PURPOSE:Treatment stratification in ALL includes diverse (cyto)genetic aberrations, requiring diverse tests to yield conclusive data. We optimized the diagnostic workflow to detect all relevant aberrations with a limited number of tests in a clinically relevant time frame. METHODS:In 467 consecutive patients with ALL (0-20 years), we compared RNA sequencing (RNAseq), fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR), karyotyping, single-nucleotide polymorphism (SNP) array, and multiplex ligation-dependent probe amplification (MLPA) for technical success, concordance of results, and turnaround time. RESULTS:To detect stratifying fusions (ETV6::RUNX1, BCR::ABL1, ABL-class, KMT2Ar, TCF3::HLF, IGH::MYC), RNAseq and FISH were conclusive for 97% and 96% of patients, respectively, with 99% concordance. RNAseq performed well in samples with a low leukemic cell percentage or low RNA quality. RT-PCR for six specific fusions was conclusive for >99% but false-negative for six patients with alternatively fused exons. RNAseq also detected gene fusions not yet used for stratification in 14% of B-cell precursor-ALL and 33% of T-ALL. For aneuploidies and intrachromosomal amplification of chromosome 21, SNP array gave a conclusive result in 99%, thereby outperforming karyotyping, which was conclusive for 64%. To identify deletions in eight stratifying genes/regions, SNP array was conclusive in 99% and MLPA in 95% of patients, with 98% concordance. The median turnaround times were 10 days for RNAseq, 9 days for FISH, 10 days for SNP array, and <7 days for MLPA and RT-PCR in this real-world prospective study. CONCLUSION:Combining RNAseq and SNP array outperformed current diagnostic tools to detect all stratifying genetic aberrations in ALL. The turnaround time is <15 days matching major treatment decision time points. Moreover, combining RNAseq and SNP array has the advantage of detecting new lesions for studies on prognosis and pathobiology.

Background and aims Treatment stratification in pediatric acute lymphoblastic leukemia (ALL) is, besides clinical parameters, guided by (cyto)genetic aberrations. These aberrations include a variety of gene fusions, aneuploidy groups, and copy number alterations (CNA), requiring multiple diagnostic assays to yield conclusive data. Over the past decade, the number and complexity of aberrations to be addressed for stratification increased whereas the turn-around-time decreased. We aimed to optimize the diagnostic workflow with a limited number of assays while allowing detection of all relevant genetic aberrations in a clinically relevant timeframe. Methods In a consecutive cohort of 467 newly diagnosed patients (0 to <19 years) immunophenotyped as ALL, we compared the diagnostic yield of RNA sequencing, 7 fluorescence in situ hybridization (FISH) probes, 7 RT-PCR targets, karyotyping, SNP array (CytoSNP-850K), and multiplex ligation-dependent probe amplification (MLPA; P335-ALL-IKZF1) for conclusiveness, concordance and turn-around-time. Results To detect stratifying fusions (ETV6::RUNX1, BCR::ABL1, ABL-class, KMT2Ar, TCF3::HLF, IGH::MYC), RNA sequencing and FISH were conclusive for 97% and 96% of patients, respectively, with 99% (433/437) concordance of test results. RT-PCR for 6 fusion transcripts was conclusive for >99%, but false-negative for 6 patients who had alternative exons in their fusion genes. RNA sequencing detected fusion genes even in samples with a leukemic cell percentage as low as 10% or RNA integrity score as low as 2. A TCF3::HLF fusion was not detected by RNA sequencing due to the insertion of intronic sequences in the chimeric transcript. A KMT2A::USP2 and a BCR::ABL1 fusion were not detected by FISH because of an inversion and probably insertion, respectively, not causing a break-apart FISH pattern. RNA sequencing detected subtype-defining fusions not (yet) used for stratification in 14% of BCP-ALL and 33% of T-ALL as well as rare fusions in 2% of BCP-ALL and 8% of T-ALL, including a targetable ETV6::NTRK3 fusion which were by definition not detected by targeted FISH or RT-PCR. Moreover, RNA sequencing showed added value for classification based on expression profile and detection of expressed mutations. The turn-around-time for RNA sequencing (7-15 days) and FISH (6-13 days) were both compatible with the demand for diagnostic reporting by day 15 of high-risk genetics and ABL-class fusions for the ALLTogether01 protocol and of BCR::ABL1 positivity for referral to the EsPhALL protocol. Because KMT2A rearrangement should be detected within 7 days to enter the Interfant-21 protocol, the shorter turn-around-time of FISH and RT-PCR were needed in infants (<1 year). For the detection of aneuploidy groups (high hyperdiploidy, low hypodiploidy and near haploidy) and intrachromosomal amplification of chromosome 21 (iAMP21), SNP array gave conclusive results for 99% of the patients, thereby outperforming karyotyping, which was conclusive for 64% and mistook 2 cases of masked hypodiploidy for high hyperdiploidy. Based on the amplification pattern on chromosome 21, 13 iAMP21 cases were identified by SNP array. Using a combination of karyotyping and RUNX1 metaphase FISH, 9/13 iAMP21 cases were identified with the remaining 4 cases lacking informative karyotypes. To identify deletions in eight genes/regions relevant for CNA risk stratification (IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, RB1, BTG1 and PAR1), SNP array was conclusive in 99% and MLPA in 95% of patients with 98% concordant CNA risk calls. SNP array was more sensitive than MLPA in aneuploid samples and samples with low leukemic cell percentage. Beyond the currently required detection of MLPA-based deletions, SNP array detected deletions in (single) exons and genes not covered by the MLPA assay as well as aberrations in low mosaicism. Conclusions Our assay conclusiveness for ≥97% of patients and concordance of results with classic methods of 99% in 467 consecutive patients has resulted in the implementation of RNA sequencing and SNP array as the primary choice in the molecular diagnostics of newly diagnosed ALL in the Netherlands with addition of FISH and RT-PCR to detect KMT2A rearrangement in the infant population only. Performing RNA sequencing and SNP array for all patients has the advantage of detecting new lesions and expressed mutations to retrospectively study their role in prognosis and pathobiology.

Background: Previous studies have shown a high prevalence of syndromes in children with cancer. We described four patterns of co-occurring morphological abnormalities indicating new tumour predisposition syndromes. These patterns were named after their key-abnormalities: blepharophimosis (BP), epicanthal folds (EF), asymmetric lower limbs (LLA) and Sydney creases (SC) pattern. The purpose of our study was to identify structural genomic variants possibly involved in these tumour predisposition syndromes.Patients and methods: In 49 probands (13 from BP, nine from EF, 20 from LLA and seven from SC patterns respectively) karyotyping was performed. Copy number variation (CNV) in genomic DNA of the probands was analysed to detect microdeletions/-duplications using SNP array. FISH and quantitative-polymerase chain reaction (q-PCR) experiments were done to validate events identified by cytogenetic and CNV analysis.Results: Cytogenetic analysis showed an inherited inversion of chromosome 15, inv(15) (q25q26) in a proband with LLA-pattern. Evaluation of the genes at the breakpoints made it unlikely that these explained the phenotype and tumour in this patient. Eleven CNV events met our inclusion criteria; three inherited CNV events involved an oncogene. A duplication involving BCL9 was identified in a proband diagnosed with Burkitt lymphoma. A duplication involving PCM1 was identified in a proband diagnosed with pre-B-ALL. Both probands showed the EF-pattern of morphological abnormalities. A deletion involving TRA@ was identified in two probands from the BP-pattern diagnosed with rhabdomyosarcoma and pre-B-ALL respectively.Conclusions: We report on structural genomic variants in paediatric cancer patients with newly recognised tumour predisposition syndromes. We identify three CNV events which we suggest to be susceptibility loci. (C) 2013 Elsevier Ltd. All rights reserved.

Background Three quarters of patients with 22q11.2 Deletion Syndrome (22q11.2DS) have congenital heart disease (CHD), typically conotruncal heart defects. Although it is currently common practice to test all children with typical CHD for 22q11.2DS, many adult patients have not been tested in the past and therefore 22q11.2DS might be under-recognised in adults.Objectives To determine the prevalence of 22q11.2DS in adults with tetralogy of Fallot (TOF) and pulmonary atresia (PA)/ventricular septal defect (VSD) and to assess the level of recognition of the syndrome in adult patients.Methods Patients were identified from CONCOR, a nationwide registry for adult patients with CHD. Inclusion criteria were diagnosis of TOF or PA/VSD and the availability of DNA. Patients with syndromes other than 22q11.2DS were excluded. Multiplex ligation-dependent probe amplification was used to detect 22q11.2 microdeletions.Results 479 patients with TOF and 79 patients with PA/VSD (56% male, median age 34.7 years) were included and analysed. Twenty patients were already known to have 22q11.2DS. A 22q11.2 microdeletion was detected in a further 24 patients. Thirty-one patients with TOF (6.5%) had 22q11.2DS, whereas 13 patients with PA/VSD had 22q11.2DS (16.5%). Of all 22q11.2 microdeletions, 54% (24/44) were unknown before this study.Conclusion This study shows that although the prevalence of 22q11.2DS in adults with TOF and PA/VSD is substantial, it is unrecognised in more than half of patients. As the syndrome has important clinical and reproductive implications, a diagnostic test should be considered in all adult patients with TOF and PA/VSD.

Septins are proteins associated with crucial steps in cell division and cellular integrity. In humans, 14 septin genes have been identified, of which five (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) are known to participate in reciprocal translocations with the MLL gene in myeloid neoplasias. We have recently shown a significant down-regulation of both SEPT2 and MLL in myeloid neoplasias with the MLL-SEPT2 fusion gene. In this study, we examined the expression pattern of the other 13 known septin genes in altogether 67 cases of myeloid neoplasia, including three patients with the MLL-SEPT2 fusion gene, four with MLL-SEPT6 fusion, and three patients with the MLL-SEPT9 fusion gene. When compared with normal controls, a statistically significant down-regulation was observed for the expression of both MLL (6.4-fold; p=0.008) and SEPT6 (1.7-fold; p=0.002) in MLL-SEPT6 leukemia. Significant down-regulation of MLL was also found in MLL-MLLT3 leukemias. In addition, there was a trend for SEPT9 down-regulation in MLL-SEPT9 leukemias (4.6-fold; p=0.077). Using hierarchical clustering analysis to compare acute myeloid leukemia genetic subgroups based on their similarity of septin expression changes, we found that MLL-SEPT2 and MLL-SEPT6 neoplasias cluster together apart from the remaining subgroups and that PML-RARA leukemia presents under-expression of most septin family genes.

We evaluated the radiation outcome and prognostic factors in a population-based study of early (T1N0M0) glottic carcinoma. Survival parameters and prognostic factors were evaluated by uni- and multivariate analysis in 316 consecutive irradiated patients with T1 glottic carcinoma in the Comprehensive Cancer Center West region of the western Netherlands. Median follow-up was 70 months (range 1–190 months). Five and ten-year local control was 86 and 84%. Disease specific survival was 97% at 5 and 10 years. In multivariate analysis, pre-existent laryngeal hypertrophic laryngitis was the only predictive factor for local control (relative risk = 3.0, P = 0.02). Comorbidity was prognostic for overall survival. No factor was predictive for disease specific survival. Pre-existent laryngeal hypertrophic laryngitis is a new risk factor associated with reduced local control in T1 glottic carcinoma treated with radiotherapy.

Background: A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature.Methods: Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient.Results: When compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023)Conclusion: We found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2.

Background: A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature. Methods: Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient. Results: When compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLLSEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023) Conclusion: We found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2. Published: 15 May 2009 BMC Cancer 2009, 9:147 doi:10.1186/1471-2407-9-147 Received: 3 December 2008 Accepted: 15 May 2009 This article is available from: http://www.biomedcentral.com/1471-2407/9/147 © 2009 Cerveira et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

OBJECTIVE:To investigate the influence of previous tumours on overall survival in patients with early glottic carcinoma.DESIGN:Retrospective, population-based cohort study.SETTING:Cancer registration area in the west Netherlands.METHODS:Population-based data on previous and subsequent tumours in patients diagnosed with early glottic carcinoma (Tis and T1) in the west Netherlands between 1982 and 1993 were collected from charts and cancer registries. The impact of previous tumours on survival was tested in multivariate analysis.MAIN OUTCOME MEASURES:Overall survival.RESULTS:Of 359 patients, 22 patients (6%) had a total of 23 previous tumours. Previous tumours had an independent impact on overall survival (hazard ratio 3.4, p < or = .001). Other determinants of survival were age and subsequent tumours. Nonmalignant comorbidity was of borderline significance.CONCLUSIONS:Previous tumors have an impact on survival in patients with early glottic carcinoma and should be considered as an additional factor in counseling and prognostication.

Treatment of acute promyelocytic leukemia (APL) with a combination of anthracycline-based chemotherapy and all-trans retinoic acid (ATRA) leads to very high rates of complete remission and survival. There are only a limited number of publications on the development of therapy-related myelodysplastic syndrome (MDS) or acute myeloid leukemia during follow-up of APL. Although drugs targeting at DNA-topoisomerase II characteristically induce translocations involving 11q23, this was seldom seen in patients treated for APL. We report on a patient initially diagnosed with APL. Response to therapy was monitored by fluorescence in situ hybridization (FISH) and reverse-transcriptase polymerase chain reaction for the PML-RARα rearrangement. Consecutive samples showed a swift and complete reduction of PML-RARα rearranged cells. Twenty months after diagnosis, however, conventional cytogenetics revealed a complex karyotype with a translocation involving 11q23 and loss of chromosomes 7q and Xq. FISH analysis with the MLL probe identified 2q37 (harboring the SEPT2 gene) as the translocation partner of chromosome 11. We consider the rather unique t(2;11)(q37;q23) as the primary event causing therapy-related MDS in our patient. This case stresses the importance of conventional karyotyping to be performed on a regular basis in all treated APL patients for the early detection of chromosomal aberrations that indicate the development of therapy-related MDS or acute myeloid leukemia.

Prospective studies on the risk of malignant transformation in patients with monoclonal gammopathy of undetermined significance (MGUS) and factors predictive of survival are lacking. The Dutch Comprehensive Cancer Centre West, comprising 1.6 million inhabitants, initiated a prospective hospital-based cohort study on 1464 patients with newly diagnosed M-proteinaemia, median age 73 (17-103) years. M-protein related diagnoses, patients' characteristics, laboratory investigations, bone marrow examinations and skeletal X-rays were registered with a yearly follow-up. Main endpoints were death, or new diagnoses of multiple myeloma and non-Hodgkin lymphoma. Kaplan-Meier survival curves were compared with age- and gender-matched survival data from the total Dutch population. Cumulative malignant transformation was corrected for death using a competing risk model. Risk factors for transformation or death were analyzed by univariate and multivariate analyses. In 1007 MGUS-patients, malignant transformation was associated with rising M-protein levels, IgA and IgM isotype and occurred at a yearly rate of 0.4%. All MGUS patients survived less than a matched cohort of the Dutch population, even in the absence of M-protein-associated comorbidity. Serum albumin levels at entry appeared highly predictive for survival. M-proteinaemia is not an innocent symptom. Although malignant transformation occurs rarely, survival is shortened irrespective of comorbidity.

PURPOSE The purpose of this study was to determine the incidence, clinical and histologic features, and patterns of outcome of thyroid lymphomas. PATIENTS AND METHODS A retrospective population-based survey of 38 patients with thyroid lymphoma was taken. Median age was 69 years (range, 33-87 years), with a 1:4 female predominance. Fifty percent of cases had a history of autoimmune thyroiditis, and coexistent thyroiditis was found in 67% of cases in which preexistent thyroid tissue was present. The most common subtype was diffuse large B-cell lymphoma (DLBCL; 63%) followed by extranodal marginal zone lymphoma (ENMZL; 29%). Ten of the patients with DLBCL showed a concomitant low-grade mucosa-associated lymphatic tissue component, and 4 cases of aggressive ENMZL were diagnosed. At diagnosis, 22 patients (58%) had localized disease, and 41% had low-risk international prognostic index scores. RESULTS Therapy was diverse and included all possible treatment modalities, none of which showed superiority. A complete clinical response was exhibited in 64% of patients, 14% exhibited a partial response, and 22% developed progressive disease. At a median follow-up of 43 months (range, 0-240 months), 15 patients had relapsed or developed progressive disease. Two-year overall survival rate was 59% for all patients, 68% for patients with localized disease, and 47% for patients with disseminated lymphoma. CONCLUSION Many thyroid lymphomas have clinical and histologic features characteristic of ENMZL and belong to this specific clinicopathologic entity.