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• ERGamp is a unique driver of myeloid malignancy with a signature genetic profile with loss of 5q, chromothripsis and TP53 loss of function variants. • Chromosome structure with copy number analysis (CNA) and loss of heterozygosity (LOH) should be standard of care to risk stratify myeloid malignancies. ETS-related gene (ERG) amplification, observed in 4–6% of acute myeloid leukemia (AML), is associated with unfavorable prognosis. To determine coincident effects of additional genomic abnormalities in AML with ERG amplification (ERG amp), we examined 11 ERG amp cases of 205 newly diagnosed AML using chromosomal microarray analysis and next generation sequencing. ERG amp cases demonstrated a distinct pattern of high genetic complexity: loss of 5q, chromothripsis and TP53 loss of function variants. Remarkably, allelic TP53 loss or loss of heterozygosity (LOH) co-occurring with TP53 inactivating mutation dramatically effected ERG amp tumor patient outcome. In the presence of homozygous TP53 loss of function, ERG amp patients demonstrated no response to induction chemotherapy with median overall survival (OS) of 3.8 months (N = 9). Two patients with heterozygous loss of TP53 function underwent alloSCT without evidence of relapse at one year. Similarly, a validation TCGA cohort, 6 of the 8 ERG amp cases with TP53 loss of function demonstrated median OS of 2.5 months. This suggests that with TP53 mutant ERG amp AML, successive loss of the second TP53 allele, typically by 17p deletion or LOH identifies a specific high-risk subtype of AML patients who are resistant to standard induction chemotherapy and need novel approaches to avert the very poor prognosis. [ABSTRACT FROM AUTHOR]

In cases of suspected intraocular malignancy, vitreous may be the preferred pathologic sample; however, cellularity may be insufficient for definitive cytopathological diagnosis. Ancillary methodology to study vitreous fluid aspiration for mutational analysis may assist in treatment decisions. Three individual patient vitreous humor samples were received in the laboratory for mutation testing. The samples were collected during standard of care and analyzed for routine cytopathology. In each case, cytopathology was inconclusive and mutational analyses to support diagnostic suspicions were clinically requested. Based on the clinically and pathologically suspected diagnoses, an appropriate massively parallel sequencing assay previously validated for clinical use was performed using DNA extracted from vitreous samples that had previously undergone various processing. Nucleic acid yield was assessed by fluorometric or spectrophotometric methods, with yield ranging from 2.7 to 86.5 ng. Library preparations were performed using standard laboratory protocols. Two of the cases were suspicious for melanoma and a 50-gene solid tumor panel was performed. The third case was worrisome for vitreoretinal lymphoma and a 49-gene myeloid panel was performed. In all cases, the molecular profiling assisted with the clinical assessment and/or management of each patient. • In this case series, the cytology of the vitreous was inconclusive and molecular profiling of the vitreous did identify variants that contributed to the clinical assessment of each patient. • In cases of inconclusive cytology, it may be possible to clarify the clinical picture using molecular profiling by massively parallel sequencing of vitreous. • In these cases, the molecular profiling assisted with diagnosis, patient management, and/or treatment. [ABSTRACT FROM AUTHOR]

• Aggressive lymphoma (DLBCL) arising in CLL/SLL is a Richter transformation. • Clonal relationship between the DLBCL and CLL/SLL carries a poor prognosis. • CMA can help establish clonality and detect high risk aberrations. • Molecular profiling with CMA has potential prognostic and therapeutic implications. Richter transformation (RT) refers to the development of an aggressive lymphoma in patients with pre-existing chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). It carries a poor prognosis secondary to poor response to therapy or rapid disease relapse. Currently there are no randomized trials to guide treatment. Therapeutic decisions are often influenced by the presence or absence of a clonal relationship between the underlying CLL/SLL and the new lymphoma given the poor prognosis of patients with clonally related RT. Chromosomal microarray analysis (CMA) can help to establish clonality while also detecting genomic complexity and clinically relevant genetic variants such as loss of CDKN2A and/or TP53. As a result, CMA has potential prognostic and therapeutic implications. For this study, CMA results from patients with Richter transformation were evaluated in paired CLL/SLL and transformed lymphoma samples. CMA revealed that 86% of patients had common aberrations in the two samples indicating evidence of common clonality. CMA was also useful in detecting aberrations associated with a poor prognosis in 71% of patients with RT. This study highlights the potential clinical utility of CMA to investigate the clonal relationship between CLL/SLL and RT, provide prognostic information, and possibly guide therapeutic decision making for patients with Richter transformation. [ABSTRACT FROM AUTHOR]

• Integrated genomic approach to diagnose myeloid neoplasms should include microarray. • MPseq precisely identifies and characterizes complicated chromosomal rearrangements. • PCM1-JAK2 fusions with aberrant tyrosine kinases have potential for targeted therapy. The 2016 World Health Organization entity 'Myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1 , or with PCM1-JAK2 ' encompasses a group of rare neoplasms that result from the formation of a fusion gene that leads to expression of an aberrant tyrosine kinase. This entity also contains variant JAK2 fusion partners, and detection of this defining event can be facilitated by various cytogenetic and molecular methods. Cryptic rearrangements of 9p24/ JAK2 can be particularly challenging to identify. We describe the use of chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH) with a probe for JAK2 , and genomic mate pair analysis to describe a complex karyotype with a t(9;22) that produced a functional BCR-JAK2 fusion, leading to the appropriate diagnosis for the patient. This case highlights the importance of using an integrated genomic approach to fully define complex aberrations to assign proper diagnoses. [ABSTRACT FROM AUTHOR]

Primary cytogenetic abnormalities in B-lymphoblastic leukemia (B-ALL) define subgroups of disease with distinct clinical implications. These subgroups are largely mutually exclusive and only rarely observed in conjunction. We describe a three-year-old female presenting with de novo B-ALL in whom dual color, dual fusion fluorescence in situ hybridization (D-FISH) evaluation suggested concurrent ETV6::RUNX1 and KMT2A::MLLT3 rearrangements. Chromosomal microarray analysis (CMA) indicated that the breakpoint within KMT2A was located outside the typical region described in KMT2A leukemogenic translocations. Owing to the unexpected identification of concurrent ETV6::RUNX1 and KMT2A translocations by D-FISH and the suspicious findings on CMA, research-based whole genome sequencing (WGS) was performed. While this analysis confirmed the ETV6::RUNX1 fusion, WGS revealed the apparent KMT2A::MLLT3 fusion detected by D-FISH resulted from a juxtaposition of the 3′ portion of KMT2A (from intron 1) to an intergenic region on chromosome 9 located 460 Kb upstream of MLLT3. The WGS results indicate the apparent KMT2A::MLLT3 rearrangement would not result in an oncogenic chimeric fusion protein, and the characteristic KMT2A::MLLT3 dual fusion identified by D-FISH testing represented a false-positive FISH result. WGS thus more precisely resolved the genomic abnormalities in this B-ALL clone in comparison to chromosomes, FISH and chromosomal microarray and enabled accurate risk stratification. This case illustrates the importance of careful interpretation of atypical cases with aberrant genomic results, comprehensively considering expected fusion product structure, assay design and integrating results in their entirety. WGS may provide a means for resolution of atypical results by standard investigation modalities. [ABSTRACT FROM AUTHOR]

Chromosomal microarray analysis (CMA) is instrumental for identification of recurrent focal deletions in patients with B cell acute lymphoblastic leukemia (B-ALL). These microdeletions include genes involved in B cell differentiation, cell cycle control and transcriptional regulation often with prognostic or therapeutic significance. Recently, a 13q12.2 microdeletion with breakpoints upstream of FLT3 and within intronic regions of the PAN3 gene was reported. Functionally, this microdeletion resulted in enhancer hijacking and constitutive activation of FLT3 in pediatric B-ALL. Seven institutions performed internal database searches to identify similar deletions. In total, 10 patients were identified, all with B-ALL, confirming the specificity for this diagnosis. This cohort included seven pediatric cases (3 male, 4 female; ages 2-14 yo, mean 5) and 3 adult cases (1 male, 2 female; ages 22, 39, 42). Subtypes included two high hyperdiploid, one hypodiploid, one iAMP21, one P2RY8::CLRF, one unbalanced rearrangement involving TFE3, one PAX5::PML, three B-ALL NOS.? Deletions ranged in size from?6.7 kb ? 136 kb (mean 76 kb); each with proximal breakpoints between FLT3 and PAN3 and 9/10 with distal breakpoints within intron 5 of the PAN3 gene. Our study is the first to report the microdeletion in adults, and unlike the discovery cohorts, our patients showed no sex bias. Our data does not confirm a strong association of the deletion with high hyperdiploidy, since 8/10 had other B-ALL subtypes. Constitutive activation of FLT3 may have significant clinical implications for prognosis and treatment; thus, detection of 13q12.2 microdeletion at diagnosis or disease relapse is paramount. [ABSTRACT FROM AUTHOR]