Sequential cancer mutations in cultured human intestinal stem cells.

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

A neonate with a unique non-Down syndrome transient proliferative megakaryoblastic disease.

Transient myeloproliferative disorder (TMD) is a leukemia type that occurs typically in newborns. In Down syndrome, TMD is referred to as transient abnormal myelopoiesis (TAM).32 Recently, transientness has also been reported in acute myeloid leukemia patients with germline trisomy 21 mosaicism, and even in cases with somatic trisomy 21, with or without GATA1 mutations. TMD cases without trisomy 21 are rare, and recurrent genetic aberrations that aid in clinical decision-making are scarcely described. We describe here a TMD patient without trisomy 21 or GATA1 mutation in whom single-nucleotide polymorphism analysis of leukemic blasts revealed a novel combined submicroscopic deletion (5q31.1-5q31.3 and 8q23.2q24).

Outcome of allogeneic transplantation in newly diagnosed and relapsed/refractory multiple myeloma: long-term follow-up in a single institution.

Allogeneic stem cell transplantation (allo-SCT) has the potential to induce long-term remission in multiple myeloma (MM), but the role of allo-SCT in MM is controversial due to the high rate of treatment-related mortality (TRM). However, although proteasome inhibitors and immunomodulatory drugs have improved the outcome of patients with MM, high-risk patients still have a very poor prognosis. This indicates the need for new treatment strategies and identification of patients who might benefit from allo-SCT. We therefore analyzed the outcome of one hundred and forty-seven patients with MM who received an allo-SCT at our institution (58 in first line, 89 in relapsed/refractory setting) after a median follow-up of 88.8 months. For the first-line setting, median progression-free survival (PFS) and overall survival (OS) were remarkably good, with a CR rate of 48.3%, median PFS of 30.2 months, and 10-yr OS of 51%. We found no difference in outcome for patients with high-risk metaphase cytogenetics or FISH del(13q14), but efficacy in current standard high-risk patients could not be determined. The outcome in the relapsed/refractory setting was poor, especially in the subgroup of patients relapsing within 18 months after auto-SCT. Therefore, if applied at all in these patients, improvement of allo-SCT is needed, focusing on reduction of TRM and more effective immunotherapy.

High cereblon expression is associated with better survival in patients with newly diagnosed multiple myeloma treated with thalidomide maintenance.

Recently, cereblon (CRBN) expression was found to be essential for the activity of thalidomide and lenalidomide. In the present study, we investigated whether the clinical efficacy of thalidomide in multiple myeloma is associated with CRBN expression in myeloma cells. Patients with newly diagnosed multiple myeloma were included in the HOVON-65/GMMG-HD4 trial, in which postintensification treatment in 1 arm consisted of daily thalidomide (50 mg) for 2 years. Gene-expression profiling, determined at the start of the trial, was available for 96 patients who started thalidomide maintenance. In this patient set, increase of CRBN gene expression was significantly associated with longerprogression-free survival (P = .005). In contrast, no association between CRBN expression and survival was observed in the arm with bortezomib maintenance. We conclude that CRBN expression may be associated with the clinical efficacy of thalidomide. This trial has been registered at the Nederlands Trial Register (www.trialregister.nl) as NTR213; at the European Union Drug Regulating Authorities Clinical Trials (EudraCT) as 2004-000944-26; and at the International Standard Randomized Controlled Trial Number (ISRCTN) as 64455289.

Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma.

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies.

Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia.

Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.

Gene expression profiling for molecular classification of multiple myeloma in newly diagnosed patients.

To identify molecularly defined subgroups in multiple myeloma, gene expression profiling was performed on purified CD138(+) plasma cells of 320 newly diagnosed myeloma patients included in the Dutch-Belgian/German HOVON-65/GMMG-HD4 trial. Hierarchical clustering identified 10 subgroups; 6 corresponded to clusters described in the University of Arkansas for Medical Science (UAMS) classification, CD-1 (n = 13, 4.1%), CD-2 (n = 34, 1.6%), MF (n = 32, 1.0%), MS (n = 33, 1.3%), proliferation-associated genes (n = 15, 4.7%), and hyperdiploid (n = 77, 24.1%). Moreover, the UAMS low percentage of bone disease cluster was identified as a subcluster of the MF cluster (n = 15, 4.7%). One subgroup (n = 39, 12.2%) showed a myeloid signature. Three novel subgroups were defined, including a subgroup of 37 patients (11.6%) characterized by high expression of genes involved in the nuclear factor kappa light-chain-enhancer of activated B cells pathway, which include TNFAIP3 and CD40. Another subgroup of 22 patients (6.9%) was characterized by distinct overexpression of cancer testis antigens without overexpression of proliferation genes. The third novel cluster of 9 patients (2.8%) showed up-regulation of protein tyrosine phosphatases PRL-3 and PTPRZ1 as well as SOCS3. To conclude, in addition to 7 clusters described in the UAMS classification, we identified 3 novel subsets of multiple myeloma that may represent unique diagnostic entities.

Improved Gene Fusion Detection in Childhood Cancer Diagnostics Using RNA Sequencing.

PURPOSE: Gene fusions play a significant role in cancer etiology, making their detection crucial for accurate diagnosis, prognosis, and determining therapeutic targets. Current diagnostic methods largely focus on either targeted or low-resolution genome-wide techniques, which may be unable to capture rare events or both fusion partners. We investigate if RNA sequencing can overcome current limitations with traditional diagnostic techniques to identify gene fusion events. METHODS: We first performed RNA sequencing on a validation cohort of 24 samples with a known gene fusion event, after which a prospective pan-pediatric cancer cohort (n = 244) was tested by RNA sequencing in parallel to existing diagnostic procedures. This cohort included hematologic malignancies, tumors of the CNS, solid tumors, and suspected neoplastic samples. All samples were processed in the routine diagnostic workflow and analyzed for gene fusions using standard-of-care methods and RNA sequencing. RESULTS: We identified a clinically relevant gene fusion in 83 of 244 cases in the prospective cohort. Sixty fusions were detected by both routine diagnostic techniques and RNA sequencing, and one fusion was detected only in routine diagnostics, but an additional 24 fusions were detected solely by RNA sequencing. RNA sequencing, therefore, increased the diagnostic yield by 38%-39%. In addition, RNA sequencing identified both gene partners involved in the gene fusion, in contrast to most routine techniques. For two patients, the newly identified fusion by RNA sequencing resulted in treatment with targeted agents. CONCLUSION: We show that RNA sequencing is sufficiently robust for gene fusion detection in routine diagnostics of childhood cancers and can make a difference in treatment decisions.

Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia.

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.

A survey of undetected, clinically relevant chromosome abnormalities when replacing postnatal karyotyping by Whole Genome Sequencing.

Whole genome sequencing (WGS) holds the potential to identify pathogenic gene mutations, copy number variation, uniparental disomy and structural rearrangements in a single genetic test. With its high diagnostic yield and decreasing costs, the question arises whether WGS can serve as a single test for all referrals to diagnostic genome laboratories ("one test fits all"). Here, we provide an estimate for the proportion of clinically relevant aberrations identified by light microscopy in postnatal referrals that would go undetected by WGS. To this end, we compiled the clinically relevant abnormal findings for each of the different referral categories in our laboratory during the period 2006-2015. We assumed that WGS would be performed on 300-500 bp DNA fragments with 150-bp paired sequence reads, and that the mean genome coverage is 30x, corresponding to current practice. For the detection of chromosomal mosaicism we set minimum thresholds of 10% for monosomy and 20% for trisomy. Based on the literature we assumed that balanced Robertsonian translocations and ∼9% of other, balanced chromosome rearrangements would not be detectable because of breakpoints in sequences of repetitive DNA. Based on our analysis of all 14,957 referrals, including 1455 abnormal cases, we show that at least 8.1% of these abnormalities would escape detection (corresponding to 0.79% of all referrals). The highest rate occurs in referrals of premature ovarian failure, as 73.3% of abnormalities would not be identified because of the frequent occurrence of low-level sex chromosome mosaicism. Among referrals of recurrent miscarriage, 25.6% of abnormalities would go undetected, mainly because of a high proportion of balanced Robertsonian translocations. In referrals of mental retardation (with or without multiple congenital anomalies) the abnormality would be missed in only 0.35% of referrals. These include cases without imbalances of unique DNA sequences but of clinical relevance, as for example, r(20) epilepsy syndrome. The expected shift to large-scale implementation of WGS ("one test fits most") as initial genetic test will be beneficial to patients and their families, since a cause for the clinical phenotype can be identified in more cases by a single genetic test at an early phase in the diagnostic process. However, a niche for genome analysis by light microscopy will remain. For example, in referrals of newborns with a suspicion of Down syndrome, karyotyping is not only a cost-effective method for providing a quick diagnosis, but also discriminates between trisomy 21 and a Robertsonian translocation involving chromosome 21. Thus, when replacing karyotyping by WGS, one must be aware of the rates and spectra of undetected abnormalities. In addition, it is equally important that requirements for cytogenetic follow-up studies are recognized.

A new subtype of MLL-SEPT2 fusion transcript in therapy-related acute myeloid leukemia with t(2;11)(q37;q23): a case report and literature review.

The t(2;11)(q37;q23) is a rare recurrent cytogenetic abnormality associated with de novo and therapy-related acute myeloid leukemia, resulting in a MLL-SEPT2 fusion gene. We report on a case of therapy-related acute myeloid leukemia M2 showing a t(2;11)(q37;q23) and resulting in a new subtype of a MLL-SEPT2 chimeric transcript. The literature on this translocation is reviewed.

Cytogenetic aberrations in neuropathy associated with IgM monoclonal gammopathy.

The occurrence and nature of cytogenetic aberrations in polyneuropathy associated with IgM monoclonal gammopathy was determined. Therefore, interphase fluorescence in situ hybridization (FISH) was applied in 22 patients with polyneuropathy associated with IgM monoclonal gammopathy, multiplex ligation-dependent probe amplification (MLPA) assay in 18 of these patients and genome-wide-array-based comparative genomic hybridization (CGH) in eight of these 18 patients. Four patients had 10-20% and one patient had 30% B cells with IgH rearrangements; one patient had additional loss of 14qter; one patient had amplification of 6p and loss of 6q. Cytogenetic aberrations may be found in one third of the patients with neuropathy associated with IgM monoclonal gammopathy and are mainly associated with indolent Waldenstrom's Macroglobulinemia.

Lymphoblastic lymphoma with a triple-hit profile: a rare but distinct and relevant entity.

Follicular lymphoma with progression to a high-grade lymphoma bears a poor prognosis. We describe a case of a 60-year-old man who presented in 2012 with an epidural mass, diagnosed as a diffuse large B-cell lymphoma (DLBCL) with concurrent low-grade follicular lymphoma. Three years later, the patient presented with a cervical mass, diagnosed as a lymphoblastic lymphoma (LBL). Both the DLBCL and LBL contained a "triple hit" with BCL2, BCL6, and cMYC translocations demonstrated by fluorescence in situ hybridization analysis and a complex karyotype by single-nucleotide polymorphism array analysis. Furthermore, the 2 lymphomas were shown to be clonally related by clonality analysis and single-nucleotide polymorphism array analysis. This case report presents a highly unusual case of an LBL with a triple hit, originating from a DLBCL, which has rarely been described in the literature and deserves further exploration.

Detection of risk-identifying chromosomal abnormalities and genomic profiling by multiplex ligation-dependent probe amplification in chronic lymphocytic leukemia.

We performed genomic profiling using multiplex ligation-dependent probe amplification (MLPA) in 54 cases with suspected or advanced chronic lymphocytic leukemia (CLL). MLPA detected abnormalities when the percentage of mutated cells was greater than approximately 35%. Loss of 9p21 CDNK2A/B was revealed. MLPA is an economically attractive, powerful tool in trial-based, centralized risk-assessment for CLL.

The MN1 oncoprotein synergizes with coactivators RAC3 and p300 in RAR-RXR-mediated transcription.

The t(12;22) creates an MN1-TEL fusion gene leading to acute myeloid leukemia. The fusion partner TEL (ETV6) is a member of the ETS family of transcription factors. The nature of the other fusion partner, MN1, has not been investigated in detail until now. We recently described that MN1 activates the transcription activity of the moloney sarcoma virus long terminal repeat, indicating that this protein itself may act as a transcription factor. We show here that MN1 comprises multiple transcription activating domains. A search for a bound DNA sequence revealed that MN1 has affinity for retinoic acid responsive elements. A DR5 retinoic acid responsive element was observed in the LTR. The combination of MN1 and ligand-activated retinoic acid receptor leads to a synergistic induction of expression directed by the LTR. Cotransfection of MN1 with RAC3 or p300, known coactivators of retinoic acid receptors, leads to a further synergistic induction of transcription. In addition, the effect of MN1 can be inhibited by the wild-type adenovirus ElA protein that inhibits p300 function, but not by an E1A mutant lacking the p300-binding site. GAL4-MN1-mediated transcription can be enhanced directly by RAC3 and p300. Taken together, our results indicate that MN1 is a transcription coactivator rather than a sequence-specific transcription factor, and that it may stimulate RAR/RXR-mediated transcription through interaction with p160 and p300.

A ZMYM2-FGFR1 8p11 myeloproliferative neoplasm with a novel nonsense RUNX1 mutation and tumor lysis upon imatinib treatment.

The 8p11 myeloproliferative neoplasm (8p11 MPN) is a rare disorder that is molecularly characterized by fusions of diverse partners to the tyrosine kinase receptor gene FGFR1. It can rapidly transform to acute myeloid leukemia. Here we report on a case with a t(8;13)(p11.2;q12.1) ZMYM2-FGFR1 fusion, with massive tumor lysis upon tyrosine kinase inhibition with imatinib. Upon reevaluation, we detected trisomy 21 in addition to the translocation. Sequencing revealed a nonsense c.958C →T RUNX1 mutation both at diagnosis and disease progression, resulting in a p.Arg320X carboxyl-terminal truncated RUNX1 protein. This is the first report on an 8p11 MPN with a trisomy 21 RUNX1 mutation.

A girl with an atypical form of ataxia telangiectasia and an additional de novo 3.14 Mb microduplication in region 19q12.

A 9-year-old girl born to healthy parents showed manifestations suggestive of ataxia telangiectasia (AT), such as short stature, sudden short bouts of horizontal and rotary nystagmus, a weak and dysarthric voice, rolling gait, unstable posture, and atactic movements. She did not show several cardinal features typical of AT such as frequent, severe infections of the respiratory tract. In contrast, she showed symptoms not generally related to AT, including microcephaly, profound motor and mental retardation, small hands and feet, severely and progressively reduced muscle tone with slackly protruding abdomen and undue drooling, excess fat on her upper arms, and severe oligoarthritis. A cranial MRI showed no cerebellar hypoplasia and other abnormalities. In peripheral blood samples she carried a de novo duplication of 3.14 Mb in chromosomal region 19q12 containing six annotated genes, UQCRFS1, VSTM2B, POP4, PLEKHF1, CCNE1, and ZNF536, and a de novo mosaic inversion 14q11q32 (96% of metaphases). In a saliva-derived DNA sample only the duplication in 19q12 was detected, suggesting that the rearrangements in blood lymphocytes were acquired. These findings reinforced the suspicion that she had AT. AT was confirmed by strongly elevated serum AFP levels, cellular radiosensitivity and two inherited mutations in the ATM gene (c.510_511delGT; paternal origin and c.2922-50_2940del69; maternal origin). This case suggest that a defective ATM-dependent DNA damage response may entail additional stochastic genomic rearrangements. Screening for genomic rearrangements appears indicated in patients suspected of defective DNA damage responses.