Profiling of Copy Number Alterations Using Low-Coverage Whole-Genome Sequencing Informs Differential Diagnosis and Prognosis in Primary Cutaneous Follicle Center Lymphoma.

Primary cutaneous follicle center lymphoma (PCFCL) has an excellent prognosis using local treatment, whereas nodal follicular lymphoma (nFL), occasionally presenting with cutaneous spread, often requires systemic therapy. Distinction of the 2 diseases based on histopathology alone might be challenging. Copy number alterations (CNAs) have scarcely been explored on a genome-wide scale in PCFCL; however, they might serve as potential biomarkers during differential diagnosis and risk stratification. Low-coverage whole-genome sequencing is a robust, high-throughput method for genome-wide copy number profiling. In this study, we analyzed 28 PCFCL samples from 20 patients and compared the copy number profiles with a cohort of diagnostic samples of 64 nFL patients. Although the copy number profile of PCFCL was similar to that of nFL, PCFCL lacked amplifications of 18q, with the frequency peaking at 18q21.33 in nFL cases involving the BCL2 locus (PCFCL: 5.0% vs nFL: 31.3%, P = .018, Fisher exact test). Development of distant cutaneous spread was significantly associated with higher genomic instability including the proportion of genome altered (0.02 vs 0.13, P = .033) and number of CNAs (2 vs 9 P = .017), as well as the enrichment of 2p22.2-p15 amplification involving REL and XPO1 (6.3% vs 60.0%, P = .005), 3q23-q24 amplification (0.0% vs 50.0%, P = .004), 6q16.1-q23.3 deletion (6.3% vs 50.0%, P = .018), and 9p21.3 deletion covering CDKN2A and CDKN2B loci (0.0% vs 40.0%, P = .014, all Fisher exact test) in PCFCL. Analysis of sequential tumor samples in 2 cases harboring an unfavorable clinical course pointed to the acquisition of 2p amplification in the earliest common progenitor underlining its pivotal role in malignant transformation. By performing genome-wide copy number profiling on the largest patient cohort to date, we identified distinctive CNA alterations conceivably facilitating the differential diagnosis of PCFCL and secondary cutaneous involvement of nFL and potentially aiding the risk stratification of patients with PCFCL in the future.

Novel, clinically relevant genomic patterns identified by comprehensive genomic profiling in ATRX-deficient IDH-wildtype adult high-grade gliomas.

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the TERT gene promoter (pTERT) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative mechanisms of telomere lengthening. In this study, we performed the first complex genomic analysis specifically concentrating on the latter subgroup. Comprehensive genomic profiling of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas revealed that ATRX and pTERT mutations are mutually exclusive. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. RAS-MAPK pathway alterations, including NF1 mutations, were more characteristic in the ATRX-deficient group. Variants of genes related to homologous recombination repair showed different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these contained a novel fusion involving the NTRK2 and LRRFIP2 genes, while the other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Genetic characteristics of ATRX-deficient IDH-wildtype adult high-grade gliomas suggest that these tumors are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA repair inhibitors.

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.

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.

Protein Kinase D3 (PKD3) Requires Hsp90 for Stability and Promotion of Prostate Cancer Cell Migration.

Prostate cancer metastasis is a significant cause of mortality in men. PKD3 facilitates tumor growth and metastasis, however, its regulation is largely unclear. The Hsp90 chaperone stabilizes an array of signaling client proteins, thus is an enabler of the malignant phenotype. Here, using different prostate cancer cell lines, we report that Hsp90 ensures PKD3 conformational stability and function to promote cancer cell migration. We found that pharmacological inhibition of either PKDs or Hsp90 dose-dependently abrogated the migration of DU145 and PC3 metastatic prostate cancer cells. Hsp90 inhibition by ganetespib caused a dose-dependent depletion of PKD2, PKD3, and Akt, which are all involved in metastasis formation. Proximity ligation assay and immunoprecipitation experiments demonstrated a physical interaction between Hsp90 and PKD3. Inhibition of the chaperone-client interaction induced misfolding and proteasomal degradation of PKD3. PKD3 siRNA combined with ganetespib treatment demonstrated a specific involvement of PKD3 in DU145 and PC3 cell migration, which was entirely dependent on Hsp90. Finally, ectopic expression of PKD3 enhanced migration of non-metastatic LNCaP cells in an Hsp90-dependent manner. Altogether, our findings identify PKD3 as an Hsp90 client and uncover a potential mechanism of Hsp90 in prostate cancer metastasis. The molecular interaction revealed here may regulate other biological and pathological functions.

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.

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.

Concomitant 1p36 deletion and TNFRSF14 mutations in primary cutaneous follicle center lymphoma frequently expressing high levels of EZH2 protein.

Primary cutaneous follicle center lymphoma (PCFCL) is an indolent variant of follicular lymphoma (FL) with limited information available on the genetic background of the disease. The genetic hallmark of nodal FL, the t(14;18) translocation, affecting the BCL2 gene, is rare in PCFCL. Loss of 1p36, the most common secondary chromosomal abnormality in nodal FL, has been recently reported in 16.7% of PCFCL cases. In order to further characterize PCFCL, 21 cases were analyzed using interphase fluorescence in situ hybridization with BCL2 break apart and 1p36/1q25 dual color probes. Sanger sequencing was used to investigate TNFRSF14 and EZH2 mutations and immunohistochemistry to assess BCL2, EZH2 protein expressions.1p36 deletion occurred in 22% (5/21), BCL2 gene break in 10% (2/20) of the PCFCL cases. Mutations of the candidate tumor suppressor gene of the 1p36 region, TNFRSF14 mutations were detected in 4/17 (23.5%) cases with 2 cases presenting with concurrent 1p36 deletion. EZH2 hotspot mutations at Y641, A682, and A692 were not found. High EZH2 protein expression associated with a BCL2 negative phenotype was observed in 43% (9/21) of the cases. BCL2 gene break or 1p36 deletion did not impact the prognosis; however, they showed association with advanced stages at diagnosis (p = 0.016) and a tendency with shorter event free survival (p = 0.052).In conclusion, 1p36 deletion co-occurs with acquired TNFRSF14 mutations, suggesting a role of this tumor suppressor gene in the development of a subgroup of PCFCL. High EZH2 protein expression associated with BCL2 negative phenotype is common and might represent an ideal therapeutic target.