaplan-Meier method and log rank test. RESULTS: A cohort of 150 patients, of which 133 received first-line treatment with a first/second gen EGFR TKI, and 17 received first-line osimertinib, was included. Median age was 63.9 years, 55% had ECOG performance score of ≥ 1. First-line osimertinib was associated with prolonged progression-free survival (P = 0.038). Since the approval of osimertinib (February 2016), 91 patients were under treatment with a 1st/2nd gen TKI. Median overall survival (OS) of this cohort was 39.3 months. At data cutoff, 87% had progressed. Of those, 92% underwent new biomarker analyses, revealing EGFR p.T790M in 51%. Overall, 91% of progressing patients received second-line therapy, which was osimertinib in 46%. Median OS with sequenced osimertinib was 50 months. Median OS of patients with p.T790M-negative progression was 23.4 months. CONCLUSION: Real-world survival outcomes of patients with EGFR-mutated lung cancer may be superior with a sequenced TKI strategy. Predictors of p.T790M-associated resistance are needed to personalize first-line treatment decisions.

Molecular Subtyping of Invasive Breast Cancer Using a PAM50-Based Multigene Expression Test-Comparison with Molecular-Like Subtyping by Tumor Grade/Immunohistochemistry and Influence on Oncologist's Decision on Systemic Therapy in a Real-World Setting.

In intermediate risk hormone receptor (HR) positive, HER2 negative breast cancer (BC), the decision regarding adjuvant chemotherapy might be facilitated by multigene expression tests. In all, 142 intermediate risk BCs were investigated using the PAM50-based multigene expression test Prosigna® in a prospective multicentric study. In 119/142 cases, Prosigna® molecular subtyping was compared with local and two central (C1 and C6) molecular-like subtypes relying on both immunohistochemistry (IHC; HRs, HER2, Ki-67) and IHC + tumor grade (IHC+G) subtyping. According to local IHC, 35.4% were Luminal A-like and 64.6% Luminal B-like subtypes (local IHC+G subtype: 31.9% Luminal A-like; 68.1% Luminal B-like). In contrast to local and C1 subtyping, C6 classified >2/3 of cases as Luminal A-like. Pairwise agreement between Prosigna® subtyping and molecular-like subtypes was fair to moderate depending on molecular-like subtyping method and center. The best agreement was observed between Prosigna® (53.8% Luminal A; 44.5% Luminal B) and C1 surrogate subtyping (Cohen's kappa = 0.455). Adjuvant chemotherapy was suggested to 44.2% and 88.6% of Prosigna® Luminal A and Luminal B cases, respectively. Out of all Luminal A-like cases (locally IHC/IHC+G subtyping), adjuvant chemotherapy was recommended if Prosigna® testing classified as Prosigna® Luminal A at high / intermediate risk or upgraded to Prosigna® Luminal B.

Rebiopsy in advanced non-small cell lung cancer, clinical relevance and prognostic implications.

INTRODUCTION: Rebiopsies of non-small cell lung cancers (NSCLC) are mainly performed to (i) cover the evolution of potentially amenable resistance mechanisms against a targeted therapy, and (ii) to identify new therapeutic targets which were not detected in the initial diagnostic biopsy. Comprehensive systematic analyses evaluating the value of rebiopsies are missing. METHODS: Clinical databases from two large comprehensive cancer center networks were queried following prespecified criteria to identify prospectively entered NSCLC cases with at least one rebiopsy at disease progression. Clinicopathological and biomarker findings including multigene sequencing were correlated with clinical outcomes. RESULTS: From a total of 17,477 stage IV NSCLC patients, a cohort of 403 evaluable patients undergoing at least one rebiopsy of a primary tumor or metastasis was retrieved. Changes in biomarker profiles as compared to baseline were observed in 48.9%. In 31.3% of cases, findings of potential therapeutic relevance were revealed, including 18 patients (4.4%) with a targetable marker only detected at rebiopsy. New findings were more frequent (greater than50%) in NSCLC with EGFR/ALK/ROS1 alterations, including mutations of the dominant oncogene, TP53 mutations, and MET or ERBB2 amplifications. Patients undergoing rebiopsy exhibited superior overall survival compared to a control group, irrespective of presence (HR 0.28) or absence (HR 0.20, both p < 0.001) of a therapeutically targetable aberration. CONCLUSIONS: Rebiopsies at progression of advanced NSCLC are strongly supported by a high rate of clinically relevant findings. Current clinical practice selects a patient population with exceptional outcomes, which merits further characterization.

Clonidine suppression test for a reliable diagnosis of pheochromocytoma: When to use.

OBJECTIVE: In clinical practice, false-positive results in biochemical testing for suspected pheochromocytoma/paraganglioma (PPGL) are not infrequent and may lead to unnecessary examinations. We aimed to evaluate the role of the clonidine suppression test (CST) in the era of analyses of plasma-free metanephrines for the diagnosis or exclusion of PPGL in patients with adrenal tumours and/or arterial hypertension. DESIGN AND METHODS: This single-centre, prospective trial investigated the use of CST in 60 patients with suspected PPGL associated with out-patient elevations of plasma normetanephrine (NMN) and/or metanephrine (MN), in most cases accompanied with hypertension or an adrenal mass. Measurements of plasma catecholamines and free metanephrines were performed by liquid chromatography with electrochemical detection and tandem mass spectrometry, respectively. RESULTS: Forty-six patients entered final analysis (n = 20 with PPGL and n = 26 with a nonfunctional adrenal mass and/or hypertension). CST reliably excluded false-positive baseline NMN results with a specificity of 100%. The sensitivity of CST improved from 85% to 94% when tumours with isolated MN increase (n = 3) were not considered. In patients with elevated baseline NMN (n = 24), CST correctly identified all patients without PPGL. Patients with falsely elevated baseline NMN results (n = 7, 26.9%) exhibited increases of baseline NMN up to 1.7-fold above the upper reference limit. CONCLUSION: CST qualifies as a useful diagnostic tool for differential diagnosis of borderline elevated plasma-free NMN in patients with suspected PPGL. In this context, CST helps to correctly identify all false-positive NMN screening results.

Age, American Thyroid Association Risk Group, and Response to Therapy Are Prognostic Factors in Children With Differentiated Thyroid Cancer.

CONTEXT: Against the background of increasing incidence, pediatric differentiated thyroid carcinoma (DTC) frequently presents with advanced disease and high recurrence rates while prognosis remains excellent. BACKGROUND: We investigated the use of a pediatric classification and an adult response to therapy risk stratification for pediatric DTC patients and their implications for adaptation of treatment and follow-up. METHODS: Data from patients aged <18 years with a diagnosis of primary DTC, registered with the German Pediatric Oncology Hematology-Malignant Endocrine Tumor registry since 1995, were analyzed. For risk prediction, patients were retrospectively assigned to the American Thyroid Association (ATA) risk groups and evaluated for response to therapy. RESULTS: By October 2019, 354 patients with DTC had been reported (median age at diagnosis 13.7 years, range 3.6-17.9) with lymph node and distant metastases in 74.3% and 24.5%. Mean follow-up was 4.1 years (range 0-20.6). Ten-year overall and event-free survival (EFS) rates were 98.9% and 78.1%. EFS was impaired for patients with lymph node and distant metastases (P < .001), positive postoperative thyroglobulin (P = .006), incomplete resection (P = .002), sequential surgeries to achieve total thyroidectomy (P = .042), invasion of capsule (P < .001) and lymph vessels (P = .005), infiltration of surrounding soft tissues (P < .001), tumor multifocality (P < .001), ATA intermediate- and high-risk group (P < .001), and age <10 years (P < .001). Multivariate analysis revealed age <10 years at diagnosis, ATA high-risk level, and poor response to therapy as significant negative prognostic factors for EFS. CONCLUSION: Age, ATA risk group, and response to therapy emerged as significant prognostic factors for EFS in pediatric patients with DTC, requiring risk-adapted individualized therapy and follow-up.

Electrochemotherapy with Bleomycin Enhances Radiosensitivity of Uveal Melanomas: First In Vitro Results in 3D Cultures of Primary Uveal Melanoma Cell Lines.

Electrochemotherapy (ECT) is emerging as a complementary treatment modality for local tumor control in various cancer entities. Irradiation is an established therapeutic option for oncologic patients, which is commonly combined with chemotherapy due to its insufficient targeting ability. The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. ECT can potentiate the radiosensitizing effect of chemotherapeutic agents such as bleomycin. The present study aims to evaluate the radiosensitizing effect of concomitant ECT with bleomycin on 3D tumor spheroids with primary and radioresistant uveal melanoma cell lines (UPMD2, UPMM3, UM92.1, Mel270) and irradiation. The changes in the spheroid growth and the cell viability as well the cytotoxic long-term effect of the combination treatment were evaluated with various combinations of electroporation settings and bleomycin concentrations as well as radiotherapy doses. A broad range of radiosensitivity was documented among the spheroids from different uveal melanoma cell lines. The primary cell lines showed a higher radiosensitivity and required lower irradiation and bleomycin doses. The maximal tumor control with a reduction of cell survival <10% was achieved with a 5 Gy irradiation only in the primary uveal melanoma cell lines and in combination with all tested ECT settings, whereas the same result could be obtained in UM92.1 spheroids only after ECT with 20 Gy irradiation. Based on the spheroid growth and the measurement of the cross-sectional area, the Mel270 spheroids, originating from a previously irradiated recurrent uveal melanoma, required higher doses of bleomycin and ECT settings after irradiation with 5 Gy in order to achieve a significant growth reduction. No significant difference could be demonstrated for the reduction of cell viability in the combination therapy with 20 Gy and 1000 V/cm between 1 and 2.5 µg/mL bleomycin even in Mel270 spheroids, underlying the importance of a drug delivery system to potentiate the radiosensitizing effect of agents in lower doses. ECT should be further assessed for its applicability in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors and a sufficient local tumor control with lower chemotherapy and irradiation doses.

Role of Tumor-Infiltrating B Cells in Clinical Outcome of Patients with Melanoma Treated With Dabrafenib Plus Trametinib.

PURPOSE: Although patients with unresectable or metastatic melanoma can experience long-term survival with BRAF- and MEK-targeted agents or immune checkpoint inhibitors over 5 years, resistance develops in most patients. There is a distinct lack of pretherapeutic biomarkers to identify which patients are likely to benefit from each therapy type. Most research has focused on the predictive role of T cells in antitumor responses as opposed to B cells. PATIENTS AND METHODS: We conducted prespecified exploratory biomarker analysis using gene expression profiling and digital pathology in 146 patients with previously untreated BRAF V600-mutant metastatic melanoma from the randomized, phase III COMBI-v trial and treated with dabrafenib plus trametinib who had available tumor specimens from screening. RESULTS: Baseline cell-cycle gene expression signature was associated with progression-free survival (P = 0.007). Patients with high T-cell/low B-cell gene signatures had improved median overall survival (not reached [95% confidence interval (CI), 33.8 months-not reached]) compared with patients with high T-cell/high B-cell signatures (19.1 months; 95% CI, 13.4-38.6 months). Patients with high B-cell signatures had high B-cell infiltration into the tumor compartment, corresponding with decreased MAPK activity and increased expression of immunosuppressive markers. CONCLUSIONS: B cells may serve as a potential biomarker to predict clinical outcome in patients with advanced melanoma treated with dabrafenib plus trametinib. As separate studies have shown an opposite effect for B-cell levels and response to immunotherapy, B cells may serve as a potential biomarker to facilitate treatment selection. Further validation in a larger patient cohort is needed.

Identification of a Prognostic Clinical Score for Patients With Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck Treated With Systemic Therapy Including Cetuximab.

Cetuximab-based chemoimmunotherapy has been the standard of care for recurrent or metastatic squamous cell carcinoma of the head and neck (r/m SCCHN) for more than a decade. To date, no predictive or prognostic biomarkers have been established to further guide the systemic treatment with cetuximab-based chemoimmunotherapy in r/m SCCHN. Against this background, we retrospectively analyzed clinical and blood-based parameters from 218 r/m SCCHN patients treated with chemoimmunotherapy including cetuximab. Multivariate Cox-regression models were used to assess their prognostic or predictive value. Eastern Co-operative Oncology Group (ECOG) performance status (≥2), older age (≥61.8 years), anemia (hemoglobin <11.80), and increased neutrophil-to-lymphocyte ratio (NLR ≥5.73) were independently and strongly associated with inferior overall survival (OS). To group patients according to risk profiles we established a prognostic clinical score (PCS) that can easily be used in clinical practice. The PCS stratified the cohort into low, intermediate, poor or very poor risk subgroups with median OS times of 23.4, 12.1, 7.5, and 4.0 months, respectively. Patients with low risk PCS had a prolonged progression-free survival (PFS) and increased overall response rate (ORR) under first-line cetuximab-based therapy. Interestingly, only patients with low and intermediate risk benefitted from the more intensive first-line cisplatin/cetuximab combination compared to carboplatin/cetuximab therapy, whereas the intensity of first-line treatment had no impact in the poor and very poor risk subgroups. Following external validation, particularly in the context of newly established first-line options, the PCS may guide clinical decision making and serve for stratification of patients with r/m SCCHN in future clinical trials.

BRAF mutations and BRAF mutation functional class have no negative impact on the clinical outcome of advanced NSCLC and associate with susceptibility to immunotherapy.

OBJECTIVE: BRAF mutations have been subtyped in three functional classes with different oncogenic modes of action. The clinical impact of BRAF mutational subtypes in non-small-cell lung cancer (NSCLC) remains to be defined. So far, ambiguous results were reported from analyses of heterogeneous patient cohorts. METHODS: We studied patients with metastatic or recurrent NSCLC who were sequentially enrolled in precision oncology programs at two large German lung cancer centres from 2009 to 2019. The study period allowed evaluating the specific impact of BRAF V600E-targeting. RESULTS: In a cohort of 72 patients, BRAF mutation subtyping revealed p.V600E mutations in 31 cases (43%), whereas 41 cases (57%) harboured 18 different BRAF mutational subtypes of functional classes II/III. Functionally relevant comutations were observed in 6.4% of class I, and 24.4% of class II/III BRAF mutations. Most patients were treated with chemotherapy. Targeted therapy was administered in 11 patients with a response rate of 72.7%. PD-1/PD-L1-immunotherapy was given in 14 patients with a response rate of 28.6%. Overall survival of patients with BRAF-mutated NSCLC was inferior (HR 1.38, p = 0.048) as compared to patients with BRAF wild-type cancers. Median time-to-treatment-failure with BRAF-targeting agents was shorter as compared to approved targeted therapy of other oncogenic drivers (HR 1.97, p = 0.05). Survival outcomes were not impacted by BRAF mutation subtype functional class. CONCLUSIONS: Patients with BRAF-mutated NSCLC have an inferior prognosis, which is not determined by BRAF mutation functional class. In contrast to NSCLC with other tractable driver mutations, BRAF-mutated NSCLC exhibit high susceptibility to immune checkpoint inhibitors.

Optimisation of the Chicken Chorioallantoic Membrane Assay in Uveal Melanoma Research.

The treatment of uveal melanoma and its metastases has not evolved sufficiently over the last decades in comparison to other tumour entities, posing a great challenge in the field of ocular oncology. Despite improvements in the conventional treatment regime and new discoveries about the genetic and molecular background of the primary tumour, effective treatment strategies to either prevent tumours or treat patients with advanced or metastatic disease are still lacking. New therapeutic options are necessary in order to achieve satisfactory local tumour control, reduce the risk of metastasis development, and preserve the eyeball and possibly the visual function of the eye. The development of in vivo model systems remains crucial for the identification and investigation of potential novel treatment modalities. The aim of this study was the optimisation of the chorioallantoic membrane (CAM) model for uveal melanoma research. We analysed the established CAM assay and its modification after the implantation of three-dimensional spheroids. The chorioallantoic membrane of a chick embryo was used to implant uveal melanoma-cell-line-derived spheroids in order to study their growth rate, angiogenic potential, and metastatic capability. Using the UM 92.1, UPMD2, UPMM3, and Mel270 cell lines, we were able to improve the viability of the embryos from 20% to >80% and to achieve up to a fourfold volume increase of the transplanted spheroid masses. The results point to the value of an optimised chicken embryo assay as an in vivo model for testing novel therapies for uveal melanoma by simplifying the research conditions and by contributing to a considerable reduction in animal experiments.

Differentiating Benign from Malignant Adrenocortical Tumors by a Single Morphological Parameter-a Clinicopathological Study on 837 Adrenocortical Neoplasias.

The morphological differentiation between benign and malignant adrenocortical tumors is an ongoing problem in diagnostic pathology. In recent decades the complex scoring systems have been widely used to calculate the probability of malignancy in adrenocortical tumors on the basis of a variety of histomorphological parameters. We herewith present a substantially simplified method to diagnose adrenocortical carcinoma by a single histomorphological parameter on a consecutive series of more than 800 adrenocortical tumors. Between January 2000 and May 2019, altogether 2305 adrenalectomies for of all types of diseases were removed, approximately 98% by minimally invasive approaches. After exclusion of pheochromocytomas, adrenal ganglioneuromas, adrenal metastases, Cushing's disease related specimens, and Conn's adenomas, the present series finally consisted of 837 adrenocortical tumors. All tumors were analyzed by experienced pathologists of a single institution using standard histopathological methods (Hematoxylin-Eosin and Ki67 stained sections). Clinical and histopathologic data were prospectively collected and retrospectively analyzed. Clinically, 385 patients had 420 functioning tumors (FT), and 417 had non-functioning adrenal tumors (NFT). The mean size of FT was 3.8 ± 1.4 cm (range 0.5-16 cm) and for NFT 4.5 ± 1.6 cm (range 1.5-18 cm). Histomorphologically, 32 adrenal tumors were classified as adrenocortical carcinoma (ACC; 3.8%). In all 32 cases (tumor size 9.1 ± 4.0 cm, range 3-18 cm), confluenting tumor necrosis could be demonstrated. The remaining 805 tumors (control group) completely lacked this highly reproducible single morphological feature. Ki67 levels above 10% were found in 31 of 32 ACCs and never in adrenocortical adenomas (ACA). With a mean follow-up of 8.2 years, 24 out of 32 patients primarily diagnosed as ACC developed distant metastases (75.0%), whereas all patients in the control group remained free of local or distant recurrence. We conclude that a single morphological parameter (confluenting tumor necrosis) is sufficient to predict a poor clinical course in adrenocortical tumors. The histomorphological diagnosis of this parameter is straightforward and highly reproducible.

Machine learning reveals a PD-L1-independent prediction of response to immunotherapy of non-small cell lung cancer by gene expression context.

OBJECTIVE: Current predictive biomarkers for PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1)-directed immunotherapy in non-small cell lung cancer (NSCLC) mostly focus on features of tumour cells. However, the tumour microenvironment and immune context are expected to play major roles in governing therapy response. Against this background, we set out to apply context-sensitive feature selection and machine learning approaches on expression profiles of immune-related genes in diagnostic biopsies of patients with stage IV NSCLC. METHODS: RNA expression levels were determined using the NanoString nCounter platform in formalin-fixed paraffin-embedded tumour biopsies obtained during the diagnostic workup of stage IV NSCLC from two thoracic oncology centres. A 770-gene panel covering immune-related genes and control genes was used. We applied supervised machine learning methods for feature selection and generation of predictive models. RESULTS: Feature selection and model creation were based on a training cohort of 55 patients with recurrent NSCLC treated with PD-1/PD-L1 antibody therapy. Resulting models identified patients with superior outcomes to immunotherapy, as validated in two subsequently recruited, separate patient cohorts (n = 67, hazard ratio = 0.46, p = 0.035). The predictive information obtained from these models was orthogonal to PD-L1 expression as per immunohistochemistry: Selecting by PD-L1 positivity at immunohistochemistry plus model prediction identified patients with highly favourable outcomes. Independence of PD-L1 positivity and model predictions were confirmed in multivariate analysis. Visualisation of the models revealed the predictive superiority of the entire 7-gene context over any single gene. CONCLUSION: Using context-sensitive assays and bioinformatics capturing the tumour immune context allows precise prediction of response to PD-1/PD-L1-directed immunotherapy in NSCLC.

A Novel Epitope Quality-Based Immune Escape Mechanism Reveals Patient's Suitability for Immune Checkpoint Inhibition.

BACKGROUND: Immune checkpoint inhibition, especially the blockade of PD-1 and PD-L1, has become one of the most thriving therapeutic approaches in modern oncology. Immune evasion caused by altered tumor epitope processing (so-called processing escapes) may be one way to explain immune checkpoint inhibition therapy failure. In the present study, we aim to demonstrate the effects of processing escapes on immunotherapy outcome in NSCLC patients. PATIENTS AND METHODS: Whole exome sequencing data of 400 NSCLC patients (AdC and SCC) were extracted from the TCGA database. The ICB cohort was composed of primary tumor probes from 48 NSCLC patients treated with nivolumab. Mutations were identified by targeted amplicon-based sequencing including hotspots and whole exomes of 22 genes. The effect of mutations on proteasomal processing was evaluated by deep learning methods previously trained on 1260 known MHC-I ligands. Cox regression modelling was used to determine the influence on overall survival. RESULTS: In the TCGA cohort, processing escapes were associated with decreased overall survival (p= 0.0140). In the ICB cohort, patients showing processing escapes in combination with high levels of PD-L1 (n=8/48) also showed significantly decreased overall survival, independently of mutational load or PD-L1 status. CONCLUSION: The concept of altered epitope processing may help to understand immunotherapy failure. Especially when combined with PD-L1 status, this method can be used as a biomarker to identify patients not suitable for immunotherapy.

Biweekly Cetuximab Plus FOLFOX6 as First-Line Therapy in Patients With RAS Wild-Type Metastatic Colorectal Cancer: The CEBIFOX Trial.

BACKGROUND: The multicenter, single-arm, phase II study CEBIFOX evaluated the efficacy of a biweekly cetuximab administration in combination with FOLFOX6 as first-line therapy in KRAS (exon 2) wild-type (wt) metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: Patients received FOLFOX6 with cetuximab (500 mg/m2) every second week. Primary endpoint was objective response rate (ORR), among others secondary endpoints were safety, progression-free survival (PFS), overall survival (OS), and patient-reported outcome (PRO). The impact on the treatment efficacy was evaluated in explorative subgroup analyses, including extended molecular profiling and primary tumor location. RESULTS: In total, 57 were included in the intention-to-treat (ITT) analyses. New RAS mutations were detected in 14.0% by post hoc next-generation sequencing analysis in 43 patients. The ORR in the all RASwt population was 70.3% with a median PFS and OS of 10.9 (95% confidence interval [CI], 9.0-12.9) and 33.8 (95% CI, 21.1-45.5) months. Grade 3-5 adverse events occurred in 66.7% of the ITT, without significant impact on the PRO. Patients with right-sided primary tumors had a reduced ORR (54.5%), and median PFS and OS (10.1 and 23.8 months). BRAF mutations were detected in 11.3%. These patients had a significantly lower ORR, and median PFS and OS. Patients with RASwt/BRAFwt tumors had a notably high median PFS and OS of 14.3 and 38.9 months. CONCLUSIONS: This study supports the efficacy and safety of biweekly cetuximab given in combination with FOLFOX6 in patients with RASwt/BRAFwt mCRC with left-sided primary tumor. CEBIFOX is the first trial reporting the complete dataset, including extended molecular profiling and tumor location of a biweekly administered cetuximab/FOLFOX6 in mCRC. Clinical trial number: NCT01051167.

HDAC Inhibition Induces PD-L1 Expression in a Novel Anaplastic Thyroid Cancer Cell Line.

While papillary thyroid cancer (PTC) has largely favorable prognosis, anaplastic thyroid cancer (ATC) is a rare but extremely aggressive malignancy with grim clinical outcome. Even though new therapeutic options are emerging for ATC, additional preclinical models and novel combinations are needed for specific subsets of patients. We established a novel cell line (PF49) from the malignant pleural effusion of a 68-year-old male patient with ATC that rapidly transformed from a BRAF and TERT promoter mutant PTC. PF49 cells demonstrated a robust migratory activity in vitro and strong invasive capacity in vivo in a pleural carcinosis model. Combined BRAF and MEK inhibition decreased the proliferation and migration of PF49 cells, however could not induce cell death. Importantly, HDAC inhibitor treatment with SAHA or valproic acid induced cell cycle arrest and strongly increased PD-L1 expression of the tumor cells. Induction of PD-L1 expression was also present when paclitaxel-cisplatin chemotherapeutic treatment was combined with HDAC inhibitor treatment. Increased PD-L1 expression after HDAC inhibition was recapitulated in an international ATC cell model. Our data suggest that HDAC inhibition alone or in combination with standard chemotherapy may potentiate anaplastic thyroid cancer cells for immunotherapy.

An Animal Model Further Uncovers the Role of Mutant BrafV600E during Papillary Thyroid Cancer Development.

Papillary thyroid carcinomas (PTCs) account for 90% of human thyroid cancer cases, which represent 1% of all cancer cases. They are likely to develop from papillary thyroid microcarcinomas (PTMCs), found in up to 36% of healthy individuals, due to rare progression events (0.01%). Although the prognosis of PTCs is excellent, 5% to 10% of tumors display an unfavorable outcome. About 45% of PTCs exhibit activating BRAFV600E mutations. Rats of the inbred BD strains postnatally exposed to the carcinogen N-ethyl-N-nitrosourea developed PTMCs, which closely resembled their human counterparts judging from their histology, size, and marginal tendency to progress. DNA sequencing revealed mutations in exon 15 of the Braf gene identical to the human BRAFV600E mutation in 82% of the cases. Predominantly a 50:50 ratio of wild-type to mutant Braf alleles was seen regardless of tumor size or animal age, indicating that the Braf mutation is an early, if not the initial, event in rat PTMC development. Surprisingly, most PTMCs carrying a confirmed BrafV600E mutation did not display BrafV600E protein expression. As the BrafV600Egene is supposed to be the driver in PTC development, down-regulation of expression should contribute to the low risk for progression of PTMC. This model system will enable further insights into the molecular mechanisms of PTMC initiation and progression to PTC, further translating into targeted tumor prevention strategies/therapies.

The mismatch repair system is not affected in medullary thyroid carcinoma independent of stromal desmoplasia or ret proto-oncogene mutation.

INTRODUCTION: Medullary thyroid carcinoma (MTC) is an aggressive neuroendocrine neoplasia of the thyroid with 10 year overall survival of 50% and limited therapeutic options. High tumor mutational burden because of microsatellite instability (MSI) seems to be a predictor of response to immune checkpoint inhibitor therapy in different tumors. Therefor in 2017 the U.S. Food and Drug Administration (FDA) permitted the therapy of solid tumors with proven Microsatellite instability (MSI) with PD1 antibody Pembrolizumab independently of their origin. As little is known about MSI in MTC and new therapeutic strategies would be eligible we tried to find out, if therapy with PD1-inhibitors could be promising. MATERIAL AND METHODS: We performed MSI-analyses of 38 cases of MTC. Included were MTCs with and without stromal desmoplasia and with/without lymph node metastases. We also checked the immunhistochemical expression of PD-L-1 and performed next generation sequencing for genetic alterations. RESULTS: All cases revealed stable conditions of the microsatellites and showed immunohistochemically positive staining of the four mismatch repair proteins. PD-L-1- Immunostaining was negative in all cases. DISCUSSION: Our data show there is no MSI in MTCs, irrespectively of their status of desmoplasia, metastases and/or ret-mutation. Therefore a positive effect of PD1 inhibitors, because of MSI-associated high tumor mutational burden, seems to be unlikely.

New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation.

BACKGROUND: Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function. METHODS: NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing. RESULTS: In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins. CONCLUSIONS: NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.

Increased Anaplastic Lymphoma Kinase Activity Induces a Poorly Differentiated Thyroid Carcinoma in Mice.

Background: Radioiodine refractory dedifferentiated thyroid cancer is a major clinical challenge. Anaplastic lymphoma kinase (ALK) mutations with increased ALK activity, especially fusion genes, have been suggested to promote thyroid carcinogenesis, leading to development of poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma. To determine the oncogenic potential of increased ALK activity in thyroid carcinogenesis in vivo, we studied mice with thyrocyte-specific expression of a constitutively active ALK mutant. Methods: Mice carrying a Cre-activated allele of a constitutively active ALK mutant (F1174L) were crossed with mice expressing tamoxifen-inducible Cre recombinase (CreERT2) under the control of the thyroglobulin (Tg) gene promoter to achieve thyrocyte-specific expression of the ALK mutant (ALKF1174L mice). Survival, thyroid hormone serum concentration, and tumor development were recorded. Thyroids and lungs were studied histologically. To maintain euthyroidism despite dedifferentiation of the thyroid, a cohort was substituted with levothyroxine (LT4) through drinking water. Results: ALKF1174L mice developed massively enlarged thyroids, which showed an early loss of normal follicular architecture 12 weeks after tamoxifen injection. A significant decrease in Tg and Nkx-2.1 expression as well as impaired thyroid hormone synthesis confirmed dedifferentiation. Histologically, the mice developed a carcinoma resembling human PDTC with a predominantly trabecular/solid growth pattern and an increased mitotic rate. The tumors showed extrathyroidal extension into the surrounding strap muscles and developed lung metastases. Median survival of ALKF1174L mice was significantly reduced to five months after tamoxifen injection. Reduced Tg expression and loss of follicular structure led to hypothyroidism with elevated thyrotropin (TSH). To test whether TSH stimulation played a role in thyroid carcinogenesis, we kept ALKF1174L mice euthyroid by LT4 substitution. These mice developed PDTC with identical histological features compared with hypothyroid mice, demonstrating that PDTC development was due to increased ALK activity and not dependent on TSH stimulation. Conclusion: Expression of a constitutively activated ALK mutant in thyroids of mice leads to development of metastasizing thyroid cancer resembling human PDTC. These results demonstrate in vivo that increased ALK activity is a driver mechanism in thyroid carcinogenesis.

The GNAS SNP c.393C>T (rs7121) as a marker for disease progression and survival in cancer.

G-protein receptor signaling plays a key role in multiple signal transduction pathways. Aberrant activity of the stimulatory Gsα subunit has been frequently associated with cancer. GNAS sequence alterations and conformational changes of Gsα can both enhance or diminish its function and change downstream effects of G-protein receptor signaling. In this review and meta-analysis, we focus on the synonymous SNP rs7121 (FokI, c.393C>T), which is associated with either tumor progression or prolonged survival in cancer patients (overall hazard ratio = 2.256; p < 0.001). We finally point out the relevance of GNAS rs7121 as a promising biomarker and a prediction tool for therapy response and the need of further experiments to implement it into routine clinical diagnostics.