Annet Simons

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

Frequent loss of 9p21 (p16(INK4A)) and other genomic imbalances in human malignant fibrous histiocytoma

To search for new recurrent genetic aberrations in malignant fibrous histiocytoma (MFH), a combination of conventional cytogenetic, comparative genomic hybridization (CGH), and Southern blot analyses was applied to a series of 34 tumors. Cytogenetic analysis revealed the presence of multiple structural and numerical aberrations, including marker chromosomes, telomeric associations, double minutes, and ring chromosomes. The most frequent genomic imbalances in this series of neoplasms as detected by CGH were gains of 1q21-q22 (69%), 17q23-qter (41%), and 20q (66%), and losses of 9p21-pter (55%), 10q (48%), 11q23-qter (55%), and 13q10-q31 (55%). Southern blot analyses with p16(INK4A) (CDKN2A; 9p21) and RB1 (13q14) probes provided clear indications for frequent deletions of these tumor suppressor genes, and as such, substantiated the CGH results. Additionally, examination of the TP53 and MDM2 genes showed frequent loss and amplification, respectively. These data indicate that genes involved in the RB1- and TP53-associated cell cycle regulatory pathways may play prominent roles in the development of human MFH.

Genome-wide arrays in routine diagnostics of hematological malignancies†

Over the last three decades, cytogenetic analysis of malignancies has become an integral part of disease evaluation and prediction of prognosis or responsiveness to therapy. In most diagnostic laboratories, conventional karyotyping, in conjunction with targeted fluorescence in situ hybridization analysis, is routinely performed to detect recurrent aberrations with prognostic implications. However, the genetic complexity of cancer cells requires a sensitive genome‐wide analysis, enabling the detection of small genomic changes in a mixed cell population, as well as of regions of homozygosity. The advent of comprehensive high‐resolution genomic tools, such as molecular karyotyping using comparative genomic hybridization or single‐nucleotide polymorphism microarrays, has overcome many of the limitations of traditional cytogenetic techniques and has been used to study complex genomic lesions in, for example, leukemia. The clinical impact of the genomic copy‐number and copy‐neutral alterations identified by microarray technologies is growing rapidly and genome‐wide array analysis is evolving into a diagnostic tool, to better identify high‐risk patients and predict patients outcomes from their genomic profiles. Here, we review the added clinical value of an array‐based genome‐wide screen in leukemia, and discuss the technical challenges and an interpretation workflow in applying arrays in the acquired cytogenetic diagnostic setting. Hum Mutat 33:941–948, 2012.

Detection of clinically relevant copy-number variants by exome sequencing in a large cohort of genetic disorders.

PURPOSEnCopy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10-20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test.nnnMETHODSnWe performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting.nnnRESULTSnIn total, 123 clinically relevant CNVs ranging in size from 727u2009bp to 15.3u2009Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to -5.8% per disorder).nnnCONCLUSIONSnThis study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016Genetics in Medicine (2016); doi:10.1038/gim.2016.163.Purpose:Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10–20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test.Methods:We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting.Results:In total, 123 clinically relevant CNVs ranging in size from 727u2009bp to 15.3u2009Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to –5.8% per disorder).Conclusions:This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016

Identification of chromosomal abnormalities relevant to prognosis in chronic lymphocytic leukemia using multiplex ligation-dependent probe amplification

B-cell chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. Characteristic genomic abnormalities provide clinically important prognostic information. Because karyotyping and fluorescence in situ hybridization (FISH) are laborious techniques, we investigated the diagnostic efficacy of the more recently developed multiplex ligation-dependent probe amplification (MLPA) technique. MLPA and interphase FISH data of 88 CLL patients were compared for loci encompassing the 13q14 region, chromosome 12, and the ATM (11q22) and TP53 (17p13) genes. We found a perfect correlation, provided that the abnormal clone was present in at least 10-20% of the cells. Because multiple loci and multiple probes per locus were included in the MLPA assay, additional abnormalities not covered by the FISH probes were detected. Furthermore, in 13 cases deletions partly covering the 13q14.3 locus were observed, including three deletions that remained undetected by FISH. All the deletions included the noncoding RNA locus DLEU1 (previously BCMS), which is considered to be the most likely CLL-associated candidate tumor suppressor gene within the 13q14 region. We conclude that MLPA serves as a comprehensive and reliable technique for the simultaneous identification of different clinically relevant and region-specific genomic aberrations in CLL.

Isolation of Osteosarcoma-Associated Amplified DNA Sequences Using Representational Difference Analysis

Comparative genomic hybridization analysis of a primary osteosarcoma and its metastasis revealed two regions of DNA amplification, one at 17p11.2‐12 and one at 19q12‐13. Subsequent representational difference analysis of the primary tumor resulted in the isolation of two distinct tumor‐amplified DNA fragments originating from chromosome 19. A YAC clone corresponding to one of the two isolated DNA fragments was used for fluorescence in situ hybridization on normal human lymphocyte metaphases and tumor‐derived nuclei. This resulted in the localization of this YAC to 19q12‐13.1 and confirmed the amplification status of the isolated fragment in the tumors. The availability of such RDA‐isolated sequences may be instrumental in the search for genes relevant for tumor development. Genes Chromosomes Cancer 20:196–200, 1997.

A novel chromosomal region of allelic loss, 4q32-q34, in human osteosarcomas revealed by representational difference analysis.

Representational difference analysis (RDA) of a human osteosarcoma xenograft resulted in the isolation of four tumor‐associated homozygously deleted DNA fragments, all originating from chromosome 4, region q32–q34. Southern blot analysis using the RDA fragments and interphase FISH analysis using PACs corresponding to these RDA fragments revealed allelic loss of the 4q32–q34 region in 17 of 27 (63%) osteosarcomas tested. These results suggest the involvement of tumor suppressor gene(s) within this chromosomal region in osteosarcoma development. The RDA fragments and corresponding PAC clones will be instrumental in the isolation of such gene(s). Genes Chromosomes Cancer 26:115–124, 1999.

Guidelines for cytogenetic investigations in tumours

Cytogenetic and molecular genetic data are of paramount importance in the diagnosis, prognosis, and risk stratification of patients with malignant diseases. Sometimes they even directly guide the choice of therapy.1 Disease-specific abnormalities, particularly translocations, can provide essential information to assist the Pathologist and/or Oncologist in assigning a diagnosis. In several diseases, tumour genetics correlate strongly with clinical risk; thus, cytogenetic information may help the Oncologist counsel the patient, choose a specific treatment, and/or modulate treatment intensity. Clinical trials may involve cytogenetic classification of patients to the appropriate treatment regimens. Currently, the provision of specific assays for acquired neoplasiaspecific genomic changes varies among and within countries as a range of laboratories offer diagnostic solid tumour genetics; these may include Cytogenetic, Pathology, Haematology, and Molecular Genetics laboratories. Technical standards and general guidelines for the analysis and the report of results on most solid tumours are lacking. To address these deficits, a tumour best practice meeting with invited tumour experts without conflict of interest was held on 23rd April 2013 in Oxford, United Kingdom. The aim was to produce professional guidelines for tumour genetic laboratories and to incorporate the standards imposed by generic European guidelines,2 regulatory bodies (ISO15189, 2012 Medical laboratories – requirements for quality and competence),3 reporting guidelines,4 ISCN,5 and acquired best practice guidelines, while taking into account the current practice in Europe. The guidelines are aimed principally at giving guidance on the minimum, standard cytogenetic analyses, which are applicable to different types of laboratories operating under different regulatory arrangements and are relevant if more specific recommendations are not available. It was universally acknowledged that information on ancillary techniques in use in most cytogenetic laboratories (eg, RTPCR) or advanced techniques not always extensively performed in all laboratories (eg, next-generation sequencing (NGS)) were considered. The process for developing these evidence-based consensus guidelines included agreement on the need of general uniform rules on solid tumour analysis and reporting, discussion on the architecture of the guidelines, working group formation with different tasks (collection, analysis and comparison of any existing guidelines on this subject, type of tumours to be included according to published data and database consultation, method of analysis to be included, report formulation), circulation of the working group activities, formulation and circulation of the initial recommendations, draft and discussion, final consensus, and approval. It is noted that some elements of the tumour diagnostic service not subject to statute may be varied according to local constraints and agreements. Therefore, these guidelines are minimum requirements and additional professional judgment may be of paramount importance under many circumstances. In addition, as new techniques, particularly NGS, as well as clinical evidence, are becoming available all the time, these should be kept under constant review. Notes: The use of ‘must’ in this document indicates a requirement and the use of ‘should’ indicates a recommendation. Where there appears to be contradiction between available guidelines, the most recently published ones should be taken to apply to all. All diagnostic laboratories should be accredited to national or internationally accepted standards (ISO15189).3,6 Laboratories should participate in an External Quality Assessment Scheme7 in all aspects of their service for which a scheme is available.

Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling

BackgroundCharacteristic genomic abnormalities in patients with B cell chronic lymphocytic leukemia (CLL) have been shown to provide important prognostic information. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA), currently used in clinical diagnostics of CLL, are targeted tests aimed at specific genomic loci. Microarray-based genomic profiling is a new high-resolution tool that enables genome-wide analyses. The aim of this study was to compare two recently launched genomic microarray platforms, i.e., the CytoScan HD Array (Affymetrix) and the HumanOmniExpress Array (Illumina), with FISH and MLPA to ascertain whether these latter tests can be replaced by either one of the microarray platforms in a clinical diagnostic setting.ResultMicroarray-based genomic profiling and FISH were performed in all 28 CLL patients. For an unbiased comparison of the performance of both microarray platforms 9 patients were evaluated on both platforms, resulting in the identification of exactly identical genomic aberrations. To evaluate the detection limit of the microarray platforms we included 7 patients in which the genomic abnormalities were present in a relatively low percentage of the cells (range 5-28%) as previously determined by FISH. We found that both microarray platforms allowed the detection of copy number abnormalities present in as few as 16% of the cells. In addition, we found that microarray-based genomic profiling allowed the identification of genomic abnormalities that could not be detected by FISH and/or MLPA, including a focal TP53 loss and copy neutral losses of heterozygosity of chromosome 17p.ConclusionFrom our results we conclude that although the microarray platforms exhibit a somewhat lower limit of detection compared to FISH, they still allow the detection of copy number abnormalities present in as few as 16% of the cells. By applying similar interpretation criteria, the results obtained from both platforms were comparable. In addition, we conclude that both microarray platforms allow the identification of additional potential prognostic relevant abnormalities such as focal TP53 deletions and copy neutral losses of heterozygosity of chromosome 17p, which would have remained undetected by FISH or MLPA. The prognostic relevance of these novel genomic alterations requires further evaluation in prospective clinical trials.

Therapy-related, donor-derived AML responding to a second allogeneic BMT

Donor cell leukemia is a rare complication of allogeneic BMT, and many hypotheses about the underlying mechanisms have been put forth. Since donor cell leukemia is such a rare phenomenon, treatment of these patients has not been standardized. In this report, we present a child with pre-B ALL who developed a secondary AML of donor origin with Mixed-lineage leukemia (MLL) gene rearrangement after treatment for a relapse of his ALL after allogeneic BMT. A second allogeneic BMT with marrow from the same donor was performed and the patient is in ongoing complete remission now for 4 years. Our data show that long-lasting remission can be achieved after donor-derived, therapy-related AML by a second transplant with marrow from the same donor. A 1-year-old, Caucasian boy with recurrent infections was diagnosed with ALL FAB L1 in 1988 and he was treated according to the ALL-BFM-86 regimen. In a period of 10 years, he relapsed twice (Figures 1a and b). Flow