Overall Survival From the EORTC LCG-1613 APPLE Trial of Osimertinib Versus Gefitinib Followed by Osimertinib in Advanced EGFR-Mutant Non-Small-Cell Lung Cancer.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Osimertinib has been established as a standard of care for patients with common sensitizing EGFR-mutant advanced non-small-cell lung cancer (NSCLC) although the sequential approach (first-generation inhibitor gefitinib followed by osimertinib) has not been formally compared. The phase II APPLE trial (ClinicalTrials.gov identifier: NCT02856893) enrolled 156 treatment-naïve patients, and two treatment strategies were evaluated: osimertinib up front or the sequential treatment approach with gefitinib up front followed by osimertinib at the time of progression, either molecular progression (detection of plasma T790M resistance mutation) regardless of the radiologic status or just at the time of radiologic progression. Patients' characteristics were well balanced, except for the higher proportion of baseline brain metastases in the sequential approach (29% v 19%). Per protocol, 73% of patients switched to osimertinib in the sequential arm. Up-front treatment with osimertinib was associated with a lower risk of brain progression versus the sequential approach (hazard ratio [HR], 0.54 [90% CI, 0.34 to 0.86]), but a comparable overall survival was observed between both strategies (HR, 1.01 [90% CI, 0.61 to 1.68]), with the 18-month survival probability of 84% and 82.3%, respectively. The APPLE trial suggests that a sequential treatment approach is associated with more frequent progression in the brain but a similar survival in advanced EGFR-mutant NSCLC.

The molecular profile in patients with polycythemia vera and essential thrombocythemia is dynamic and correlates with disease's phenotype.

Introduction: Polycythemia vera (PV) and essential thrombocythemia (ET) are diseases driven by canonical mutations in JAK2, CALR, or MPL gene. Previous studies revealed that in addition to driver mutations, patients with PV and ET can harbor other mutations in various genes, with no established impact on disease phenotype. We hypothesized that the molecular profile of patients with PV and ET is dynamic throughout the disease. Methods: In this study, we performed a 37-gene targeted next-generation sequencing panel on the DNA samples collected from 49 study participants in two-time points, separated by 78-141 months. We identified 78 variants across 37 analyzed genes in the study population. Results: By analyzing the change in variant allele frequencies and revealing the acquisition of new mutations during the disease, we confirmed the dynamic nature of the molecular profile of patients with PV and ET. We found connections between specific variants with the development of secondary myelofibrosis, thrombotic events, and response to treatment. We confronted our results with existing conventional and mutation-enhanced prognostic systems, showing the limited utility of available prognostic tools. Discussion: The results of this study underline the significance of repeated molecular testing in patients with PV and ET and indicate the need for further research within this field to better understand the disease and improve available prognostic tools.

Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants.

Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (LDLR) gene, leading to an increased risk of premature cardiovascular diseases. Tremendous advances in sequencing techniques have resulted in the discovery of more than 3000 variants of the LDLR gene, but not all of them are clinically relevant. Therefore, functional studies of selected variants are needed for their proper classification. Here, a single-cell, kinetic, fluorescent LDL uptake assay was applied for the functional analysis of LDLR variants in a model of an LDLR-deficient human cell line. An LDLR-defective HEK293T cell line was established via a CRISPR/Cas9-mediated luciferase-puromycin knock-in. The expressing vector with the LDLR gene under the control of the regulated promoter and with a reporter gene has been designed to overproduce LDLR variants in the host cell. Moreover, an LDLR promoter-luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the LDLR gene, which can serve as a simple tool for screening and testing new HMG CoA reductase-inhibiting drugs for atherosclerosis therapy. The data presented here demonstrate that the obtained LDLR-deficient human cell line HEK293T-ldlrG1 and the dedicated pTetRedLDLRwt expression vector are valuable tools for studying LDL internalization and functional analysis of LDLR and its genetic variants. Using appropriate equipment, LDL uptake to a single cell can be measured in real time. Moreover, the luciferase gene knock-in downstream of the LDLR promotor allows the study of promoter regulation in response to diverse conditions or drugs. An analysis of four known LDLR variants previously classified as pathogenic and benign was performed to validate the LDLR-expressing system described herein with the dedicated LDLR-deficient human cell line, HEK293T-ldlrG1.

Analysis of BRCA1 and BRCA2 alternative splicing in predisposition to ovarian cancer.

BACKGROUND: The mRNA splicing is regulated on multiple levels, resulting in the proper distribution of genes' transcripts in each cell and maintaining cell homeostasis. At the same time, the expression of alternative transcripts can change in response to underlying genetic variants, often missed during routine diagnostics. AIM: The main aim of this study was to define the frequency of aberrant splicing in BRCA1 and BRCA2 genes in blood RNA extracted from ovarian cancer patients who were previously found negative for the presence of pathogenic alterations in the 25 most commonly analysed ovarian cancer genes, including BRCA1 and BRCA2. MATERIAL AND METHODS: Frequency and spectrum of splicing alterations in BRCA1 and BRCA2 genes were analysed in blood RNA from 101 ovarian cancer patients and healthy controls (80 healthy women) using PCR followed by gel electrophoresis and Sanger sequencing. The expression of splicing events was examined using RT-qPCR. RESULTS: We did not identify any novel, potentially pathogenic splicing alterations. Nevertheless, we detected six naturally occurring transcripts, named BRCA1ΔE9-10, BRCA1ΔE11, BRCA1ΔE11q, and BRCA2ΔE3, BRCA2ΔE12 and BRCA2ΔE17-18 of which three (BRCA1ΔE11q, BRCA1ΔE11 and BRCA2ΔE3) were significantly higher expressed in the ovarian cancer cohort than in healthy controls (p ≤ 0.0001). CONCLUSIONS: This observation indicates that the upregulation of selected naturally occurring transcripts can be stimulated by non-genetic mechanisms and be a potential systemic response to disease progression and/or treatment. However, this hypothesis requires further examination.

Genomic Profiling Identifies Putative Pathogenic Alterations in NSCLC Brain Metastases.

Introduction: Brain metastases (BM) severely affect the prognosis and quality of life of patients with NSCLC. Recently, molecularly targeted agents were found to have promising activity against BM in patients with NSCLC whose primary tumors carry "druggable" mutations. Nevertheless, it remains critical to identify specific pathogenic alterations that drive NSCLC-BM and that can provide novel and more effective therapeutic targets. Methods: To identify potentially targetable pathogenic alterations in NSCLC-BM, we profiled somatic copy number alterations (SCNAs) in 51 matched pairs of primary NSCLC and BM samples from 33 patients with lung adenocarcinoma and 18 patients with lung squamous cell carcinoma. In addition, we performed multiregion copy number profiling on 15 BM samples and whole-exome sequencing on 40 of 51 NSCLC-BM pairs. Results: BM consistently had a higher burden of SCNAs compared with the matched primary tumors, and SCNAs were typically homogeneously distributed within BM, suggesting BM do not undergo extensive evolution once formed. By comparing focal SCNAs in matched NSCLC-BM pairs, we identified putative BM-driving alterations affecting multiple cancer genes, including several potentially targetable alterations in genes such as CDK12, DDR2, ERBB2, and NTRK1, which we validated in an independent cohort of 84 BM samples. Finally, we identified putative pathogenic alterations in multiple cancer genes, including genes involved in epigenome editing and 3D genome organization, such as EP300, CTCF, and STAG2, which we validated by targeted sequencing of an independent cohort of 115 BM samples. Conclusions: Our study represents the most comprehensive genomic characterization of NSCLC-BM available to date, paving the way to functional studies aimed at assessing the potential of the identified pathogenic alterations as clinical biomarkers and targets.

Personalized health risk assessment based on single-cell RNA sequencing analysis of a male with 45, X/48, XYYY karyotype.

Numeric sex chromosome abnormalities are commonly associated with an increased cancer risk. Here, we report a 14-year-old boy with a rare mosaic 45, X/48, XYYY karyotype presenting with subtle dysmorphic features and relative height deficiency, requiring growth hormone therapy. As only 12 postnatal cases have been described so far with very limited follow-up data, to assess the proband's long-term prognosis, including cancer risk, we performed high-throughput single-cell RNA sequencing (scRNA-seq) analysis. Although comprehensive cytogenetic analysis showed seemingly near perfect balance between 45, X and 48, XYYY cell populations, scRNA-seq revealed widespread differences in genotype distribution among immune cell fractions, specifically in monocytes, B- and T-cells. These results were confirmed at DNA level by digital-droplet PCR on flow-sorted immune cell types. Furthermore, deregulation of predominantly autosomal genes was observed, including TCL1A overexpression in 45, X B-lymphocytes and other known genes associated with hematological malignancies. Together with the standard hematological results, showing increased fractions of monocytes and CD4+/CD8+T lymphocytes ratio, long-term personalized hemato-oncological surveillance was recommended in the reported patient.

Alterations in key signaling pathways in sinonasal tract melanoma. A molecular genetics and immunohistochemical study of 90 cases and comprehensive review of the literature.

Sinonasal mucosal melanoma is a rare tumor arising within the nasal cavity, paranasal sinuses, or nasopharynx (sinonasal tract). This study evaluated 90 cases diagnosed in 29 males and 61 females with median age 68 years. Most tumors involved the nasal cavity and had an epithelioid morphology. Spectrum of research techniques used in this analysis includes targeted-DNA and -RNA next-generation sequencing, Sanger sequencing, fluorescence in situ hybridization and immunohistochemistry. Sinonasal melanomas were commonly driven by RAS (38/90, 42%), especially NRAS (n = 36) mutations and rarely (4/90, 4%) displayed BRAF pathogenic variants. BRAF/RAS mutants were more frequent among paranasal sinuses (10/14, 71%) than nasal (26/64, 41%) tumors. BRAF/RAS-wild type tumors occasionally harbored alterations of the key components and regulators of Ras-MAPK signaling pathway: NF1 mutations (1/17, 6%) or NF1 locus deletions (1/25, 4%), SPRED1 (3/25, 12%), PIK3CA (3/50, 6%), PTEN (4/50, 8%) and mTOR (1/50, 2%) mutations. These mutations often occurred in a mutually exclusive manner. In several tumors some of which were NRAS mutants, TP53 was deleted (6/48, 13%) and/or mutated (5/90, 6%). Variable nuclear accumulation of TP53, mirrored by elevated nuclear MDM2 expression was seen in >50% of cases. Furthermore, sinonasal melanomas (n = 7) including RAS/BRAF-wild type tumors (n = 5) harbored alterations of the key components and regulators of canonical WNT-pathway: APC (4/90, 4%), CTNNB1 (3/90, 3%) and AMER1 (1/90, 1%). Both, TERT promoter mutations (5/53, 9%) and fusions (2/40, 5%) were identified. The latter occurred in BRAF/RAS-wild type tumors. No oncogenic fusion gene transcripts previously reported in cutaneous melanomas were detected. Eight tumors including 7 BRAF/RAS-wild type cases expressed ADCK4::NUMBL cis-fusion transcripts. In summary, this study documented mutational activation of NRAS and other key components and regulators of Ras-MAPK signaling pathway such as SPRED1 in a majority of sinonasal melanomas.

Synchronous bilateral multifocal basal cell adenomas of the parotid gland-a case report.

BACKGROUND: Bilateral parotid gland tumors account for up to 3% of cases. In this group, the vast majority are Warthin's tumors. However, bilateral presentations of other parotid gland tumor entities is also possible, an example of which is a basal cell adenoma (BCA). Bilateral BCA is extremely rare, which could cause misdiagnosing it as a Warthin tumor. CASE PRESENTATION: The current study reports the unique case of a 48-year-old woman who presented with a 6-month history of slowly growing masses located bilaterally in the parotid region, surgically treated with 5-year follow-up (no recurrence, normal facial nerve function). Magnetic resonance imaging (MRI) revealed three lesions: two in the superficial and deep lobes of the right parotid gland, and one in the superficial lobe of the left parotid gland. A total parotidectomy with facial nerve preservation was performed on the right side, and superficial parotidectomy on the left side 6 months later. Histopathological examination confirmed that all three tumors were BCAs. Molecular analysis didn't show any strong, potential of unknown clinical significance in the studied sample. CONCLUSIONS: Multifocal bilateral lesions of the parotid gland are usually Warthin tumors. Detailed preoperative diagnostics including MRI and histopathological examination is essential to avoid misdiagnosing BCA and Warthin tumors. To our best knowledge, no case of synchronous bilateral multifocal basal cell adenomas of the parotid gland has been reported in English literature so far.

Impact of Activation of EGFL7 within Microenvironment of High Grade Ovarian Serous Carcinoma on Infiltration of CD4+ and CD8+ Lymphocytes.

Background: It has been demonstrated that Egfl7 promotes tumor cell escape from immunity by downregulating the activation of tumor blood vessels. Aim: to analyze mRNA expression of EGFL7 within the tumor microenvironment of high-grade ovarian serous carcinoma and its association with a number of intraepithelial CD4+/CD8+ lymphocytes and ICAM-1 expression. Methods: qPCR analysis of EGFL7 mRNA in cancer cells and adjacent stromal endothelium microdissected from formalin-fixed paraffin-embedded tumors of 59 high-grade ovarian serous carcinoma patients, was performed. Infiltration of intraepithelial lymphocytes (CD4+/CD8+) and expression of ICAM-1 were evaluated by immunohistochemistry and compared between tumors with different statuses of EGFL7 expression. Results: EGFL7 was expressed in cancer cells (9/59, 15.25%), endothelium (8/59, 13.56%), or both cancer cells and adjacent endothelium (4/59, 6.78%). ICAM-1 was expressed on cancer cells (47/59, 79.66%), stromal endothelium (46/59, 77.97%), or both epithelium and endothelium (40 of 59, 67.8%). EGFL7-positivity of cancer cells and endothelium was associated with lower intraepithelial inflow of CD4+ (p = 0.022 and p = 0.029, respectively) and CD8+ lymphocytes (p = 0.004 and p = 0.031, respectively) but impact neither epithelial nor endothelial ICAM-1 expression (p = 0.098 and p = 0.119, respectively). The patients' median follow-up was 23.83 months (range 1.07-78.07). Lack of prognostic significance of EGFL7-status and ICAM-1 expression was notified. Conclusion: EGFL7 is activated in the cancer cells as frequently as in the endothelium of human high-grade ovarian serous carcinoma. Activation of EGFL7 in cancer cells and/or endothelial cells could negatively impact diapedesis regardless of localization.

Secondary chronic myeloid leukemia in a patient with CALR and ASXL1-mutated primary myelofibrosis.

Development of secondary CML has only been casually described, with few reports attempting to analyze and explain the mechanisms behind this phenomenon. Reported cases vary with regard to presumed pathogenesis and clinical characteristics, but similarities can be observed. This report presents the case of a patient diagnosed with CALR and ASXL1-mutated primary myelofibrosis who developed CML 13 years after the initial diagnosis. In contrast with previously reported cases, this patient did not have JAK2 or ABL1 gene mutations, and also exhibited primary resistance to tyrosine kinase inhibitor (TKI) treatment. Here, we analyze the molecular evolution of CML and describe successful treatment with concomitant therapy including a TKI and JAK inhibitor. This report aims to deepen clinical experience and further broaden knowledge about chronic myeloproliferative neoplasms.

Variant Identification in BARD1, PRDM9, RCC1, and RECQL in Patients with Ovarian Cancer by Targeted Next-generation Sequencing of DNA Pools.

Several ovarian cancer susceptibility genes have been discovered, but more are likely to exist. In this study, we aimed to analyze knowledge-based selected genes, that is, BARD1, PRDM9, RCC1, and RECQL, in which pathogenic germline variants have been reported in patients with breast and/or ovarian cancer. As deep sequencing of DNA samples remains costly, targeted next-generation sequencing of DNA pools was utilized to screen the exons of BARD1, PRDM9, RCC1, and RECQL in approximately 400 Polish ovarian cancer cases. A total of 25 pools of 16 samples (including several duplicated samples with known variants) were sequenced on the NovaSeq6000 and analyzed with SureCall (Agilent) application. The set of variants was filtrated to exclude spurious variants, and, subsequently, the identified rare genetic variants were validated using Sanger sequencing. No pathogenic mutation was found within the analyzed cohort of patients with ovarian cancer. Validation genotyping of filtered rare silent and missense variants revealed that the majority of them were true alterations, especially those with a higher mutation quality value. The high concordance (R2 = 0.95) of population allele frequency for 44 common SNPs in the European control population (gnomAD) and our experiment confirmed the reliability of pooled sequencing. Mutations in BARD1, PRDM9, RCC1, and RECQL do not contribute substantially to the risk of ovarian cancer. Pooled DNA sequencing is a cost-effective and reliable method for the initial screening of candidate genes; however, it still requires validation of identified rare variants. PREVENTION RELEVANCE: BARD1, PRDM9, RCC1, and RECQL are not high/moderate-risk ovarian cancer susceptibility genes. Pooled sequencing is a reliable and cost-effective method to detect rare variants in candidate genes.

Intrasalivary Thymic Carcinoma: A Case Report and Literature Review.

Ectopic thymic carcinomas are rarely diagnosed in the thyroid gland, let alone in extrathyroid tissues. In the currently available literature, only five cases of extrathyroidal malignancies with thymic differentiation have been reported as arising in the major salivary glands. A 69-year-old female presented with a slow-growing palpable mass in the left parotid gland. Fine needle aspiration biopsy suggested metastatic cancer, whereas core needle biopsy revealed high-grade squamous cell carcinoma. The patient underwent left radical parotidectomy with selective ipsilateral lymph node dissection and subsequent radiation therapy. The surgical specimen was taken for histopathological examination. Microscopically, the tumor resembled thymic carcinoma. It was composed of large nests of squamoid cells with smooth contours, focally with a syncytial growth pattern, and accompanied by abundant lymphocytes with reactive lymphoid follicles. This appearance resembled a micronodular thymic carcinoma with lymphoid hyperplasia. Moreover, the tumor displayed expression of squamous markers (p40 and p63) and markers of thymic carcinoma (CD5 and CD117). Therefore, the final diagnosis of intrasalivary thymic carcinoma was rendered. The molecular analysis including next-generation sequencing demonstrated no variants of the strong, potential, or unknown clinical significance. The patient remains disease-free at 1-year follow-up. In the current case, we comprehensively present a clinical, microscopic, molecular, and radiological picture of CD5-positive squamous cell carcinoma of the parotid. We postulate that similar cases should be designated as intrasalivary thymic carcinoma analogically to similar thyroid tumors. Our case and the limited literature data indicate they should be distinguished from conventional squamous cell carcinoma of major salivary glands due to their rather favorable prognosis.

Therapeutic options in inoperable ROS1-rearranged inflammatory myofibroblastic tumor of the tongue in a child: a case report and literature review.

Inflammatory myofibroblastic tumor (IMT) is a rare borderline malignancy, usually treated with surgery only. Exceedingly rare cases of inoperable, recurrent, or metastatic IMTs pose a therapeutic challenge. We report successful treatment of a 7-year-old girl with an inoperable anaplastic lymphoma kinase (ALK)-negative IMT of the tongue. The patient underwent various anti-inflammatory (steroids, nonsteroidal anti-inflammatory drugs, clarithromycin) and antiproliferative (chemotherapy) therapies to enable tumor regression and complete resection. Ultimately, next-generation sequencing of the tumor revealed a TFG-ROS-1 translocation, allowing for an off-label targeted therapy with crizotinib. Crizotinib treatment caused slight tumor regression but evident change of its structure, allowing for complete non-mutilating resection. Two histopathology examinations revealed complete disappearance of neoplastic cells following therapy. The patient remains disease-free 22 months after the delayed surgery. In children with inoperable ALK-negative IMTs, molecular testing must be performed to identify other targetable oncogenic fusions, including TFG-ROS1.

The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer.

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.

The Role of Butyrylcholinesterase and Iron in the Regulation of Cholinergic Network and Cognitive Dysfunction in Alzheimer's Disease Pathogenesis.

Alzheimer's disease (AD), the most common form of dementia in elderly individuals, is marked by progressive neuron loss. Despite more than 100 years of research on AD, there is still no treatment to cure or prevent the disease. High levels of amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs) in the brain are neuropathological hallmarks of AD. However, based on postmortem analyses, up to 44% of individuals have been shown to have high Aß deposits with no clinical signs, due to having a "cognitive reserve". The biochemical mechanism explaining the prevention of cognitive impairment in the presence of Aß plaques is still unknown. It seems that in addition to protein aggregation, neuroinflammatory changes associated with aging are present in AD brains that are correlated with a higher level of brain iron and oxidative stress. It has been shown that iron accumulates around amyloid plaques in AD mouse models and postmortem brain tissues of AD patients. Iron is required for essential brain functions, including oxidative metabolism, myelination, and neurotransmitter synthesis. However, an imbalance in brain iron homeostasis caused by aging underlies many neurodegenerative diseases. It has been proposed that high iron levels trigger an avalanche of events that push the progress of the disease, accelerating cognitive decline. Patients with increased amyloid plaques and iron are highly likely to develop dementia. Our observations indicate that the butyrylcholinesterase (BChE) level seems to be iron-dependent, and reports show that BChE produced by reactive astrocytes can make cognitive functions worse by accelerating the decay of acetylcholine in aging brains. Why, even when there is a genetic risk, do symptoms of the disease appear after many years? Here, we discuss the relationship between genetic factors, age-dependent iron tissue accumulation, and inflammation, focusing on AD.

Carotid intima-media thickness (IMT) in patients with severe familial and non-familial hypercholesterolemia: The effect of measurement site on the IMT correlation with traditional cardiovascular risk factors and calcium scores.

BACKGROUND: The carotid intima-media thickness (IMT) measurement may be carried out proximally (pIMT) or distally (dIMT) in relation to the bulb of the common carotid artery which has significant implications on the results and correlation with risk factors. The aim of the study was to compare the pIMT and dIMT in patients with familial hypercholesterolemia confirmed by genetic testing (FH group) and patients with severe non-familial hypercholesterolemia, with negative results of genetic testing (NFH group) and to determine the correlation of results with traditional atherosclerotic risk factors and calcium scores. METHODS: A total of 86 FH and 50 NFH patients underwent pIMT and dIMT measurements of both carotid arteries as well as computed tomography (CT) with coronary and thoracic aorta calcium scoring. RESULTS: The meanpIMT of both right and left common carotid artery were significantly higher in patients with FH compared to the NFH group (meanpRIMT 0.721 ± 0.152 vs. 0.644 ± 0.156, p < 0.01, meanpLIMT 0.758 ± 0.173 vs. 0.670 ± 0.110, p < 0.01). Patient age, pre-treatment lowdensity lipoprotein (LDL) cholesterol levels (LDLmax) at baseline and systolic blood pressure were independent predictors of pIMT increases in both carotid arteries. Smoking history, age and LDLmax were independent predictors of dIMT increase. There was a significant correlation between the calcium scores of the ascending aorta, coronary artery and aortic valve and all IMT parameters. CONCLUSIONS: The IMT measured proximally better between patients with familial and non-familial hypercholesterolemia. The association between IMT and traditional cardiovascular risk factors varies between measurement sites. IMT values correlate CT calcium scores in all patients with hypercholesterolaemia regardless of genetic etiology.

Colorectal Adenocarcinomas Harboring ALK Fusion Genes: A Clinicopathologic and Molecular Genetic Study of 12 Cases and Review of the Literature.

This study determined the frequency and the clinicopathologic and genetic features of colorectal carcinomas driven by oncogenic fusions of the anaplastic lymphoma kinase gene (ALK). Of the 8150 screened tumors, 12 (0.15%) were immunohistochemically ALK-positive with D5F3 antibody. These cancers harbored CAD-ALK (n=1), DIAPH2-ALK (n=2), EML4-ALK (n=2), LOC101929227-ALK (n=1), SLMAP-ALK (n=1), SPTBN1-ALK (n=4), and STRN-ALK (n=1) fusions, as detected by an RNA-based next-generation sequencing assay. ALK fusion carcinomas were diagnosed mostly in older patients with a 9:3 female predominance (median age: 72 y). All tumors, except a rectal one, occurred in the right colon. Most tumors were stage T3 (n=7) or T4 (n=3). Local lymph node and distant metastases were seen at presentation in 9 and 2 patients. These tumors showed moderate (n=6) or poor (n=3) glandular differentiation, solid medullary growth pattern (n=2), and pure mucinous morphology (n=1). DNA mismatch repair-deficient phenotype was identified in 10 cases. Tumor-infiltrating lymphocytes were prominent in 9 carcinomas. In 4 carcinomas, tumor cells showed strong, focal (n=3), or diffuse programmed death-ligand 1 immunoreactivity. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 tumors. Four patients died of disease within 3 years, and 7 were alive with follow-up ranging from 1 to 8 years. No mutations in BRAF, RAS, and in genes encoding components of PI3K-AKT/MTOR pathway were identified. However, 1 tumor had a loss-of-function PTEN mutation. Aberration of p53 signaling, TP53 mutations, and/or nuclear accumulation of p53 protein was seen in 9 cases. ALK fusion colorectal carcinomas are a distinct and rare subtype of colorectal cancers displaying some features of mismatch repair-deficient tumors.

New Approach to Paediatric Mastocytosis: Implications of KIT D816V Mutation Detection in Peripheral Blood.

is missing (Short communication).

Cell-free DNA BRAF V600E measurements during BRAF inhibitor therapy of metastatic melanoma: long-term analysis.

OBJECTIVE: We assessed the status of the BRAF V600E mutation in cell-free circulating tumor DNA (cfDNA) isolated from the plasma of patients with metastatic melanoma treated with the BRAF inhibitor vemurafenib, collected at different time points during therapy to evaluate the sensitivity and specificity of quantitative polymerase chain reaction and droplet digital polymerase chain reaction (ddPCR) and the correlation between the level of plasma cfDNA p.V600E and the long-term clinical outcome. METHODS: cfDNA in patients with BRAF-mutated melanoma (n = 62) was analyzed at baseline and at 4-8 weeks from the start of vemurafenib therapy. BRAF mutations were assessed using tumor tissue-derived DNA and circulating cfDNA from plasma samples. Quantification of BRAF V600E was performed in cfDNA using ddPCR. RESULTS: cfDNA V600E was detected in the plasma of 48/62 (77%) patients at baseline and in 18/62 (29%) patients after 4-8 weeks of treatment. Patients positive for BRAF mutations in cfDNA at baseline had shorter progression-free survival (PFS) and overall survival (OS) compared with patients with undetectable cfDNA BRAF mutations. Undetectable cfDNA p.V600E at baseline and after 4-8 weeks of therapy was associated with the best prognosis. When treated as a continuous variable, the log-transformed concentration of baseline cfDNA p.V600E was significantly associated with both PFS and OS. This effect was retained in the multivariate OS Cox model adjusted for Eastern Cooperative Oncology Group performance status, the presence of brain metastases, patient age, and previous systemic treatment. CONCLUSIONS: Monitoring of plasma BRAF p.V600E cfDNA concentrations in patients with metastatic melanoma on targeted therapy may have prognostic value. Undetectable cfDNA p.V600E before and during treatment was associated with a favorable prognosis.

Colonic Adenocarcinomas Harboring NTRK Fusion Genes: A Clinicopathologic and Molecular Genetic Study of 16 Cases and Review of the Literature.

This study was undertaken to determine the frequency, and the clinicopathologic and genetic features, of colon cancers driven by neurotrophic receptor tyrosine kinase (NTRK) gene fusions. Of the 7008 tumors screened for NTRK expression using a pan-Trk antibody, 16 (0.23%) had Trk immunoreactivity. ArcherDx assay detected TPM3-NTRK1 (n=9), LMNA-NTRK1 (n=3), TPR-NTRK1 (n=2) and EML4-NTRK3 (n=1) fusion transcripts in 15 cases with sufficient RNA quality. Patients were predominantly women (median age: 63 y). The tumors involved the right (n=12) and left colon unequally and were either stage T3 (n=12) or T4. Local lymph node and distant metastases were seen at presentation in 6 and 1 patients, respectively. Lymphovascular invasion was present in all cases. Histologically, tumors showed moderate to poor (n=11) differentiation with a partly or entirely solid pattern (n=5) and mucinous component (n=10), including 1 case with sheets of signet ring cells. DNA mismatch repair-deficient phenotype was seen in 13 cases. Tumor-infiltrating CD4/CD8 lymphocytes were prominent in 9 cases. Programmed death-ligand 1 positive tumor-infiltrating immune cells and focal tumor cell positivity were seen in the majority of cases. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 cases. No mutations in BRAF, RAS, and PIK3CA were identified. However, other genes of the PI3K-AKT/MTOR pathway were mutated. In several cases, components of Wnt/ß-catenin (APC, AMER1, CTNNB1), p53, and TGFß (ACVR2A, TGFBR2) pathways were mutated. However, no SMAD4 mutations were found. Two tumors harbored FBXW7 tumor suppressor gene mutations. NTRK fusion tumors constitute a distinct but rare subgroup of colorectal carcinomas.