Low-burden TP53 mutations represent frequent genetic events in CLL with an increased risk for treatment initiation.

TP53 aberrations predict chemoresistance and represent a contraindication for the use of standard chemoimmunotherapy in chronic lymphocytic leukaemia (CLL). Recent next-generation sequencing (NGS)-based studies have identified frequent low-burden TP53 mutations with variant allele frequencies below 10%, but the clinical impact of these low-burden TP53 mutations is still a matter of debate. In this study, we aimed to scrutinise the subclonal architecture and clinical impact of TP53 mutations using a sensitive, NGS-based mutation analysis in a 'real-world' cohort of 901 patients with CLL. In total, 225 TP53 mutations were identified in 17.5% (158/901) of the patients; 48% of these alterations represented high-burden mutations, while 52% were low-burden TP53 mutations. Low-burden mutations as sole alterations were identified in 39% (62/158) of all mutated cases with 82% (51/62) of these being represented by a single low-burden TP53 mutation. Patients harbouring low-burden TP53 mutations had significantly lower time to first treatment compared to patients with wild-type TP53. Our study has expanded the knowledge on the frequency, clonal architecture, and clinical impact of low-burden TP53 mutations. By demonstrating that patients with sole low-burden TP53 variants represent more than one-third of patients with TP53 mutations and have an increased risk for treatment initiation, our findings strengthen the need to redefine the threshold of TP53 variant reporting to below 10% in the routine diagnostic setting.

PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. METHODS: Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. RESULTS: CNAs were detected in 93.8% (n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status (IKZF1normal, IKZF1del and IKZF1plus) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low (n = 215): 86.3%, IKAROS-medium (n = 27): 57.4% and IKAROS-high (n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients (n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients (n = 103) showed high (n = 74) or ultra-poor (n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). CONCLUSIONS: PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Parallel testing of liquid biopsy (ctDNA) and tissue biopsy samples reveals a higher frequency of EZH2 mutations in follicular lymphoma.

BACKGROUND: Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain-of-function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of patients. However, these analyses relied on single-site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations. OBJECTIVES: We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies [TB]) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study. METHODS: Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in-house-designed, highly sensitive multiplex droplet digital PCR assay. RESULTS: EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild-type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones. CONCLUSION: The in-depth spatio-temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.

Next-Generation Sequencing-Based Genomic Profiling of Children with Acute Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality, with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML Berlin-Frankfurt-Münster protocols. Targeted next-generation sequencing of 54 genes revealed 17 genes that were recurrently mutated in >5% of patients. Considerable differences were observed in the mutational profiles compared with previous studies, as BCORL1, CUX1, KDM6A, PHF6, and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in the BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, and WT1) mutations were found to be associated with induction failure and shorter event-free survival, suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% versus 44.4%) and transcription factors (35.1% versus 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identify previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.

Landscape of BCL2 Resistance Mutations in a Real-World Cohort of Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia Treated with Venetoclax.

The oral, highly selective Bcl2 inhibitor venetoclax has substantially improved the therapeutic landscape of chronic lymphocytic leukemia (CLL). Despite the remarkable response rates in patients with relapsed/refractory (R/R) disease, acquired resistance is the leading cause of treatment failure, with somatic BCL2 mutations being the predominant genetic drivers underpinning venetoclax resistance. To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10-4) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax-rituximab combination therapy. With a median follow-up time of 23 months, BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with four patients harboring both resistance mutations. Ten out of eleven patients carrying BCL2 G101V and/or D103Y experienced relapse during the follow-up period, representing 43.5% of the cases (10/23) showing clinical signs of disease progression. All BCL2 G101V or D103Y variants were detected in patients receiving venetoclax as a continuous single-agent treatment while these mutations were not observed during or after fixed-duration venetoclax therapy. Targeted ultra-deep sequencing of BCL2 uncovered three additional variants in four patient samples obtained at relapse, suggesting convergent evolution and implying a cooperating role of BCL2 mutations in driving venetoclax resistance. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.

Morphologic and molecular analysis of Richter syndrome in chronic lymphocytic leukaemia patients treated with ibrutinib or venetoclax.

Richter syndrome (RS) represents the development of high-grade lymphoma in patients with chronic lymphocytic leukaemia (CLL) or small lymphocytic lymphoma (SLL) and presents a diagnostic and therapeutic challenge with an adverse prognosis. The genetic background and morphology of RS in CLL patients treated with chemoimmunotherapy is extensively characterised; however, our knowledge about RS in patients treated with targeted oral therapies should be extended. To understand the morphologic and molecular changes leading to RS in CLL patients treated with the Bruton's tyrosine kinase inhibitor, ibrutinib, and the BCL2 inhibitor, venetoclax, sequential samples from six CLL/SLL patients undergoing RS were collected in both the CLL and RS phases. A detailed immunophenotypic analysis of formalin-fixed, paraffin-embedded tissue specimens of RS phase was performed, followed by extensive molecular characterisation of CLL and RS samples, including the immunoglobulin heavy chain gene (IGH) rearrangement, TP53 mutations, drug-induced resistance mutations in BTK and BCL2 genes and various copy number changes and point mutations detectable with multiplex ligation-dependent probe amplification (MLPA). Rare, non-diffuse large B-cell lymphoma phenotypes of RS were observed in 3/6 cases, including plasmablastic lymphoma and a transitory entity between diffuse large B-cell lymphoma and classical Hodgkin lymphoma. The majority of cases were clonally related and harboured an unmutated variable region of the immunoglobulin heavy chain gene. Abnormalities affecting the TP53 gene occurred in all patients, and every patient carried at least one genetic abnormality conferring susceptibility to RS. In the background of RS, 2/5 patients treated with ibrutinib showed a BTK C481S resistance mutation. One patient developed a BCL2 G101V mutation leading to venetoclax resistance and RS. In conclusion, our findings contribute to better understanding of RS pathogenesis in the era of targeted oral therapies. Rare phenotypic variants of RS do occur under the treatment of ibrutinib or venetoclax, and genetic factors leading to RS are similar to those identified in patients treated with chemoimmunotherapy. To our best knowledge, we have reported the first BCL2 G101V mutation in an RS patient treated with venetoclax.

Limitations of VS38c labeling in the detection of plasma cell myeloma by flow cytometry.

Plasma cell myeloma (multiple myeloma [MM]) is a malignant neoplasm originating from the plasma cells. Besides other methods, flow cytometric analysis of the patient's bone marrow aspirate has an important role in the diagnosis and also in the response assessment. Since the cell surface markers, used for identifying abnormal plasma cells, are expressed diversely and the treatment can also alter the phenotype of the plasma cells, there is an increasing demand for new plasma cell markers. VS38c is a monoclonal antibody that recognizes the CLIMP-63 protein in the membrane of the endoplasmic reticulum. CLIMP-63 is known to be expressed at high levels in normal and pathologic plasma cells in the bone marrow, thus VS38c antibody can be used to identify them. Although VS38c staining of plasma cells is reported to be constant and strong even in myeloma, we were wondering whether sample preparation can affect the staining. We have investigated the effect of different permeabilization agents and washing of the cells on the quality of the VS38c staining and found that in many cases the staining is inadequate to identify the plasma cells. We measured the VS38c staining of the bone marrow aspirates of 196 MM patients and observed that almost all cases showed bright staining with VS38c. However, permeabilization with mild detergent resulted in the appearance of a significant VS38cdim subpopulation, which showed increased sensitivity to mechanical stress (centrifugation). Our results indicate that VS38cdim MM cells can appear due to the improper permeabilization of the endoplasmic reticulum and this finding raises the possibility of the existence of a plasma cell subpopulation with different membrane properties. The significance of this population is unclear yet, but these cells can be easily missed with VS38c staining and can be lost due to centrifugation-induced lysis during sample preparation.

Distinct miRNA Expression Signatures of Primary and Secondary Central Nervous System Lymphomas.

Central nervous system (CNS) lymphoma is a rare and aggressive non-Hodgkin lymphoma that might arise in the CNS (primary CNS lymphoma) or disseminates from a systemic lymphoma to the CNS (secondary CNS lymphoma). Dysregulated expression of miRNAs is associated with various pathologic processes, and miRNA expression patterns may have diagnostic, prognostic, and therapeutic implications. However, miRNA expression is understudied in CNS lymphomas. We performed expression analysis of 798 miRNAs in 73 CNS lymphoma samples using the NanoString platform, followed by an analysis to identify potential diagnostic biomarkers characterizing subgroups and to examine differences based on their primary and secondary nature, molecular subtype, mutational patterns, and survival. Thirty-one differentially expressed miRNAs were identified between primary and secondary groups. In addition, 7 more miRNAs were identified associated with a molecular subtype and 25 associated with mutation status. Using unsupervised clustering methods, a small but distinct primary CNS lymphoma subgroup, with characteristically different expression patterns compared with the rest of the cases was defined. Finally, differentially regulated pathways were identified in the above comparisons and the utility of miRNA expression patterns in predicting survival was assessed. Our study identifies a novel CNS lymphoma subgroup defined by distinct miRNAs, proves the importance of specific miRNAs and pathways in the pathogenesis of CNS lymphomas, and provides the basis for future research in defining potential biomarkers.

The cryptic gonadotropin-releasing hormone neuronal system of human basal ganglia.

Human reproduction is controlled by ~2000 hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Here, we report the discovery and characterization of additional ~150,000-200,000 GnRH-synthesizing cells in the human basal ganglia and basal forebrain. Nearly all extrahypothalamic GnRH neurons expressed the cholinergic marker enzyme choline acetyltransferase. Similarly, hypothalamic GnRH neurons were also cholinergic both in embryonic and adult human brains. Whole-transcriptome analysis of cholinergic interneurons and medium spiny projection neurons laser-microdissected from the human putamen showed selective expression of GNRH1 and GNRHR1 autoreceptors in the cholinergic cell population and uncovered the detailed transcriptome profile and molecular connectome of these two cell types. Higher-order non-reproductive functions regulated by GnRH under physiological conditions in the human basal ganglia and basal forebrain require clarification. The role and changes of GnRH/GnRHR1 signaling in neurodegenerative disorders affecting cholinergic neurocircuitries, including Parkinson's and Alzheimer's diseases, need to be explored.

Lenalidomide abrogates the survival effect of bone marrow stromal cells in chronic lymphocytic leukemia.

In the pathogenesis of chronic lymphocytic leukemia (CLL) the microenvironment plays an important role, as it produces survival signals and mediates drug resistance. Lenalidomide, which has immunomodulatory effect, can enhance the activation of T-, NK-cells and endothelial cells, however there are no data available whether it can modulate bone marrow stromal cells (BMSCs). In our study, we investigated the effects of lenalidomide on BMSCs and CLL cells. CLL cells were cultured alone or with BMSCs and were treated with lenalidomide. Apoptosis, immunophenotype, and cytokine secretion of BMSCs and CLL cells were determined by flow cytometry. Lenalidomide slightly increased the apoptosis of CLL cells and abrogated the anti-apoptotic effect of BMSCs on CLL cells. Lenalidomide treatment decreased the expression of antigens on CLL cells, which mediate the interactions with the microenvironment. Interestingly, lenalidomide enhanced the expression of IRF4 and the co-stimulatory molecule CD86. The secretion of several cytokines was not changed significantly by lenalidomide. CD49d-negative CLL cases were more sensitive to lenalidomide treatment. Our results suggest that lenalidomide has a limited effect on BMSCs, but it renders CLL cells more immunogenic and unresponsive to survival signals provided by BMSCs.

Screening and monitoring of the BTKC481S mutation in a real-world cohort of patients with relapsed/refractory chronic lymphocytic leukaemia during ibrutinib therapy.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTKC481S , sensitive (10-4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTKC481S was detected in 48·2% (40/83) of the patients, with 80·0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72·7% (32/44) of all patients experiencing relapse, emergence of the BTKC481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTKC481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTKC481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression.

Graft survival using cadaver and multiorgan donors between 2008 and 2017 in our department / Cadaverbol és multiorgan donorból származó szaruhártyák túlélése 2008 és 2017 között klinikánkon.

Összefoglaló. Bevezetés: Az elso szaruhártya-bank 1944-es alapítása óta jelentos változásokon ment át. A szaruhártya túlélését számos tényezo befolyásolja, így a tárolási mód, melynek a szövet lejárati ideje szerint rövid, közép és hosszú távú módszereit fejlesztették ki. Célkituzés: Retrospektív vizsgálatunk célja a 2008. január 1. és 2017. december 31. között perforáló és lamelláris keratoplasztika során felhasznált cadaverbol és multiorgan donorból származó szaruhártyák túlélésének vizsgálata volt a Semmelweis Egyetem Szemészeti Klinikáján. Módszer: Feljegyeztük a recipiens nemét, életkorát, a mutétet indikáló klinikai diagnózist, a mutét idopontját, a szövettani vizsgálat eredményét, valamint, hogy a beültetett szaruhártya cadaverbol vagy multiorgan donorból származott. Meghatároztuk, hogy a recipiens életkora korrelált-e a rekeratoplasztikáig eltelt idovel. Eredmények: 1451 szaruhártya-átültetés történt 1088 beteg (44,6% férfi) 1159 szemén (életkor 62,8 ± 18,5 év), melyek között 938 (64,6%) cadaver és 262 (18,0%) multiorgan donor került felhasználásra, 251 esetben (17,2%) nem állt rendelkezésre adat. A leggyakoribb primer diagnózis a szaruhártya-dekompenzáció volt (325 eset, 28%). A primer keratoplasztikák során felhasznált szaruhártyák 740 esetben (63,8%) cadaverbol, 212 esetben (18,2%) multiorgan donorból származtak, 207 esetben (17,8%) nem állt rendelkezésre adat. Elso rekeratoplasztika a primer keratoplasztikák közül 217 esetben (18,7%) történt. A leggyakoribb szövettani diagnózis az endothelsejt-degeneráció volt (130 esetben, 60,4%). 146 esetben (67,2%) korábban cadaver, 31 esetben (14,2%) multiorgan donor esetén került sor ismételt mutétre, 40 esetben (18,4%) nem állt rendelkezésre adat. Következtetés: Klinikánkon elsosorban cadaverbol származó donorok biztosítják a szaruhártya átültetésekhez szükséges szövetet. Cadaverbol vagy multiorgan donorból származó szaruhártyák esetén nem kerül gyakrabban sor rekeratoplasztikára. A szaruhártya-banki tevékenység további fejlesztésével növelheto a donorok túlélése hazánkban. Orv Hetil. 2021; 162(13): 488-496. INTRODUCTION: Corneal banking methods have been changing since the foundation of the first corneal bank in 1944. Corneal graft survival may be affected by several factors, among others the storage method, which may be short-, middle- and long-term storage. OBJECTIVE: To investigate corneal graft survival at the Department of Ophthalmology, Semmelweis University between 1 January 2008 and 31 December 2017, using cadaver and multiorgan donors for penetrating and lamellar keratoplasty, retrospectively. METHOD: Recipient sex, age, clinical diagnosis, date of surgery, histological examination results and origin of donors (cadaver or multiorgan donor) were recorded. Correlation between recipient age and time to repeat keratoplasty was also analyzed. RESULTS: There were 1451 keratoplasties in 1159 eyes (age 62.8 ± 18.5 years) of 1088 patients (44.6% male) using 938 (64.6%) cadaver and 262 (18.0%) multiorgan donors, data was not available in 251 (17.2%) cases. There was repeat keratoplasty in 217 patients (18.7% of first keratoplasties). The most common histological diagnosis was endothelial decompensation (130 cases, 60.4%) in these cases. In patients with a first repeat keratoplasty, in 146 cases (67.2%) the first donor originated from cadavers, in 31 cases (14.2%) from multiorgan donors and in 40 cases (18.4%) data were not available. CONCLUSION: Corneal donors mainly originate from cadavers at our Department. The necessity of repeat keratoplasties does not differ using cadaver or multiorgan donors. With further development of corneal banking, donor survival may be increased in Hungary. Orv Hetil. 2021; 162(13): 488-496.

Characterization of Kisspeptin Neurons in the Human Rostral Hypothalamus.

BACKGROUND: Kisspeptin (KP) neurons in the rostral periventricular region of the 3rd ventricle (RP3V) of female rodents mediate positive estrogen feedback to gonadotropin-releasing hormone neurons and, thus, play a fundamental role in the mid-cycle luteinizing hormone (LH) surge. The RP3V is sexually dimorphic, and male rodents with lower KP cell numbers are unable to mount estrogen-induced LH surges. OBJECTIVE: To find and characterize the homologous KP neurons in the human brain, we studied formalin-fixed post-mortem hypothalami. METHODS: Immunohistochemical techniques were used. RESULTS: The distribution of KP neurons in the rostral hypothalamus overlapped with distinct subdivisions of the paraventricular nucleus. The cell numbers decreased after menopause, indicating that estrogens positively regulate KP gene expression in the rostral hypothalamus in humans, similarly to several other species. Young adult women and men had similar cell numbers, as opposed to rodents reported to have more KP neurons in the RP3V of females. Human KP neurons differed from the homologous rodent cells as well, in that they were devoid of enkephalins, galanin and tyrosine hydroxylase. Further, they did not contain known KP neuron markers of the human infundibular nucleus, neurokinin B, substance P and cocaine- and amphetamine-regulated transcript, while they received afferent input from these KP neurons. CONCLUSIONS: The identification and positive estrogenic regulation of KP neurons in the human rostral hypothalamus challenge the long-held view that positive estrogen feedback may be restricted to the mediobasal part of the hypothalamus in primates and point to the need of further anatomical, molecular and functional studies of rostral hypothalamic KP neurons.

Changing trends in penetrating keratoplasty indications at a tertiary eye care center in Budapest, Hungary between 2006 and 2017.

AIM: To analyze the changing trends in penetrating keratoplasty (PKP) indications. METHODS: This retrospective study included all patients with PKP between 2006 and 2017. Patients were classified using histological diagnoses. Our groups were as the following: pseudophakic or aphakic bullous keratopathy, regraft, acute necrotizing and ulcerative keratitis, keratoconus, Fuchs' dystrophy, corneal dystrophy other than Fuchs', corneal scar, other diagnoses and failed endothelial keratoplasty graft. Additionally, two different time-periods (2006-2012 and 2013-2017) were analysed. RESULTS: Totally 1721 histological analyses of 1214 patients were available for review. The diagnoses were pseudophakic or aphakic bullous keratopathy in 487 (28.3%), regraft in 443 (25.7%), acute necrotizing and ulcerative keratitis in 313 (18.2%), corneal scar in 153 (8.9%), keratoconus in 140 (8.1%). Fuchs' dystrophy in 61 (3.5%), corneal dystrophy other than Fuchs' in 46 (2.7%), other diagnoses in 44 (2.6%) and failed endothelial keratoplasty graft in 34 (2.0%) cases. From the first to the second analysed time-period, incidence of acute necrotizing and ulcerative keratitis, corneal scar, Fuchs' dystrophy increased (P≤0.032 for all) and incidence of keratoconus significantly decreased (P=0.015). CONCLUSION: Pseudophakic or aphakic bullous keratopathy is the leading indication for PKP, followed by regraft and acute necrotizing and ulcerative keratitis.

Quantitative Analysis and Monitoring of EZH2 Mutations Using Liquid Biopsy in Follicular Lymphoma.

Recent advances in molecular technologies enable sensitive and quantitative assessment of circulating tumor DNA, offering a noninvasive disease monitoring tool for patients with malignant disorders. Here, we demonstrated on four follicular lymphoma cases that circulating tumor DNA based EZH2 mutation analysis performed by a highly sensitive droplet digital PCR method may be a valuable treatment monitoring approach in EZH2 mutant follicular lymphoma. EZH2 variant allele frequencies changed in parallel with the volume of metabolically active tumor sites observed on 18F-fluorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. Variant allele frequencies of EZH2 mutations decreased or were eliminated rapidly upon successful treatment, with treatment failure being associated with elevated EZH2 variant allele frequencies. We also demonstrated spatial heterogeneity in a patient with two different EZH2 mutations in distinct anatomical sites, with both mutations simultaneously detected in the liquid biopsy specimen. In summary, circulating tumor DNA based EZH2 mutation analysis offers a rapid, real-time, radiation-free monitoring tool for sensitive detection of EZH2 mutations deriving from different anatomical sites in follicular lymphoma patients receiving immunochemotherapy.

Grade I, II and III Follicular Lymphomas Express Ig VH Genes with Different Patterns of Somatic Mutation.

Follicular lymphoma (FL) is an indolent, B-cell, non-Hodgkin's lymphoma with varying cytological appearance and clinical behavior. The genetic hallmark of FL is the t(14;18) translocation, and as a germinal center derived entity it is also characterized by somatic hypermutation of the immunoglobulin heavy chain (IgH) gene. In an attempt to correlate this molecular signature with the cytological grading of FL, we have analyzed the IgH variable (IgVH), regions in all cytological grades of FL. Four FL cases showing t(14;18) translocation were classified into grade I-III categories according to the current WHO guidelines. The IgVH gene segments were PCR-amplified, sequenced, and compared to their respective germline IgVH sequences. The neoplastic cells of grade I and II FLs revealed clonally related, but highly divergent IgVH gene sequences indicating the ongoing nature of somatic hypermutation. Grade III FL also showed extensive presence of somatic hypermutation, but these mutations were not associated with intraclonal divergence. Thus, these results suggest that grade I-II and grade III FL may represent different biological entities. The presence of ongoing somatic hypermutation of IgVH sequences in grade I and II FLs is compatible with direct follicular origin of these tumor cells, contrasting the homogenous, stable clones of grade III FL resembling a post-follicular stage of B-cell development. Our findings demonstrate that contrary to the three tiered cytological grading, molecular features of IgH genes classify FL into two distinct subcategories. These studies also suggest that with progression FL gains post-follicular-like molecular features and becomes independent of the germinal center microenvironment.

Molecular Subtypes and Genomic Profile of Primary Central Nervous System Lymphoma.

Primary central nervous system lymphomas (PCNSL) are aggressive non-Hodgkin lymphomas affecting the central nervous system (CNS). Although immunophenotyping studies suggested an uniform activated B-cell (ABC) origin, more recently a spectrum of ABC and germinal center B-cell (GC) cases has been proposed, with the molecular subtypes of PCNSL still being a matter of debate. With the emergence of novel therapies demonstrating different efficacy between the ABC and GC patient groups, precise assignment of molecular subtype is becoming indispensable. To determine the molecular subtype of 77 PCNSL and 17 secondary CNS lymphoma patients, we used the NanoString Lymphoma Subtyping Test (LST), a gene expression-based assay representing a more accurate technique of subtyping compared with standard immunohistochemical (IHC) algorithms. Mutational landscapes of 14 target genes were determined using ultra-deep next-generation sequencing. Using the LST-assay, a significantly lower proportion (80% vs 95%) of PCNSL cases displayed ABC phenotype compared with the IHC-based characterization. The most frequently mutated genes included MYD88, PIM1, and KMT2D. In summary, we successfully applied the LST-assay for molecular classification of PCNSL, reporting higher proportion of cases with GC phenotype compared with IHC analyses, leading to a more precise patient stratification potentially applicable in the diagnostic algorithm of PCNSL.

Dissection of subclonal evolution by temporal mutation profiling in chronic lymphocytic leukemia patients treated with ibrutinib.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is inducing durable responses in chronic lymphocytic leukemia (CLL) patients with refractory/relapsed disease or with TP53 defect, with BTK and phospholipase C gamma 2 (PLCG2) mutations representing the predominant mechanisms conferring secondary ibrutinib resistance. To understand the landscape of genomic changes and the dynamics of subclonal architecture associated with ibrutinib treatment, an ultra-deep next-generation sequencing analysis of 30 recurrently mutated genes was performed on sequential samples of 20 patients, collected before and during single-agent ibrutinib treatment. Mutations in the SF3B1, MGAand BIRC3 genes were enriched during ibrutinib treatment, while aberrations in the BTK, PLCG2, RIPK1, NFKBIE and XPO1 genes were exclusively detected in posttreatment samples. Besides the canonical mutations, four novel BTK mutations and three previously unreported PLCG2 variants were identified. BTK and PLCG2 mutations were backtracked in five patients using digital droplet PCR and were detectable on average 10.5 months before clinical relapse. With a median follow-up time of 36.5 months, 7/9 patients harboring BTK mutations showed disease progression based on clinical and/or laboratory features. In conclusion, subclonal heterogeneity, dynamic clonal selection and various patterns of clonal variegation were identified with novel resistance-associated BTK mutations in individual patients treated with ibrutinib.

Comprehensive profiling of disease-relevant copy number aberrations for advanced clinical diagnostics of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPATM). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPATM-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPATM provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.

Calreticulin mutation specific CAL2 immunohistochemistry accurately identifies rare calreticulin mutations in myeloproliferative neoplasms.

Mutations of the multifunctional protein calreticulin (CALR) are recognised as one of the main driver alterations involved in the pathogenesis of Philadelphia negative myeloproliferative neoplasms (Ph- MPN) and also represent a major diagnostic criterion in the most recent World Health Organization classification of myeloid neoplasms. Nowadays, quantitative assessment of the driver mutations is gaining importance, as recent studies demonstrated the clinical relevance of the mutation load reflecting the size of the mutant clone. Here, we performed for the first time a manual and automated quantitative assessment of the CALR mutation load at protein level using CAL2, a recently developed CALR mutation specific monoclonal antibody, on a cohort of 117 patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) and compared the CALR protein mutation loads with the CALR mutation load values established by a molecular assay. Eighteen different CALR mutations were detected in the cohort of the 91 CALR mutant cases. Mutation loads of the CALR mutations were between 13% and 94% with mean value in PMF cases significantly higher than ET cases (49.94 vs 41.09; t-test, p=0.004). Cases without CALR mutation (n=26) showed no or only minimal labelling with the CAL2 antibody, while all 18 different types of CALR mutations were associated with CAL2 labelling. The CALR mutation load showed a significant correlation (p=0.03) with the occurrence of major thrombotic events, with higher mutation load in patients presenting with these complications. We report a 100% concordance between the mutation status determined by immunohistochemistry and the CALR molecular assay, and we extend the applicability of this approach to 16 rare CALR mutations previously not analysed at protein level.