Clonal Translocations Research Articles

Ossifying Fibromyxoid Tumor with Atypical Features: A Case Report and Review of Literature

Abstract Introduction/Objective Ossifying fibromyxoid tumor (OFMT) is a soft tissue neoplasm usually behaving in a benign fashion, however, it can rarely show atypical or malignant features. It most frequently occurs within the extremities and trunk. Although OFMTs are translocation-associated tumors, and very commonly associated with recurrent gene rearrangements involving the PHF1 gene, their morphologic and immunophenotypic profile can overlap with other soft tissue tumors posing a diagnostic challenge. Methods/Case Report Herein we present a case of a 23-year-old woman who had an excision of a subcutaneous shoulder mass. Grossly, the 5.3 cm mass had a thin translucent capsule and a pink, soft-cut surface with scattered calcifications. Microscopic examination revealed a well-circumscribed lesion composed of plump spindle cells with uniform oval nuclei arranged in short fascicles within a mildly collagenous background. There was prominent hemangiopericytoma-like vasculature and foci of ossification. The tumor cells contained moderate cytoplasm and showed a mild degree of nuclear atypia. Mitotic activity was up to 6 per 10 high-power fields. Immunohistochemical stains showed tumor cells are positive for Desmin, SMA, AE1/AE3 (patchy), TLE, and SATB2, while negative for CD31, CD34, S-100, C-kit, DOG-1, CD117, HMB-45, Melan-A, ALK-1, SOX10, Inhibin, and STAT6. Ki-67 proliferation index was low (1-2%). Fluorescence in situ hybridization (FISH) testing was negative for SYT and MDM2 gene amplification. FISH testing for PHF1 gene (using experimental probes) showed a break apart signal in a subset (~ 25%) of tumor cells, which is equivocal but insufficient to establish clonal interchromosomal translocation involving PHF1. Results (if a Case Study enter NA) NA Conclusion There is a histologic spectrum and morphologic overlap of OFMTs with several other soft tissue neoplasms. OFMT with atypical features (increased cellularity and mitotic activity), as in this case, may harbor the unpredictable biologic potential for recurrence and metastasis. Additional studies are required to discover a morphologic, immunohistochemical, and possibly molecular pattern that will help to differentiate OFMT subtypes.

Retrospective Evaluation of Cytogenetic Effects Induced by Internal Radioiodine Exposure: A 27-Year Follow-Up Study

Radioiodine (131I) is widely used in the treatment of hyperthyroidism and as an effective ablative therapy for differentiated thyroid cancer. Radioiodine (131I) constitutes 90% of the currently used therapies in the field of nuclear medicine. Here, we report the cytogenetic findings of a long-term follow-up study of 27 years on a male patient who received two rounds of radioiodine treatment within a span of 26 months between 1992 and 1994 for his papillary thyroid cancer. A comprehensive cytogenetic follow-up study utilizing cytokinesis blocked micronucleus assay, dicentric chromosome assay, genome wide translocations and inversions was initiated on this patient since the first administration of radioiodine in 1992. Frequencies of micronuclei (0.006/cell) and dicentric chromosomes (0.008/cell) detected in the current study were grossly similar to that reported earlier in 2019. The mFISH analysis detected chromosome aberrations in 8.6% of the cells in the form of both unbalanced and balanced translocations. Additionally, a clonal translocation involving chromosomes 14p; 15q was observed in 2 of the 500 cells analyzed. Out of the 500 cells examined, one cell showed a complex translocation (involving chromosomes 9, 10, and 16) besides 5 other chromosome rearrangements. Collectively, our study indicates that the past radioiodine exposure results in long-lasting chromosome damage and that the persistence of translocations can be useful for both retrospective biodosimetry and for monitoring chromosome instability in the lymphocytes of radioiodine exposed individuals.

Targeted Next Generation Sequencing Improves Diagnosis in Unclassifiable Leukemic Indolent B-Cell Non-Hodgkin Lymphoma and Identifies a Subset with Recurrent MYD88 Mutations in a Prospective Multicentre Study

Introduction: Patients with leukemic indolent B-cell Non-Hodgkin Lymphomas (LI B-NHL) that do not easily fall into a distinct pathological category with standard diagnostics can pose a challenge as their optimal management is uncertain. Lack of a clear diagnostic label can deny patients access to novel therapies as regulatory approval of drugs is largely granted within histological categories. Moreover, these patients may be excluded from participation in clinical trials. We report findings from a prospective study evaluating targeted next generation sequencing (NGS) of circulating malignant B-cells in this setting. Methods: Over a period of 4 years, 108 patients with LI B-NHL, deemed unclassifiable on standard peripheral blood (PB) diagnostics, were prospectively enrolled from 14 centres around the UK including ours. Patients with non-clonal lymphocytosis, confirmed CLL-type MBL, CLL (Matutes score 4 or 5), MCL, HCL, high grade lymphoma on standard PB diagnostics were excluded. Clinical and morphological characteristics were analysed. PB immunophenotyping was used for characterisation including ROR1 expression. CD15+ cells were positively selected with magnetic beads for germline DNA. Tumor-germline pairs were analysed with theEuroclonality-NGS DNA capture panel, and ARResT/Interrogate and complementary pipelines, to characterise clonal immunoglobulin rearrangements, translocations and somatic mutations (Stewart et al. ASH annual meeting 2019). Germline variants were subtracted to improve somatic mutation detection. Results: Median age was 72yrs with M:F ratio of 2:1. Incidental lymphocytosis on a routine blood count was the commonest mode of presentation with only a third of patients symptomatic at presentation. Median lymphocyte count was 12.4 x 109/L at the time of sampling. Just under 50% of cases had partial or complete CD5 expression, CD19 and 20 expression was moderate to strong in most cases while ROR1 was negative in most cases. NGS was able to assign a diagnostic category in 11/90 (12%) cases based on detection of IGH;CCND1 (6), IGH;BCL2 (2), IGH;BCL3 (2) translocations and in one case a BRAF mutation with confirmation of hairy cell leukaemia on immuno-morphology. These tests were not triggered by standard diagnostic pathways at initial presentation. Two novel translocations of uncertain significance were detected - RAD51B:BIRC3 and IGHMswitch-MICALL2/INTS1. Clonal IGHV-IGHD-IGHJ rearrangements were detected by NGS and/or Sanger sequencing with predominantly IGHV3 and IGHV4 gene usage, IGHV4-34 being the most common with 15 cases. The majority of rearrangements showed somatic hypermutations above 2% and none could be assigned to the 19 major CLL-associated stereotyped subsets (using ARResT/AssignSubsets). Somatic mutations were detected in 74/108 cases. The most frequently mutated genes were TP53 in 16/108 (15%) and the hotspot MYD88 L265P mutation in 14/108 (13%) with VAF of 20-46%. Other variants detected were distributed among genes associated with NFkB signalling and chromatin remodelling (Figure 1). The MYD88 L265P mutation was present in a fifth of cases with mutations detected (14/74) and was the sole change seen in 7 cases. Accompanying mutations in the remaining cases included CD79B (2), POT1 (1), NFKBIE (1). No concomitant CXCR4 mutations were detected. Median age of the MYD88 L265P cohort was 68 years with male predominance (M:F=10:4). 9/14 presented with an incidental lymphocytosis and median count of 11.18 x 109/L. Lymphadenopathy was present in 3/14 (21%) while 7/14 (50%) had splenomegaly. A paraprotein was detected in 6/14 cases (4 IgM, 1 IgG, 1 IgA). CD5 was expressed in 6/14 cases by flow cytometry. CD19 and 20 were uniformly positive, CD79b was variable and 12/14 cases tested did not express ROR1. Conclusion: This prospective study outlines the value of a targeted NGS panel in enhancing the diagnosis of unclassifiable LI B-NHL thereby improving patient access to novel therapies and clinical trials. It highlights recurrent MYD88 mutations in a subset of patients that have splenomegaly as a frequent feature. 5 year clinical follow up data, currently being gathered in both the MYD88 mutated and overall cohort, will be valuable in further characterisation and risk stratification to inform management of these patients. Disclosures Furtado: Abbvie: Other: Conference Support. El-Sharkawi:Abbvie: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Innate: Consultancy. Iyengar:Abbvie: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Janssen: Honoraria; Beigene: Consultancy.

Clinicopathological analysis of mucosa associated lymphoid tissue lymphoma secondary to Sjögren' s syndrome in salivary gland

OBJECTIVE To analyze the clinicopathological characteristics of mucosa associated lymphoid tissue (MALT) lymphoma secondary to Sjogren' s syndrome (SS) (SS-MALT lymphoma) in salivary gland and to explore the value of the combined application of histopathological morphology, protein expression and molecular phenotype in pathological diagnosis and prognostic evaluation of SS-MALT lymphoma. METHODS Sixteen patients with SS-MALT lymphoma were collected from 260 patients who were diagnosed with SS in Peking University School and Hospital of Stomatology from January 1997 to December 2016. Twelve patients with non-MALT lymphoma secondary to SS (non-SS-MALT lymphoma) in salivary gland were selected as controls. The clinical data of the patients were retrospectively reviewed and analyzed. All the patients were followed up until December 20, 2019. Hematoxylin-eosin staining, immunohistochemistry, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques were used to observe the histologic characteristics and to detect the manifestations of light chain restrictive expression, immunoglobulin (Ig) gene clonal rearrangement, chromosome translocation and gene abnormality, so as to evaluate their values in pathological diagnosis and prognostic evaluation. RESULTS The malignant transformation rate of SS to MALT lymphoma was about 6.15%, ranged from 3 to 240 months, during which 2 patients died due to high-level deterioration. Microscopically, the acini of the glandular tissue were atrophied and destroyed. The tumor cells dominated by central cell-like lymphocytes grew diffusely, destroying the epithelial islands. All SS-MALT lymphoma cases were positive in CD20 and Pax5. Half of them had the Ki-67 proliferation index of 10% or less, and half greater than 10%. 93.75% cases expressed AE1/AE3 protein, which showed the residual glandular epithelium. All the tumor cells were negative in CD3e, and the plasma cells were detected by CD138 antigen. The light chain restrictive expression of κ and λ was 37.5% in SS-MALT lymphoma group. The positive detection rates of immunoglobulin heavy chain (IgH)-FR1, IgH-FR2, IgH-FR3, immunoglobulin kappa chain (IgK)-A, and IgK-B in SS-MALT lymphoma group were 33.3%, 53.3%, 33.3%, 20.0%, and 26.7%, respectively, and 93.3% when together used with IgH and IgK. The positive rates of the MALT1, IGH and BCL6 genes with dual color break-apart probes were 36.4%, 27.3% and 27.3%, and the detection rate of chromosome translocation and gene abnormality by applying the three probes was 72.7%. CONCLUSION There are no specific histological characteristics and protein phenotypes in the histologic diagnosis of SS-MALT lymphoma in salivary gland. The combined application of histopathological manifestations, immunohistochemistry, PCR and FISH techniques helps the accurate pathologic diagnosis of the disease. Although SS-MALT lymphoma is considered as an indolent lymphoma with a relatively favorable prognosis, the regular return visit and long-term follow-up should be conducted to detect the clues of recurrence and advanced deterioration.

Spontaneous and Radiation-Induced Chromosome Aberrations in Primary Fibroblasts of Patients With Pediatric First and Second Neoplasms.

The purpose of the present study was to investigate whether former childhood cancer patients who developed a subsequent secondary primary neoplasm (SPN) are characterized by elevated spontaneous chromosomal instability or cellular and chromosomal radiation sensitivity as surrogate markers of compromised DNA repair compared to childhood cancer patients with a first primary neoplasm (FPN) only or tumor-free controls. Primary skin fibroblasts were obtained in a nested case-control study including 23 patients with a pediatric FPN, 22 matched patients with a pediatric FPN and an SPN, and 22 matched tumor-free donors. Clonogenic cell survival and cytogenetic aberrations in Giemsa-stained first metaphases were assessed after X-irradiation in G1 or on prematurely condensed chromosomes of cells irradiated and analyzed in G2. Fluorescence in situ hybridization was applied to investigate spontaneous transmissible aberrations in selected donors. No significant difference in clonogenic survival or the average yield of spontaneous or radiation-induced aberrations was found between the study populations. However, two donors with an SPN showed striking spontaneous chromosomal instability occurring as high rates of numerical and structural aberrations or non-clonal and clonal translocations. No correlation was found between radiation sensitivity and a susceptibility to a pediatric FPN or a treatment-associated SPN. Together, the results of this unique case-control study show genomic stability and normal radiation sensitivity in normal somatic cells of donors with an early and high intrinsic or therapy-associated tumor risk. These findings provide valuable information for future studies on the etiology of sporadic childhood cancer and therapy-related SPN as well as for the establishment of predictive biomarkers based on altered DNA repair processes.

Inflammatory Myofibroblastic Tumor of the Breast: A Case Report

Inflammatory myofibroblastic tumors (IMTs) of the breast are extremely uncommon lesions, usually labelled as a subgroup of inflammatory pseudotumors. They are composed of inflammatory cells and bland spindle cells without nuclear atypia. Nearly half of all IMTs of the breast include clonal translocation of the anaplastic lymphoma kinase (ALK) gene, located at the chromosome band 2p23, and generally present with a palpable lump, swelling, and pain. Herein, we present a 66-year-old female patient with pain, swelling and a palpable lump in her right breast. A 40×26 mm sized T1A hypointense and T2A hyperintense mass with slightly lobulated margins was detected at breast magnetic resonance imaging. A mass associated with an intramammary lymph node was evaluated in the upper inner quadrant of the right breast via mammography. The results were considered as BI-RADS 3. Breast-conserving surgery with sentinel lymph node biopsy was performed. During pathological evaluation, cytoplasm with poorly-defined margins, and large-nucleoli tumor cells with benign ducts between these tumor cells, were observed. Intensive inflammatory cell infiltration and sclerotic changes in different areas were also noted. The lesion stained positive for caldesmon, smooth muscle actin, vimentin, CD10, and S100; however, it was negative for ALK on immunohistochemistry. The patient remained disease-free after the surgical procedure.International Journal of Human and Health Sciences Vol. 04 No. 04 October’20 Page : 305-308

Detection of Simple, Complex, and Clonal Chromosome Translocations Induced by Internal Radioiodine Exposure: A Cytogenetic Follow-Up Case Study after 25 Years

Here, we report the findings of a 25-year cytogenetic follow-up study on a male patient who received 2 rounds of radioiodine treatment within a span of 26 months (1.78 GBq in 1992 and 14.5 GBq in 1994). The patient was 34 years old with a body mass index of 25 at the time of the first radioiodine treatment. Multicolor FISH and multicolor banding (mBAND) techniques performed on the patient detected inter- and intrachromosomal exchanges. Although the frequency of chromosome translocations remained essentially the same as reported in our earlier study (0.09/cell), the percentage of reciprocal (balanced) translocations increased from 54.38 to 80.30% in the current study. In addition to simple chromosome translocations, complex exchanges (0.29%) involving more than 2 chromosomes were detected for the first time in this patient. Strikingly, a clonal translocation involving chromosomes 14 and 15, t(14p;15q), was found in 7 of the 677 cells examined (1.03%). The presence of complex and clonal translocations indicates the onset of chromosomal instability induced by internal radioiodine exposure. mBAND analysis using probes specific for chromosomes 1, 2, 4, 5, and 10 revealed 5 inversions in a total of 717 cells (0.69%), and this inversion frequency is several-fold higher than the baseline frequency reported in healthy individuals using the classical G-banding technique. Collectively, our study suggests that stable chromosome aberrations such as translocations and inversions can be useful not only for retrospective biodosimetry but also for long-term monitoring of chromosomal instability caused by past radioiodine exposure.

Factors and Clinical Scenarios Possibly Related to Endometrial Micropolyps and Chronic Endometritis

Objectives: To study factors and clinical scenarios possibly related to the development of endometrial micropolyps, hence chronic endometritis. Materials and methods: 197 patients with abnormal uterine bleeding, infertility or recurrent miscarriages were studied by early follicular phase transvaginal ultrasound scan examinations and hysteroscopy. Presence of endometrial micropolyps was assessed in relation to sexual intercourse frequency, regular vaginal washing, parity, previous pelvic infection and previous uterine surgery. The likelihood of patients to use vaginal washing was examined against the other four factors. Furthermore, the association of micropolyps with other uterine pathologies was examined. Cross tabulation with chi square test, binary logistic regression analysis and odds ratio was used as appropriate to examine the data. Results: Hysteroscopic examination revealed 81 cases with micropolyps not diagnosed previously by transvaginal ultrasound scan examinations. 56 of them (69.14%) presented with abnormal uterine bleeding, 13 with infertility (16.04%) and 12 with repeated miscarriages (14.81%), p=0.012. Cross tabulation and chi square test showed micropolyps were significantly associated to parity, sexual intercourse frequency, previous pelvic infection and regular vaginal washing, but not previous uterine surgery. Furthermore, binary logistic regression analysis showed vaginal washing was the most significant factor in this group. It also showed that micropolyps were significantly associated to the presence of endometrial polyps (p=0.022) and symptomatic patients with negative ultrasound findings (p=0.049). Patients who had more frequent sexual intercourse showed high odds ratio to use regular vaginal washing, but not the other three groups. Conclusion: Women should be advised against vaginal washing, especially those at risk of chronic endometritis. Micropolyps might be precursors to endometrial polyps because of the close association between the two. Chronic endometritis might trigger clonal rearrangements and translocations between the four stromal chromosomes already suspected of initiating endometrial polyps’ development.

Deciphering the Chronology of Copy Number Alterations in Multiple Myeloma (MM): What Comes First?

Deciphering the Chronology of Copy Number Alterations in Multiple Myeloma (MM): What Comes First?

RARE-23. PRIMARY INTRA-AXIAL CENTRAL NERVOUS SYSTEM INFLAMMATORY MYOFIBROBLASTIC TUMOR, ALK NEGATIVE: A RARE ENTITY

Inflammatory myofibroblastic tumor (IMT) is a rare spindle cell neoplasm with admixed inflammatory cells which occurs mainly in the abdomen or thoracic cavity of children or young adults. Primary CNS IMT is exceedingly rare with roughly 100 reported cases in the world literature, most of which are extra-axial and occur as a dural-based mass. Herein, we describe a rare case of intra-axial primary CNS IMT. A 24 year-old healthy female presented to the ER after falling and striking her head. A CT scan revealed an acute intraparenchymal hemorrhage, as well as a mass within the right frontoparietal lobe. Subsequent MRI was performed further characterizing the lesion as a 3.0 cm intra-axial tumor. Craniotomy was performed displaying a circumscribed neoplasm with relatively bland spindle cells arranged in fascicles with an admixed lymphoplasmacytic infiltrate. Mitotic activity was present but limited. Immunohistochemistry (IHC) was positive for TLE1 and vimentin but negative for GFAP, ALK, SMA, MUC4, KIT and β-catenin. Additional molecular testing by FISH for ALK (2p23) rearrangement was negative. We report a rare case of intra-axial primary CNS ALK Negative IMT. Approximately half of all IMTs harbor a clonal translocation that activates the anaplastic lymphoma kinase (ALK)-receptor tyrosine kinase at the 2p23 locus. As a result, ALK is overexpressed and can be detected by IHC or via molecular diagnostics (FISH, RNA sequencing or RT-PCR). Since this case was processed, additional novel anomalies involving rearrangements in ROS1, RET, ETV6 and/or NTRK3 genes have been described and could lead to promising therapeutic targets in the future.

The Role for the DSB Response Pathway in Regulating Chromosome Translocations.

In response to DNA double strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that coordinate their detection, signaling and repair. Central to this network is the ATM kinase and its substrates at chromatin surrounding DSBs H2AX, MDC1 and 53BP1. In humans, germline inactivation of ATM causes Ataxia Telangiectasia (A-T), an autosomal recessive syndrome of increased proneness to hematological malignancies driven by clonal chromosomal translocations. Studies of cancers arising in A-T patients and in genetically engineered mouse models (GEMM) deficient for ATM and its substrates have revealed complex, multilayered roles for ATM in translocation suppression and identified functional redundancies between ATM and its substrates in this context. "Programmed" DSBs at antigen receptor loci in developing lymphocytes employ ubiquitous DDR factors for signaling and repair and have been particularly useful for mechanistic studies because they are region-specific and can be monitored in vitro and in vivo. In this context, murine thymocytes deficient for ATM recapitulate the molecular events that lead to transformation in T cells from A-T patients and provide a widely used model to study the mechanisms that suppress RAG recombinase-dependent translocations. Similarly, analyses of the fate of Activation induced Cytidine Deaminase (AID)-dependent DSBs during mature B cell Class Switch Recombination (CSR) have defined the genetic requirements for end-joining and translocation suppression in this setting. Moreover, a unique role for 53BP1 in the promotion of synapsis of distant DSBs has emerged from these studies.

Novel Clonal t(2;4) (q23;p14) Secondary Cytogenetic Abnormality in a Primary Myxoid Liposarcoma

Myxoid liposarcomas are malignant lipomatous tumors with a predilection for young adults. They are characterized by the presence of reciprocal translocation between the CHOP (DDIT3) gene on chromosome 12 and the FUS gene on chromosome 16, t(12;16)(q13;p11.2) in >95% of cases, or less commonly, a translocation between the DDIT3 and EWSR1 genes, t(12;22)(q13;q12). Secondary aberrations involving trisomy 8 and chromosomes 1 and 16 have been reported. Herein, we report for the first time a novel secondary clonal translocation, t(2;4) (q23;p14) in addition to t(12;16)(q13; p11.2) in a 30-year-old woman with myxoid liposarcoma on the left posterior thigh region without any prior chemoradiation therapy. The significance of this translocation remains to be established.

Does clonal resource translocation relate to invasiveness of Typha taxa? Results from a common garden experiment

Plant species traits such as leaf and seed characteristics are increasingly used to understand drivers of species dominance and invasiveness. Because many plants are clonal, traits that influence the connectivity of clonal ramets might also be important in community assembly, species dominance, and invasion. We investigated the resource translocation (both amount of translocation and translocation distance) in three different native and non-native cattail (Typha) taxa that differ in their invasiveness by varying the initial amount of maternal resources available to clonal fragments and measuring their growth response in a field experiment. We found the non-native invasive taxa Typha angustifolia and Typha x glauca responded most strongly to increased maternal resource availability, and grew significantly more than the native Typha latifolia. T. angustifolia translocated resources to the largest number of daughter ramets, while T. latifolia translocated resources to the fewest and their hybrid (T. x glauca) was intermediate. These results are consistent with the hypothesis that resource translocation confers advantages for invasion and that the invasiveness of Typha taxa is related to clonal traits. Future studies and meta-analyses relating invasiveness to plant traits should consider clonal traits in addition to leaf and seed traits.

Tcrδ translocations that delete the Bcl11b haploinsufficient tumor suppressor gene promote atm-deficient T cell acute lymphoblastic leukemia

ATM is the master regulator of the cellular response to DNA double strand breaks (DSBs). Deficiency of ATM predisposes humans and mice to αβ T lymphoid cancers with clonal translocations between the T cell receptor (TCR) α/δ locus and a 450 kb region of synteny on human chromosome 14 and mouse chromosome 12. While these translocations target and activate the TCL1 oncogene at 14q32 to cause T cell pro-lymphocytic leukemia (T-PLL), the TCRα/δ;14q32 translocations in ATM-deficient T cell acute lymphoblastic leukemia (T-ALL) have not been characterized and their role in cancer pathogenesis remains unknown. The corresponding lesion in Atm-deficient mouse T-ALLs is a chromosome t(12;14) translocation with Tcrδ genes fused to sequences on chromosome 12; although these translocations do not activate Tcl1, they delete the Bcl11b haploinsufficient tumor suppressor gene. To assess whether Tcrδ translocations that inactivate one copy of Bcl11b promote transformation of Atm-deficient cells, we analyzed Atm−/− mice with mono-allelic Bcl11b deletion initiating in thymocytes concomitant with Tcrδ recombination. Inactivation of one Bcl11b copy had no effect on the predisposition of Atm−/− mice to clonal T-ALLs. Yet, none of these T-ALLs had a clonal chromosome t(12;14) translocation that deleted Bcl11b indicating that Tcrδ translocations that inactivate a copy of Bcl11b promote transformation of Atm-deficient thymocytes. Our data demonstrate that antigen receptor locus translocations can cause cancer by deleting a tumor suppressor gene. We discuss the implications of these findings for the etiology and therapy of T-ALLs associated with ATM deficiency and TCRα/δ translocations targeting the 14q32 cytogenetic region.

Somatic inactivation of Tp53 in hematopoietic stem cells or thymocytes predisposes mice to thymic lymphomas with clonal translocations

TP53 protects cells from transformation by responding to stresses including aneuploidy and DNA double-strand breaks (DSBs). TP53 induces apoptosis of lymphocytes with persistent DSBs at antigen receptor loci and other genomic loci to prevent these lesions from generating oncogenic translocations. Despite this critical function of TP53, germline Tp53−/− mice succumb to immature T-cell (thymic) lymphomas that exhibit aneuploidy and lack clonal translocations. However, Tp53−/− mice occasionally develop B lineage lymphomas and Tp53 deletion in pro-B cells causes lymphomas with oncogenic immunoglobulin (Ig) locus translocations. In addition, human lymphoid cancers with somatic TP53 inactivation often harbor oncogenic IG or T-cell receptor (TCR) locus translocations. To determine whether somatic Tp53 inactivation unmasks translocations or alters the frequency of B lineage tumors in mice, we generated and analyzed mice with conditional Tp53 deletion initiating in hematopoietic stem cells (HSCs) or in lineage-committed thymocytes. Median tumor-free survival of each strain was similar to the lifespan of Tp53−/− mice. Mice with HSC deletion of Tp53 predominantly succumbed to thymic lymphomas with clonal translocations not involving Tcr loci; however, these mice occasionally developed mature B-cell lymphomas that harbored clonal Ig translocations. Deletion of Tp53 in thymocytes caused thymic lymphomas with aneuploidy and/or clonal translocations, including oncogenic Tcr locus translocations. Our data demonstrate that the developmental stage of Tp53 inactivation affects karyotypes of lymphoid malignancies in mice where somatic deletion of Tp53 initiating in thymocytes is sufficient to cause thymic lymphomas with oncogenic translocations.

Abstract 1776: Lymphomas associated with aberrant DNA rearrangements are suppressed by Mre11 mutation.

Abstract Many translocations associated with spontaneous human lymphoid malignancies are mediated by short regions of sequence homology (microhomology) at the breakpoints. The hallmark features of this error prone pathway are deletions of flanking sequence and short homologies of 1-25 bases at the junctions that serve to align the DNA ends prior to joining. In addition to its importance in the etiology of human cancers, microhomology-mediated joining or alternative NHEJ (aNHEJ) also appears to play a significant role in therapy-induced oncogenic translocations. Studies have provided compelling evidence that the DNA end-binding complex composed of Mre11 (nuclease), Rad50 (tetherer) and Nbs1 (signaler)–MRN–functions in the context of the aNHEJ pathway. In addition, in vivo and cell based studies have identified that deficiencies in components of the MRN complex reduce aNHEJ activity. However, it is not known whether the MRN complex participates in the generation of aNHEJ catalyzed translocations that can predispose to cancer. Therefore, our studies seek to better understand the contribution of Mre11/MRN to the aNHEJ pathway and thus, in generation of translocations. We have obtained mice harboring an Mre11 conditionally targeted null allele as well as an Mre11 knock-in mutation that abrogates nuclease activity. We have included mutant mice in this study that succumb to early onset Pro-B lymphomas associated with clonal translocations containing microhomologies of 2-8 nucleotides. Thus, the spontaneously arising tumors in this mutant background represent a valuable in vivo system to assess the involvement of Mre11/MRN in aNHEJ mediated translocations. We have determined that Mre11/MRN inactivation in the B-cell lineage suppresses Pro-B lymphoma in this mutant background. In future experiments we are investigating the impact of Mre11/MRN inactivation on various cancer phenotypes. Together, these studies will provide insights into the mechanisms underlying human lymphoma and may ultimately provide insights into targets for improved treatment of lymphoid malignancies. Citation Format: Cheryl J. Smith, Jeffrey Xie, David O. Ferguson, JoAnn M. Sekiguchi. Lymphomas associated with aberrant DNA rearrangements are suppressed by Mre11 mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1776. doi:10.1158/1538-7445.AM2013-1776

Conditional inactivation of p53 in mature B cells promotes generation of nongerminal center-derived B-cell lymphomas

The p53 tumor suppressor exerts a central role in protecting cells from oncogenic transformation. Accordingly, the p53 gene is mutated in a large number of human cancers. In mice, germ-line inactivation of p53 confers strong predisposition to development of different types of malignancies, but the early onset of thymic lymphomas in the majority of the animals prevents detailed studies of tumorigenesis in other tissues. Here, we use the Cre/Lox approach to inactivate p53 in mature B cells in mice (referred to as "CP" B cells) and find that such p53 inactivation results in the routine development of IgM-positive CP peripheral B-cell lymphomas. The CP lymphomas generally appear to arise, even in mice subjected to immunization protocols to activate germinal center reaction, from naive B cells that had not undergone immunoglobulin (Ig) heavy chain gene class switching or somatic hypermutation. In contrast to thymic lymphomas that arise in p53-deficient mice, which generally lack clonal translocations, nearly all analyzed CP B-cell tumors carried clonal translocations. However, in contrast to spontaneous translocations in other mouse B-cell tumor models, CP B-cell tumor translocations were not recurrent and did not involve Ig loci. Therefore, CP tumors might provide models for human lymphomas lacking Ig translocations, such as splenic marginal zone B-cell lymphoma or Waldenstrom macroglobulinemia. Our studies indicate that deletion of p53 is sufficient to trigger transformation of mature B cells and support the notion that p53 deficiency may allow accumulation of oncogenic translocations in B cells.

ATM and 53BP1 Function in Nonoverlapping Pathways to Suppress T Lineage Lymphomagenesis.

Abstract 2374 Background:In response to DNA double-strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that promotes their detection, signaling and repair. Among DDR factors, the Ataxia-Telangiectasia Mutated (ATM) kinase and the 53BP1 adaptor localize to chromatin surrounding DSBs, where 53BP1 is thought to transduce a subset of ATM-dependent reactions. In addition, 53BP1 may have ATM-independent functions via its interactions with the MRE11/RAD50/NBS1 (MRN) complex and others to protect DNA ends from degradation and to promote synapsis of distant DSBs. Both ATM and 53BP1 are tumor suppressors in humans and mice, but whether the combined mutation may aggravate the DSB repair defect and/or accelerate oncogenesis in vivo is currently unknown. Objectives:1) To generate a compound mouse model deficient for both ATM and 53BP1 and investigate their genetic interaction during growth and development; 2) To determine whether 53BP1 plays ATM-independent functions in DSB repair and cell cycle checkpoint activation in primary thymocytes; 3) To determine the effect of 53BP1 deficiency on the penetrance and latency of T cell lymphomas in ATM−/− mice. Design/Methods:We have generated 53BP1/ATM double knock out (DKO) mice and assessed the kinetics of DSB signaling and repair in primary thymocytes and mature B cells. We have also followed cohorts of DKO and control mice for tumor development and evaluated mechanisms of tumorigenesis using a combination of molecular, biochemical and cytogenetic approaches. Results:When compared to ATM−/− mice, DKO mice showed no differences in organismal growth and development, or in lymphoid differentiation. However, DKO mice developed T cell lymphomas with higher penetrance and at younger age than ATM−/− mice, and their survival was markedly reduced (p=0.0001; log-rank test). Like ATM−/− lymphomas, DKO lymphomas contained clonal translocations with breakpoints at the TCRα/δ locus and amplification of upstream DNA sequences, indicating that 53BP1 deficiency markedly accelerates the acquisition of oncogenic translocations in an ATM−/− background. In primary cells, loss of 53BP1 further increases the frequency of ionizing irradiation (IR)-induced chromosomal aberrations in ATM−/− cells. We will present a model for how 53BP1 actions at DSBs suppress genomic instability in ATM-deficient thymocytes, including its effects on checkpoint activation and DSB repair. Conclusion:Our studies in a mouse model of T cell lymphomagenesis suggest that mutations in 53BP1 and ATM may be nonmutually exclusive in human lymphomas. The combined mutation may define a subgroup of patients with more aggressive hematological malignancies, and may have implications for their response to therapy with DNA damaging agents. Disclosures:No relevant conflicts of interest to declare.

Deletion of Atm in the Hematopoetic Stem Cell As a Mouse Model for Human T Cell Acute Lymphoblastic Leukemia/Lymphoma.

Abstract 2418Acute lymphoblastic leukemia (ALL) is diagnosed in approximately 2500 children per year. Although high cure rates have been achieved for ALL, these cancers account for the highest number of non-brain tumor cancer-related deaths in children. T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature TCRβ−CD4+/CD8+ T-cells that represents ∼15% of pediatric ALL diagnoses, comprises most of the therapy-resistant ALL tumors, and exhibits a high frequency of relapse.The Ataxia Telangiectasia mutated (ATM) protein kinase activates the cellular response to DNA double strand breaks (DSBs) to coordinate DNA repair with cell survival, proliferation, and differentiation. Somatic inactivating ATM mutations occur in 10–20% of T-ALL and T cell lymphoblastic lymphoma (T-LL) tumors and are associated with resistance to genotoxic chemotherapy drugs and therapy relapse, likely driven by increased genomic instability in cells lacking functional ATM. The impaired DSB response of ATM-deficient cells can be exploited to design combinations of genotoxic drugs that specifically kill these cells in vitro. However, the in vivo potential of such drug combinations to treat T-ALL have not been reported.We sought to develop a pre-clinical mouse model that could be used to test effectiveness of such drug combinations to treat T-ALLs and T-LLs with somatic ATM inactivation. Although germline ATM-deficient (Atm−/−) mice succumb by six months of age to immature CD4+/CD8+ T-cell lymphomas containing genomic instability analogous to human T-ALL tumors, we sought a more physiologic model that would avoid potential complications due to ATM-deficiency in thymic epithelial cells. Thus, we generated and characterized VavCre:Atmflox/flox mice with conditional Atm inactivation restricted to hematopoietic cell lineages. These mice contain reduced numbers of TCRβ−CD4+/CD8+, TCRβ+CD4+/CD8−, and TCRβ+CD4−/CD8+ thymocytes and of TCRβ+CD4+ and TCRb+CD8+ splenic T-cells, mirroring the phenotype of Atm−/− mice. We have found that VavCre:Atmflox/flox mice succumb at an average of 95 days (range 53–183 days) to clonal TCRβ−CD4+/CD8+ or TCRβ+CD4−/CD8+ thymic lymphomas. Evaluation of the bone marrow in a subset of these mice indicates that the lymphoma has disseminated and are classified as leukemia. Our initial cytogenetic analyses of these tumors indicate that they contain both clonal translocations involving chromosome 12 and/or chromosome 14 and deletion of one allelic copy of the haploinsufficient Bcl11b tumor suppressor gene. Hemizygous BCL11B inactivation occurs in ∼20% of human T-ALL tumors, indicating the clinical relevance of VavCre:Atmflox/flox mice as a model for human T-ALL. Our ongoing studies include complete cytogenetic and molecular characterization of VavCre:Atmflox/flox tumors and in vivo testing of chemotherapeutics targeting the Atm pathway in this mouse model of T-ALL/T-LL. Disclosures:No relevant conflicts of interest to declare.

Redundant and Nonredundant Functions of ATM and H2AX in αβ T-Lineage Lymphocytes

The ataxia telangiectasia mutated (ATM) kinase and H2AX histone tumor suppressor proteins are each critical for maintenance of cellular genomic stability and suppression of lymphomas harboring clonal translocations. ATM is the predominant kinase that phosphorylates H2AX in chromatin around DNA double-strand breaks, including along lymphocyte Ag receptor loci cleaved during V(D)J recombination. However, combined germline inactivation of Atm and H2ax in mice causes early embryonic lethality associated with substantial cellular genomic instability, indicating that ATM and H2AX exhibit nonredundant functions in embryonic cells. To evaluate potential nonredundant roles of ATM and H2AX in somatic cells, we generated and analyzed Atm-deficient mice with conditional deletion of H2ax in αβ T-lineage lymphocytes. Combined Atm/H2ax inactivation starting in early-stage CD4(-)/CD8(-) thymocytes resulted in lower numbers of later-stage CD4(+)/CD8(+) thymocytes, but led to no discernible V(D)J recombination defect in G1 phase cells beyond that observed in Atm-deficient cells. H2ax deletion in Atm-deficient thymocytes also did not affect the incidence or mortality of mice from thymic lymphomas with clonal chromosome 14 (TCRα/δ) translocations. Yet, in vitro-stimulated Atm/H2ax-deficient splenic αβ T cells exhibited a higher frequency of genomic instability, including radial chromosome translocations and TCRβ translocations, compared with cells lacking Atm or H2ax. Collectively, our data demonstrate that both redundant and nonredundant functions of ATM and H2AX are required for normal recombination of TCR loci, proliferative expansion of developing thymocytes, and maintenance of genomic stability in cycling αβ T-lineage cells.