The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation.

Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:

Guiding the global evolution of cytogenetic testing for hematologic malignancies.

Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing.

ROS1 Alterations as a Potential Driver of Gliomas in Infant, Pediatric, and Adult Patients.

Gliomas harboring oncogenic ROS1 alterations are uncommon and primarily described in infants. Our goal was to characterize the clinicopathological features and molecular signatures of the full spectrum of ROS1 fusion-positive gliomas across all age groups. Through a retrospective multi-institutional collaboration, we report a collection of unpublished ROS1 fusion gliomas along with the characterization and meta-analysis of new and published cases. A cohort of 32 new and 58 published cases was divided into the following 3 age groups: 19 infants, 40 pediatric patients, and 31 adults with gliomas. Tumors in infants and adults showed uniformly high-grade morphology; however, tumors in pediatric patients exhibited diverse histologic features. The GOPC::ROS1 fusion was prevalent (61/79, 77%) across all age groups, and 10 other partner genes were identified. Adult tumors showed recurrent genomic alterations characteristic of IDH wild-type glioblastoma, including the +7/-10/CDKN2A deletion; amplification of CDK4, MDM2, and PDGFRA genes; and mutations involving TERTp, TP53, PIK3R1, PIK3CA, PTEN, and NF1 genes. Infant tumors showed few genomic alterations, whereas pediatric tumors showed moderate genomic complexity. The outcomes were significantly poorer in adult patients. Although not statistically significant, tumors in infant and pediatric patients with high-grade histology and in hemispheric locations appeared more aggressive than tumors with lower grade histology or those in nonhemispheric locations. In conclusion, this study is the largest to date to characterize the clinicopathological and molecular signatures of ROS1 fusion-positive gliomas from infant, pediatric, and adult patients. We conclude that ROS1 likely acts as a driver in infant and pediatric gliomas and as a driver or codriver in adult gliomas. Integrated comprehensive clinical testing might be helpful in identifying such patients for possible targeted therapy.

Quantification of aneuploidy in targeted sequencing data using ASCETS.

SUMMARY: The expansion of targeted panel sequencing efforts has created opportunities for large-scale genomic analysis, but tools for copy-number quantification on panel data are lacking. We introduce ASCETS, a method for the efficient quantitation of arm and chromosome-level copy-number changes from targeted sequencing data. AVAILABILITY AND IMPLEMENTATION: ASCETS is implemented in R and is freely available to non-commercial users on GitHub: https://github.com/beroukhim-lab/ascets, along with detailed documentation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Standards for the classification of pathogenicity of somatic variants in cancer (oncogenicity): Joint recommendations of Clinical Genome Resource (ClinGen), Cancer Genomics Consortium (CGC), and Variant Interpretation for Cancer Consortium (VICC).

PURPOSE: Several professional societies have published guidelines for the clinical interpretation of somatic variants, which specifically address diagnostic, prognostic, and therapeutic implications. Although these guidelines for the clinical interpretation of variants include data types that may be used to determine the oncogenicity of a variant (eg, population frequency, functional, and in silico data or somatic frequency), they do not provide a direct, systematic, and comprehensive set of standards and rules to classify the oncogenicity of a somatic variant. This insufficient guidance leads to inconsistent classification of rare somatic variants in cancer, generates variability in their clinical interpretation, and, importantly, affects patient care. Therefore, it is essential to address this unmet need. METHODS: Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group and ClinGen Germline/Somatic Variant Subcommittee, the Cancer Genomics Consortium, and the Variant Interpretation for Cancer Consortium used a consensus approach to develop a standard operating procedure (SOP) for the classification of oncogenicity of somatic variants. RESULTS: This comprehensive SOP has been developed to improve consistency in somatic variant classification and has been validated on 94 somatic variants in 10 common cancer-related genes. CONCLUSION: The comprehensive SOP is now available for classification of oncogenicity of somatic variants.

Re-evaluating tumors of purported specialized prostatic stromal origin reveals molecular heterogeneity, including non-recurring gene fusions characteristic of uterine and soft tissue sarcoma subtypes.

Tumors of purported specialized prostatic stromal origin comprise prostatic stromal sarcomas (PSS) and stromal tumors of uncertain malignant potential (STUMP). Prior studies have described their clinicopathologic characteristics, but the molecular features remain incompletely understood. Moreover, these neoplasms are morphologically heterogeneous and the lack of specific adjunctive markers of prostatic stromal lineage make precise definition more difficult, leading some to question whether they represent a specific tumor type. In this study, we used next-generation DNA and RNA sequencing to profile 25 primary prostatic mesenchymal neoplasms of possible specialized prostatic stromal origin, including cases originally diagnosed as PSS (11) and STUMP (14). Morphologically, the series comprised 20 cases with solid architecture (11 PSS and 9 STUMP) and 5 cases with phyllodes-like growth pattern (all STUMP). Combined DNA and RNA sequencing results demonstrated that 19/22 (86%) cases that underwent successful sequencing (either DNA or RNA) harbored pathogenic somatic variants. Except for TP53 alterations (6 cases), ATRX mutations (2 cases), and a few copy number variants (-13q, -14q, -16q and +8/8p), the findings were largely nonrecurrent. Eight gene rearrangements were found, and 4 (NAB2-STAT6, JAZF1-SUZ12, TPM3-NTRK1 and BCOR-MAML3) were useful for reclassification of the cases as specific entities. The present study shows that mesenchymal neoplasms of the prostate are morphologically and molecularly heterogeneous and include neoplasms that harbor genetic aberrations seen in specific mesenchymal tumors arising in other anatomic sites, including soft tissue and the uterus. These data suggest that tumors of purported specialized prostatic stromal origin may perhaps not represent a single diagnostic entity or specific disease group and that alternative diagnoses should be carefully considered.

Correlation of methylthioadenosine phosphorylase (MTAP) protein expression with MTAP and CDKN2A copy number in malignant pleural mesothelioma.

AIMS: Methylthioadenosine phosphorylase (MTAP) immunohistochemical expression is a specific marker of CDKN2A deletion in malignant mesothelioma. However, the relationship of MTAP expression with MTAP copy number remains unexplored. METHODS AND RESULTS: Forty malignant pleural mesotheliomas were characterised by targeted next-generation sequencing (29), single-nucleotide polymorphism microarray (seven), or both (four). MTAP and CDKN2A copy numbers were correlated with MTAP expression. Twenty-seven (68%) tumours showed CDKN2A deletion (14 heterozygous; 13 homozygous), of which 20 (74%) showed MTAP codeletion (15 heterozygous; five homozygous). No tumours showed MTAP deletion without CDKN2A codeletion. Loss of MTAP expression was seen in 16 (40%) tumours, and was 75% sensitive and 95% specific for MTAP deletion, and 59% sensitive and 100% specific for CDKN2A deletion. Nine of 40 (23%) tumours showed heterogeneous MTAP staining, and the percentage of tumour cells with MTAP loss correlated with molecular detection of MTAP deletion. CONCLUSIONS: MTAP is frequently codeleted with CDKN2A in pleural mesothelioma. However, homozygous deletion of both genes occurs in a minority of tumours (5/40; 13%); CDKN2A deletion often co-occurs with heterozygous MTAP deletion or neutral MTAP copy number; and MTAP expression correlates inconsistently with heterozygous MTAP deletion. Correspondingly, MTAP immunohistochemistry is a highly specific but only moderately sensitive assay for CDKN2A deletion.

Characterization of molecular signatures of supratentorial ependymomas.

Ependymomas show poor correlation between World Health Organization grade and clinical outcome. A subgroup of supratentorial ependymomas are characterized by C11orf95-RELA fusions, presumed to be secondary to chromothripsis of chromosome 11, resulting in constitutive activation of the NF-κB signaling pathway and overexpression of cyclin D1, p65, and L1 cell adhesion molecule (L1CAM). These RELA-fused ependymomas are recognized as a separate, molecularly defined World Health Organization entity and might be associated with poor clinical outcome. In this study, we show that immunohistochemistry for NF-κB signaling components, such as L1CAM, p65, and cyclin D1, can help distinguish RELA-fused from non-RELA-fused supratentorial ependymomas. Furthermore, these three markers can reliably differentiate RELA-fused ependymomas from a variety of histologic mimics. Lastly, we report that RELA-fused ependymomas may be associated with different chromosomal copy number changes and molecular alterations compared to their non-RELA-fused counterparts, providing additional insight into the genetic pathogenesis of these tumors and potential targets for directed therapies.

Molecular characterization of localized pleural mesothelioma.

Localized pleural mesothelioma is a rare solitary circumscribed pleural tumor that is microscopically similar to diffuse malignant pleural mesothelioma. However, the molecular characteristics and nosologic relationship with its diffuse counterpart remain unknown. In a consecutive cohort of 1110 patients with pleural mesotheliomas diagnosed in 2005-2018, we identified six (0.5%) patients diagnosed with localized pleural mesotheliomas. We gathered clinical history, evaluated the histopathology, and in select cases performed karyotypic analysis and targeted next-generation sequencing. The cohort included three women and three men (median age 63; range 28-76), often presenting incidentally during radiologic evaluation for unrelated conditions. Neoadjuvant chemotherapy was administered in two patients. All tumors (median size 5.0 cm; range 2.7-13.5 cm) demonstrated gross circumscription (with microscopic invasion into lung, soft tissue, and/or rib in four cases), mesothelioma histology (four biphasic and two epithelioid types), and mesothelial immunophenotype. Of four patients with at least 6-month follow-up, three were alive (up to 8.9 years). Genomic characterization identified several subgroups: (1) BAP1 mutations with deletions of CDKN2A and NF2 in two tumors; (2) TRAF7 mutations in two tumors, including one harboring trisomies of chromosomes 3, 5, 7, and X; and (3) genomic near-haploidization, characterized by extensive loss of heterozygosity sparing chromosomes 5 and 7. Localized pleural mesotheliomas appear genetically heterogeneous and include BAP1-mutated, TRAF7-mutated, and near-haploid subgroups. While the BAP1-mutated subgroup is similar to diffuse malignant pleural mesotheliomas, the TRAF7-mutated subgroup overlaps genetically with adenomatoid tumors and well-differentiated papillary mesotheliomas, in which recurrent TRAF7 mutations have been described. Genomic near-haploidization, identified recently in a subset of diffuse malignant pleural mesotheliomas, suggests a novel mechanism in the pathogenesis of both localized pleural mesothelioma and diffuse malignant pleural mesothelioma. Our findings describe distinctive genetic features of localized pleural mesothelioma, with both similarities to and differences from diffuse malignant pleural mesothelioma.

Differential Expression of PLAG1 in Apocrine and Eccrine Cutaneous Mixed Tumors: Evidence for Distinct Molecular Pathogenesis.

Cutaneous mixed tumors, also known as chondroid syringomas, are benign adnexal neoplasms that share histomorphologic features with pleomorphic adenomas of the salivary gland. Recent work suggests that the similarity between these 2 tumor types extends to the molecular level because both harbor identical chromosomal rearrangements involving the PLAG1 gene. The resulting nuclear PLAG1 overexpression can be detected by immunohistochemistry and has become a useful diagnostic adjunct for both tumor types. In the skin, however, there are 2 morphologically distinct types of mixed tumor, which have been referred to as apocrine-type cutaneous mixed tumor (AMT) and eccrine-type cutaneous mixed tumor (EMT). Previous studies of PLAG1 expression in cutaneous mixed tumor did not distinguish between these types. Here, we evaluated PLAG1 expression by immunohistochemistry in a cohort of 25 cutaneous mixed tumors stratified by type. PLAG1 was overexpressed in the majority of AMT cases (14 of 16) but in none of the EMT cases (0 of 9). A second gene, HMGA2, known to be upregulated in a subset of salivary gland pleomorphic adenomas, was overexpressed in only 1 case of AMT (1 of 16) and in none of the cases of EMT (0 of 9). Our results indicate that apocrine- and eccrine-type mixed tumors are associated with different pathways of molecular pathogenesis and suggest that the emerging relationship between skin and salivary gland mixed tumors is likely limited to those of apocrine type.

Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studies. Mechanistically, we demonstrate that EAG2 protein is confined intracellularly during interphase but is enriched in the plasma membrane during late G2 phase and mitosis. Disruption of EAG2 expression results in G2 arrest and mitotic catastrophe associated with failure of premitotic cytoplasmic condensation. While the tumor suppression function of EAG2 knockdown is independent of p53 activation, DNA damage checkpoint activation, or changes in the AKT pathway, this defective cell volume control is specifically associated with hyperactivation of the p38 MAPK pathway. Inhibition of the p38 pathway significantly rescues the growth defect and G2 arrest. Strikingly, ectopic membrane expression of EAG2 in cells at interphase results in cell volume reduction and mitotic-like morphology. Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which activates the tumor suppressor p38 MAPK pathway, and provides clinical relevance for targeting this ion channel in human MBs.

Technical laboratory standards for interpretation and reporting of acquired copy-number abnormalities and copy-neutral loss of heterozygosity in neoplastic disorders: a joint consensus recommendation from the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC).

The detection of acquired copy-number abnormalities (CNAs) and copy-neutral loss of heterozygosity (CN-LOH) in neoplastic disorders by chromosomal microarray analysis (CMA) has significantly increased over the past few years with respect to both the number of laboratories utilizing this technology and the broader number of tumor types being assayed. This highlights the importance of standardizing the interpretation and reporting of acquired variants among laboratories. To address this need, a clinical laboratory-focused workgroup was established to draft recommendations for the interpretation and reporting of acquired CNAs and CN-LOH in neoplastic disorders. This project is a collaboration between the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC). The recommendations put forth by the workgroup are based on literature review, empirical data, and expert consensus of the workgroup members. A four-tier evidence-based categorization system for acquired CNAs and CN-LOH was developed, which is based on the level of available evidence regarding their diagnostic, prognostic, and therapeutic relevance: tier 1, variants with strong clinical significance; tier 2, variants with some clinical significance; tier 3, clonal variants with no documented neoplastic disease association; and tier 4, benign or likely benign variants. These recommendations also provide a list of standardized definitions of terms used in the reporting of CMA findings, as well as a framework for the clinical reporting of acquired CNAs and CN-LOH, and recommendations for how to deal with suspected clinically significant germline variants.

Yield of additional genetic testing after chromosomal microarray for diagnosis of neurodevelopmental disability and congenital anomalies: a clinical practice resource of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: Chromosomal microarray (CMA) is recommended as the first-tier test in evaluation of individuals with neurodevelopmental disability and congenital anomalies. CMA may not detect balanced cytogenomic abnormalities or uniparental disomy (UPD), and deletion/duplications and regions of homozygosity may require additional testing to clarify the mechanism and inform accurate counseling. We conducted an evidence review to synthesize data regarding the benefit of additional testing after CMA to inform a genetic diagnosis. METHODS: The review was guided by key questions related to the detection of genomic events that may require additional testing. A PubMed search for original research articles, systematic reviews, and meta-analyses was evaluated from articles published between 1 January 1983 and 31 March 2017. Based on the key questions, articles were retrieved and data extracted in parallel with comparison of results and discussion to resolve discrepancies. Variables assessed included study design and outcomes. RESULTS: A narrative synthesis was created for each question to describe the occurrence of, and clinical significance of, additional diagnostic findings from subsequent testing performed after CMA. CONCLUSION: These findings may be used to assist the laboratory and clinician when making recommendations about additional testing after CMA, as it impacts clinical care, counseling, and diagnosis.

PHOX2B is a reliable immunomarker in distinguishing peripheral neuroblastic tumours from CNS embryonal tumours.

AIMS: The PHOX2B gene regulates neuronal maturation in the brain stem nuclei associated with cardiorespiratory function and in the autonomic sympathetic and enteric nervous system. PHOX2B expression is a reliable immunomarker for peripheral neuroblastic tumours; however, no systematic evaluation of central nervous system (CNS) embryonal tumours was included in the studies. We encountered two cases in which the differential diagnosis included neuroblastoma and CNS embryonal tumour, and we hypothesised that PHOX2B immunostain would be helpful in establishing the diagnosis. METHODS AND RESULTS: PHOX2B immunostain was performed on 29 paediatric cases, with adequate controls: one retroperitoneal embryonal tumour in a child with retinoblastoma (index 1), one posterior fossa embryonal tumour in a child with a neuroblastoma (index 2), seven medulloblastomas, four atypical teratoid/rhabdoid tumours (ATRT), four retinoblastomas, six pineoblastomas, four embryonal tumours with multilayered rosettes (ETMR) and two CNS embryonal tumours, not elsewhere classified. Cell lineage immunomarkers (GFAP, OLIG2, synaptophysin, NeuN, CRX, PGP 9.5), immunosurrogates for molecular alterations (beta-catenin, INI1, Lin-28), array CGH and OncoPanel were performed as needed. Medulloblastomas, ATRTs, ETMRs, retinoblastomas and CNS embryonal tumours not elsewhere classified were essentially negative for PHOX2B. Two of six pineoblastomas had significant PHOX2B expression, while the rest were negative. Index 1 was negative for PHOX2B and PGP 9.5 and positive for CRX, consistent with retinoblastoma. Index 2 had diffuse PHOX2B expression, MYCN amplification and no copy number changes of medulloblastoma, in keeping with neuroblastoma. CONCLUSION: PHOX2B antibody is helpful in distinguishing between peripheral neuroblastic and CNS embryonal tumours, which are immunonegative, with the caveat that a subset of pineoblastomas has significant expression.

Clonal selection drives genetic divergence of metastatic medulloblastoma.

Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord. Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and in most children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system. The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour. Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-ß signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

Somatic cell transfer of c-Myc and Bcl-2 induces large-cell anaplastic medulloblastomas in mice.

A highly aggressive subgroup of the pediatric brain tumor medulloblastoma is characterized by overexpression of the proto-oncogene c-Myc, which encodes a transcription factor that normally maintains neural progenitor cells in an undifferentiated, proliferating state during embryonic development. Myc-driven medulloblastomas typically show a large-cell anaplastic (LCA) histological pattern, in which tumor cells display large, round nuclei with prominent nucleoli. This subgroup of medulloblastoma is therapeutically challenging because it is associated with a high rate of metastatic dissemination, which is a powerful predictor of short patient survival times. Genetically engineered mouse models have revealed important insights into the pathogenesis of medulloblastoma and served as preclinical testing platforms for new therapies. Here we report a new mouse model of Myc-driven medulloblastoma, in which tumors arise in situ after retroviral transfer and expression of Myc in Nestin-expressing neural progenitor cells in the cerebella of newborn mice. Tumor induction required concomitant loss of Tp53 or overexpression of the antiapoptotic protein Bcl-2. Like Myc-driven medulloblastomas in humans, the tumors induced in mice by Myc + Bcl-2 and Myc - Tp53 showed LCA cytoarchitecture and a high rate of metastatic dissemination to the spine. The fact that Myc - Tp53 tumors arose only in Tp53(-/-) mice, coupled with the inefficient germline transmission of the Tp53-null allele, made retroviral transfer of Myc + Bcl-2 a more practical method for generating LCA medulloblastomas. The high rate of spinal metastasis (87% of brain tumor-bearing mice) will be an asset for testing new therapies that target the most lethal aspect of medulloblastoma.

FISHing in the dark: How the combination of FISH and conventional karyotyping improves the diagnostic yield in CpG-stimulated chronic lymphocytic leukemia.

Despite significant advances in molecular genetic approaches, fluorescence in situ hybridization (FISH) remains the gold standard for the diagnostic evaluation of genomic aberrations in patients with chronic lymphocytic leukemia (CLL). Efforts to improve the diagnostic utility of molecular cytogenetic testing have led to the expansion of the traditional 4-probe CLL FISH panel. Not only do these efforts increase the cost of testing, they remain hindered by the inherent limitations of FISH studies - namely the inability to evaluate genomic changes outside of the targeted loci. While array-based profiling and next generation sequencing (NGS) have critically expanded our understanding of the molecular pathogenesis of CLL, these methodologies are not routinely used by diagnostic laboratories to evaluate copy number changes or the mutational profile of this disease. Mitogenic stimulation of CLL specimens with CpG-oligonucleotide (CpG-ODN) has been identified as a reliable and reproducible means of obtaining a karyotype, facilitating a low-resolution genome-wide analysis. Across a cohort of 1255 CpG-ODN-stimulated CLL specimens, we describe the clinical utility associated with the combinatorial use of FISH and karyotyping. Our testing algorithm achieves a higher diagnostic yield (∼10%) through the detection of complex karyotypes, well-characterized chromosomal aberrations not covered by the traditional CLL FISH panel and through the detection of concurrent secondary malignancies. Moreover, the single cell nature of this approach permits the evaluation of emerging new clinical concepts including clonal dynamics and clonal evolution. This approach can be broadly applied by diagnostic laboratories to improve the utility of traditional and molecular cytogenetic studies of CLL. Am. J. Hematol. 91:978-983, 2016. © 2016 Wiley Periodicals, Inc.