Sequential antigen loss and branching evolution in lymphoma after CD19- and CD20-targeted T-cell-redirecting therapy.

ABSTRACT: CD19 chimeric antigen receptor (CAR) T cells and CD20 targeting T-cell-engaging bispecific antibodies (bispecs) have been approved in B-cell non-Hodgkin lymphoma lately, heralding a new clinical setting in which patients are treated with both approaches, sequentially. The aim of our study was to investigate the selective pressure of CD19- and CD20-directed therapy on the clonal architecture in lymphoma. Using a broad analytical pipeline on 28 longitudinally collected specimen from 7 patients, we identified truncating mutations in the gene encoding CD20 conferring antigen loss in 80% of patients relapsing from CD20 bispecs. Pronounced T-cell exhaustion was identified in cases with progressive disease and retained CD20 expression. We also confirmed CD19 loss after CAR T-cell therapy and reported the case of sequential CD19 and CD20 loss. We observed branching evolution with re-emergence of CD20+ subclones at later time points and spatial heterogeneity for CD20 expression in response to targeted therapy. Our results highlight immunotherapy as not only an evolutionary bottleneck selecting for antigen loss variants but also complex evolutionary pathways underlying disease progression from these novel therapies.

Detection of wildtype Merkel cell polyomavirus genomic sequence and VP1 transcription in a subset of Merkel cell carcinoma.

AIMS: Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV). Characteristic for these virus-positive (VP) MCC is MCPyV integration into the host genome and truncation of the viral oncogene Large T antigen (LT), with full-length LT expression considered as incompatible with MCC growth. Genetic analysis of a VP-MCC/trichoblastoma combined tumour demonstrated that virus-driven MCC can arise from an epithelial cell. Here we describe two further cases of VP-MCC combined with an adnexal tumour, i.e. one trichoblastoma and one poroma. METHODS AND RESULTS: Whole-genome sequencing of MCC/trichoblastoma again provided evidence of a trichoblastoma-derived MCC. Although an MCC-typical LT-truncating mutation was detected, we could not determine an integration site and we additionally detected a wildtype sequence encoding full-length LT. Similarly, Sanger sequencing of the combined MCC/poroma revealed coding sequences for both truncated and full-length LT. Moreover, in situ RNA hybridization demonstrated expression of a late region mRNA encoding the viral capsid protein VP1 in both combined as well as in a few cases of pure MCC. CONCLUSION: The data presented here suggest the presence of wildtype MCPyV genomes and VP1 transcription in a subset of MCC.

Oncogenic alterations in KIR3DL1 in cutaneous acral CD8+ lymphoproliferative disorder.

BACKGROUND: Primary cutaneous acral CD8+ T-cell lymphoproliferative disorder (TLPD) is a rare and indolent lymphoma entity. Although known for years, the molecular pathogenesis is still unknown. OBJECTIVES: In order to better understand the molecular pathogenesis of cutaneous acral CD8+ TLPD and to identify further discriminatory markers to discern this lymphoma subtype from further CD8+ cutaneous lymphomas, we analysed five cases of cutaneous acral CD8+ TLPD for putative molecular alterations. METHODS: Somatic alterations were assessed by whole exome and targeted sequencing of paraffin-embedded tissue. Results were evaluated by immunohistochemical staining of respective relevant proteins. CD8+ cutaneous T-cell lymphomas (n = 12) served as control for KIR3DL1-staining. RESULTS: Copy number variations (CNV) analysis revealed a homozygous deletion of the KIR3DL1 gene in two of the analysed cases. This resulted in loss of KIR3DL1 protein expression which was observed in all cases of cutaneous acral CD8+ TLPD. In contrast, KIR3DL1 expression was more variable in other CD8+ cutaneous T-cell lymphomas with 50% of analysed cases (n = 12) being positive. In addition, one further case of acral CD8+ TLPD harboured a loss of function mutation in the PIK3R1 gene presumably activating the PI3K-AKT pathway. CONCLUSION: Alterations of KIR3DL1 gene may be of pathogenetic relevance for acral CD8+ TLPD. Loss of KIR3DL1 protein expression may support the diagnosis of this indolent lymphoma entity, albeit not being a subtype-specific discriminative feature.

Tracheal brush cells release acetylcholine in response to bitter tastants for paracrine and autocrine signaling.

For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter "taste" substances are most probably initiated by tracheal brush cells (BC). Our single-cell RNA-seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca2+]i in BC and subsequent ACh-release. ACh-release is regulated in an autocrine manner. While the muscarinic ACh-receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca2+]i in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5- and M3R-mediated. We show that ACh-release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.

hnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons.

Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance.

The identification of patient-specific mutations reveals dual pathway activation in most patients with melanoma and activated receptor tyrosine kinases in BRAF/NRAS wild-type melanomas.

BACKGROUND: Increasing knowledge of cancer genomes has triggered the development of specific targeted inhibitors, thus providing a valuable therapeutic pool. METHODS: In this report, the authors analyze the presence of targetable alterations in 136 tumor samples from 92 patients with melanoma using a comprehensive approach based on targeted DNA sequencing and supported by RNA and protein analysis. Three topics of high clinical relevance are addressed: the identification of rare, activating alterations; the detection of patient-specific, co-occurring single nucleotide variants (SNVs) and copy number variations (CNVs) in parallel pathways; and the presence of cancer-relevant germline mutations. RESULTS: The analysis of patient-matched blood and tumor samples was done with a custom-designed gene panel that was enriched for genes from clinically targetable pathways. To detect alterations with high therapeutic relevance for patients with unknown driver mutations, genes that are untypical for melanoma also were included. Among all patients, CNVs were identified in one-third of samples and contained amplifications of druggable kinases, such as CDK4, ERBB2, and KIT. Considering SNVs and CNVs, 60% of patients with metastases exhibited co-occurring activations of at least 2 pathways, thus providing a rationale for individualized combination therapies. Unexpectedly, 9% of patients carry potentially protumorigenic germline mutations frequently affecting receptor tyrosine kinases. Remarkably two-thirds of BRAF/NRAS wild-type melanomas harbor activating mutations or CNVs in receptor tyrosine kinases. CONCLUSIONS: The results indicate that the integrated analysis of SNVs, CNVs, and germline mutations reveals new druggable targets for combination tumor therapy.

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation-negative early-onset and high-risk breast cancer patients.

To evaluate the role of constitutive epigenetic changes in normal body cells of BRCA1/BRCA2-mutation negative patients, we have developed a deep bisulfite sequencing assay targeting the promoter regions of 8 tumor suppressor (TS) genes (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) and the estrogene receptor gene (ESR1), which plays a role in tumor progression. We analyzed blood samples of two breast cancer (BC) cohorts with early onset (EO) and high risk (HR) for a heterozygous mutation, respectively, along with age-matched controls. Methylation analysis of up to 50,000 individual DNA molecules per gene and sample allowed quantification of epimutations (alleles with >50% methylated CpGs), which are associated with epigenetic silencing. Compared to ESR1, which is representative for an average promoter, TS genes were characterized by a very low (< 1%) average methylation level and a very low mean epimutation rate (EMR; < 0.0001% to 0.1%). With exception of BRCA1, which showed an increased EMR in BC (0.31% vs. 0.06%), there was no significant difference between patients and controls. One of 36 HR BC patients exhibited a dramatically increased EMR (14.7%) in BRCA1, consistent with a disease-causing epimutation. Approximately one third (15 of 44) EO BC patients exhibited increased rates of single CpG methylation errors in multiple TS genes. Both EO and HR BC patients exhibited global underexpression of blood TS genes. We propose that epigenetic abnormalities in normal body cells are indicative of disturbed mechanisms for maintaining low methylation and appropriate expression levels and may be associated with an increased BC risk.

Whole transcriptome profiling reveals the RNA content of motor axons.

Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.

Immunoglobulin rearrangement analysis from multiple lesions in the same patient using next-generation sequencing.

AIMS: For patients who have multiple lymphomas with discordant pathology, it is relevant to determine whether there is one disseminated lymphoma or two unrelated lymphomas. Patients with disseminated, clonally related lymphomas are usually treated with the most powerful drugs available, while patients with unrelated (primary) lymphomas receive mainly standard first-line therapies. METHODS AND RESULTS: We used next-generation sequencing on the Ion Torrent Personal Genome Machine to characterize the immunoglobulin heavy gene V-D-J rearrangements in two diagnostic tissue samples, including formalin-fixed and paraffin-embedded tissue, of two patients with iatrogenic immunodeficiency-associated Epstein-Barr virus lymphoproliferative disorder, with ulcerative colitis as underlying disease. The immunoglobulin rearrangement sequences obtained by next-generation sequencing revealed undoubtedly clonally related lesions in two tissue biopsies that were taken over time in the first patient, which is concordant with disseminated lymphoma. The other patient showed two clonally unrelated lesions, which is incompatible with clonal dissemination. This information was not inferred from evaluation of the heavy and light chain rearrangements by fragment analysis, which is currently the gold standard. CONCLUSION: Our study demonstrates the diagnostic application of next-generation sequencing of immunoglobulin rearrangement assessment in pathology for clinical decision-making in patients with several simultaneous or subsequent lymphoproliferations.

Circulating cell-free DNA-based biomarkers for prognostication and disease monitoring in adrenocortical carcinoma.

OBJECTIVE: Adrenocortical carcinoma (ACC) is a rare aggressive cancer with heterogeneous behaviour. Disease surveillance relies on frequent imaging, which comes with significant radiation exposure. The aim of the study was to investigate the role of circulating cell-free DNA (ccfDNA)-related biomarkers (BMs) for prognostication and monitoring of ACC. DESIGN AND METHODS: We investigated 34 patients with ACC and 23 healthy subjects (HSs) as controls. Circulating cell-free DNA was extracted by commercial kits and ccfDNA concentrations were quantified by fluorimeter (BM1). Targeted sequencing was performed using a customized panel of 27 ACC-specific genes. Leucocyte DNA was used to discriminate somatic variants (BM2), while tumour DNA was sequenced in 22/34 cases for comparison. Serial ccfDNA samples were collected during follow-up in 19 ACC patients (median period 9 months) and analysed in relationship with standard radiological imaging. RESULTS: Circulating cell-free DNA concentrations were higher in ACC than HS (mean ± SD, 1.15 ± 1.56 vs 0.05 ± 0.05 ng/µL, P < .0001), 96% of them being above the cut-off of 0.146 ng/µL (mean HS + 2 SD, positive BM1). At ccfDNA sequencing, 47% of ACC showed at least 1 somatic mutation (positive BM2). A combined ccfDNA-BM score was strongly associated with both progression-free and overall survival (hazard ratio [HR] = 2.63; 95% CI, 1.13-6.13; P = .010, and HR = 5.98; 95% CI, 2.29-15.6; P = .0001, respectively). During disease monitoring, positive BM2 showed the best specificity (100%) and sensitivity (67%) to detect ACC recurrence or progress compared with BM1. CONCLUSION: ccfDNA-related BMs are frequently detected in ACC patients and represent a promising, minimally invasive tool to predict clinical outcome and complement surveillance imaging. Our findings will be validated in a larger cohort of ACCs with long-term follow-up.

Autosomal-dominant striatal degeneration is caused by a mutation in the phosphodiesterase 8B gene.

Autosomal-dominant striatal degeneration (ADSD) is an autosomal-dominant movement disorder affecting the striatal part of the basal ganglia. ADSD is characterized by bradykinesia, dysarthria, and muscle rigidity. These symptoms resemble idiopathic Parkinson disease, but tremor is not present. Using genetic linkage analysis, we have mapped the causative genetic defect to a 3.25 megabase candidate region on chromosome 5q13.3-q14.1. A maximum LOD score of 4.1 (Theta = 0) was obtained at marker D5S1962. Here we show that ADSD is caused by a complex frameshift mutation (c.94G>C+c.95delT) in the phosphodiesterase 8B (PDE8B) gene, which results in a loss of enzymatic phosphodiesterase activity. We found that PDE8B is highly expressed in the brain, especially in the putamen, which is affected by ADSD. PDE8B degrades cyclic AMP, a second messenger implied in dopamine signaling. Dopamine is one of the main neurotransmitters involved in movement control and is deficient in Parkinson disease. We believe that the functional analysis of PDE8B will help to further elucidate the pathomechanism of ADSD as well as contribute to a better understanding of movement disorders.

Epithelioid and spindle cell rhabdomyosarcoma with EWSR1::TFCP2 fusion mimicking metastatic lung cancer: A case report and literature review.

Rhabdomyosarcoma (RMS) with EWSR1/FUS::TFCP2 fusion is an emerging, molecularly defined, rare subtype of RMS. It can affect patients in a wide age range and follows an aggressive clinical course according to the reported cases. Due to its unusual clinical and pathohistological features, with a typical intraosseous presentation and common cytokeratin expression, the diagnosis is challenging, and metastatic undifferentiated/sarcomatoid carcinoma can be an important differential diagnosis. We report here a case of a 55-year-old woman with an RMS with EWSR1::TFCP2 fusion mimicking metastatic lung cancer in view of the clinical and microscopic presentation. However, further molecular workup, including RNA sequencing, led to the proper diagnosis. Although these tumors are rare, knowledge of their unique features is essential for correct diagnosis as a basis for clinical management and optimization of therapeutic approaches.

Performance of DNA-based biomarkers for classification of adrenocortical carcinoma: a prognostic study.

OBJECTIVE: Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with heterogeneous clinical outcomes. Recent studies proposed a combination of clinical/histopathological parameters (S-GRAS score) or molecular biomarkers (BMs) to improve prognostication. We performed a comparative analysis of DNA-based BMs by evaluating their added prognostic value to the S-GRAS score. DESIGN AND METHODS: A total of 194 formalin-fixed, paraffin-embedded (FFPE) ACC samples were analysed, including a retrospective training cohort (n = 107) and a prospective validation cohort (n = 87). Targeted DNA sequencing and pyrosequencing were used to detect somatic single-nucleotide variations in ACC-specific genes and methylation in the promoter region of paired box 5 (PAX5). The European Network for the Study of Adrenocortical Tumors (ENSAT) tumour stage, age, symptoms at presentation, resection status, and Ki-67 were combined to calculate S-GRAS. Endpoints were overall (OS), progression-free (PFS), and disease-free survival (DFS). Prognostic role was evaluated by multivariable survival analysis and their performance compared by Harrell's concordance index (C index). RESULTS: In training cohort, an independent prognostic role was confirmed at multivariate analysis for two DNA-based BMs: alterations in Wnt/ß-catenin and Rb/p53 pathways and hypermethylated PAX5 (both P< .05 for PFS and DFS, hazard ratio [HR] 1.47-2.33). These were combined to S-GRAS to obtain a combined (COMBI) score. At comparative analysis, the best discriminative prognostic model was COMBI score in both cohorts for all endpoints, followed by S-GRAS score (C index for OS 0.724 and 0.765, PFS 0.717 and 0.670, and DFS 0.699 and 0.644, respectively). CONCLUSIONS: Targeted DNA-based BM evaluated on routinely available FFPE samples improves prognostication of ACC beyond routinely available clinical and histopathological parameters. This approach may help to better individualise patient's management.

Pontine autosomal dominant microangiopathy with leukoencephalopathy: Col4A1 gene variants in the original family and sporadic stroke.

BACKGROUND: (1) Description of clinical and cranial MRI features in the original Pontine Autosomal Dominant Microangiopathy with Leukoencephalopathy (PADMAL) family and correlation with the segregation analysis of the causative collagen 4A1 gene (COL4A1) variant. (2) Sequence analysis of the COL4A1 miRNA-binding site containing the causative variant in two independent cross-sectional samples of sporadic stroke patients. PATIENTS AND METHODS: Sanger sequencing of the COL4A1 miRNA-binding site in the PADMAL family and 874 sporadic stroke patients. RESULTS: PADMAL shows adult-onset usually between 30 and 50 years of age with initial brainstem-related symptoms most commonly dysarthria, with progression to dementia and tetraparesis. Radiologically pontine lacunes are followed by supratentorial white matter involvement. Radiological onset may precede clinical symptoms. We found no variants in the COL4A1 miRNA-binding site of sporadic stroke patients. CONCLUSION: Our results allow an early diagnosis of PADMAL based on cranial MRI, clinical signs, and confirmatory sequencing of the COL4A1 miRNA-29-binding site. COL4A1 miRNA-29-binding site variants do not contribute to a sizeable proportion of sporadic stroke.

Plastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy.

Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degeneration. Despite the observed beneficial modifying effect of PLS3, the mechanism of how it supports F-actin-mediated cellular processes in motoneurons is not yet well understood. Our data reveal disturbed F-actin-dependent translocation of the Tropomyosin receptor kinase B (TrkB) to the cell surface of Smn-deficient motor axon terminals, resulting in reduced TrkB activation by its ligand brain-derived neurotrophic factor (BDNF). Improved actin dynamics by overexpression of hPLS3 restores membrane recruitment and activation of TrkB and enhances spontaneous calcium transients by increasing Cav2.1/2 "cluster-like" formations in SMA axon terminals. Thus, our study provides a novel role for PLS3 in supporting correct alignment of transmembrane proteins, a key mechanism for (moto)-neuronal development.

Febrile infection-related epilepsy syndrome (FIRES) is not caused by SCN1A, POLG, PCDH19 mutations or rare copy number variations.

AIM: Febrile infection-related epilepsy syndrome (FIRES) is an enigmatic seizure disorder in childhood with an innocuous febrile infection triggering severe and intractable multifocal epilepsy, mostly with status epilepticus. FIRES shares several phenotypic features with epilepsies seen in patients with protocadherin 19 (PCDH19), sodium channel protein type 1 subunit alpha (SCN1A), and DNA polymerase subunit gamma-1 (POLG) mutations. The aim of the study was the mutation analysis of these prime candidate genes in a cohort of patients with FIRES. Additionally, given that rare copy number variations (CNVs) have recently been established as important risk factors for epilepsies, we performed a genome-wide CNV analysis. METHOD: We analysed the protein coding region, including splice sites of the three candidate genes in 15 patients (eight males, seven females) with FIRES (age at onset 3-15 y, median 6) using Sanger sequencing. Inclusion criteria were a status epilepticus without identifiable cause and a preceding febrile infection in previously healthy children. In addition, we performed genome-wide human single-nucleotide polymorphism 6.0 arrays in a subset of 10 patients to identify pathological CNVs. RESULTS: We could not identify the most likely pathogenic mutations or CNVs in FIRES. INTERPRETATION: Mutations in PCDH19, SCN1A, POLG, or CNVs are not responsible for FIRES.

Panel Sequencing of Primary Cutaneous B-Cell Lymphoma.

BACKGROUND: Primary cutaneous follicular B-cell lymphoma (PCFBCL) represents an indolent subtype of Non-Hodgkin's lymphomas, being clinically characterized by slowly growing tumors of the skin and common cutaneous relapses, while only exhibiting a low propensity for systemic dissemination or fatal outcome. Up to now, only few studies have investigated underlying molecular alterations of PCFBCL with respect to somatic mutations. OBJECTIVES: Our aim was to gain deeper insight into the pathogenesis of PCFBCL and to delineate discriminatory molecular features of this lymphoma subtype. METHODS: We performed hybridization-based panel sequencing of 40 lymphoma-associated genes of 10 cases of well-characterized PCFBCL. In addition, we included two further ambiguous cases of atypical B-cell-rich lymphoid infiltrate/B-cell lymphoma of the skin for which definite subtype attribution had not been possible by routine investigations. RESULTS: In 10 out of 12 analyzed cases, we identified genetic alterations within 15 of the selected 40 target genes. The most frequently detected alterations in PCFBCL affected the TNFRSF14, CREBBP, STAT6 and TP53 genes. Our analysis unrevealed novel mutations of the BCL2 gene in PCFBCL. All patients exhibited an indolent clinical course. Both the included arbitrary cases of atypical B-cell-rich cutaneous infiltrates showed somatic mutations within the FAS gene. As these mutations have previously been designated as subtype-specific recurrent alterations in primary cutaneous marginal zone lymphoma (PCMZL), we finally favored the diagnosis of PCMZL in these two cases based on these molecular findings. CONCLUSIONS: To conclude, our molecular data support that PCFBCL shows distinct somatic mutations which may aid to differentiate PCFBCL from pseudo-lymphoma as well as from other indolent and aggressive cutaneous B-cell lymphomas. While the detected genetic alterations of PCFBCL did not turn out to harbor any prognostic value in our cohort, our molecular data may add adjunctive discriminatory features for diagnostic purposes on a molecular level.

Merkel Cell Polyomavirus‒Negative Merkel Cell Carcinoma Originating from In Situ Squamous Cell Carcinoma: A Keratinocytic Tumor with Neuroendocrine Differentiation.

Although virus-negative Merkel cell carcinoma (MCC) is characterized by a high frequency of UV-induced mutations, the expression of two viral oncoproteins is regarded as a key mechanism driving Merkel cell polyomavirus‒positive MCC. The cells in which these molecular events initiate MCC oncogenesis have yet not been identified for both MCC subsets. A considerable proportion of virus-negative MCC is found in association with squamous cell carcinoma (SCC), suggesting (i) coincidental collision, (ii) one providing a niche for the other, or (iii) one evolving from the other. Whole-exome sequencing of four combined tumors consisting of SCC in situ and Merkel cell polyomavirus‒negative MCC showed many mutations shared between SCC and MCC in all cases, indicating a common ancestry and thereby a keratinocytic origin of these MCCs. Moreover, analyses of the combined cases as well as of pure SCC and MCC suggest that RB1 inactivation in SCC facilitates MCC development and that epigenetic changes may contribute to the SCC/MCC transition.

Novel genomic techniques open new avenues in the analysis of monogenic disorders.

The molecular genetic cause of over 3,000 monogenic disorders is currently unknown. This review discusses how novel genomic techniques like Next-Generation DNA Sequencing (NGS) and genotyping arrays open new avenues in the elucidation of genetic defects causing monogenic disorders. They will not only speed up disease gene identification but will enable us to systematically tackle previously intractable monogenic disorders. These are mainly disorders not amenable to classic linkage analysis, for example, due to insufficient family size. Most monogenic diseases are caused by exonic mutations or splice-site mutations changing the amino acid sequence of the affected gene. These mutations can be identified by sequencing of all exons in the human genome (exome sequencing) rendering whole genome sequencing unnecessary in most cases. Genotyping arrays containing 105 -2×106 single nucleotide polymorphisms (SNPs) and nonpolymorphic markers allow highly accurate mapping of genomic deletions and duplications not detectable by exome sequencing, which are the second most common cause of monogenic disorders. However, several hundred rare, previously unknown sequence variants affecting the amino acid sequence of the encoded protein are found in the exome of every human individual. Therefore, the main challenge will be the differentiation between the many rare benign variants detected by novel genomic techniques and disease causing mutations.

Oncogenic Mutations and Gene Fusions in CD30-Positive Lymphoproliferations and Clonally Related Mycosis Fungoides Occurring in the Same Patients.

The emergence of a common progenitor cell has been postulated for the association of CD30-positive lymphoproliferative disease (LPD) and mycosis fungoides (MF) within the same patient. Up to now, no comprehensive analysis has yet addressed the genetic profiles of such concurrent lymphoma subtypes. We aimed to delineate the molecular alterations of clonally related CD30-positive LPD and MF occurring in the same two patients. We analyzed the molecular profile of 16 samples of two patients suffering both from CD30-positive LPD and MF being obtained over a time course of at least 5 years. To detect oncogenic mutations, we applied targeted sequencing technologies with a hybrid capture-based DNA library preparation approach, and for the identification of fusion transcripts, an anchored multiplex PCR enrichment kit was used. In all samples of CD30-positive LPD and MF, oncogenic fusions afflicting the Jak/signal transducer and activator of transcription signaling pathway were present, namely NPM1‒TYK2 in patient 1 and ILF3‒JAK2 in patient 2. Additional signal transducer and activator of transcription 5A gene STAT5A mutations exclusively occurred in lesions of CD30-positive LPD in one patient. CD30-positive LPD and MF may share genetic events when occurring within the same patients. Constitutive activation of the Jak/signal transducer and activator of transcription signaling pathway may play a central role in the molecular pathogenesis of both entities.