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Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell–specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell–specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor–related signaling molecules, including CD3ϵ, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALKK210R, downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling. [Cancer Res 2009;69(22):8611–9]

Supplementary Figure 5 from NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

Supplementary Figure 3 from NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

Supplementary Figure 1 from NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

Supplementary Figure 4 from NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

Supplementary Figure Legends 1-5 from NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

Kaposiform hemangioendothelioma (KHE) is a rare locally aggressive mixed vascular tumor, with typical onset in early childhood and characterized by progressive angio- and lymphangiogenesis. Its etiopathogenesis and molecular bases are still unclear. Here, we report the first case of congenital KHE harboring a PIK3CA mosaic pathogenic variant (c.323G > A, p.Arg108His) in a boy with very subtle PIK3CA-related overgrowth spectrum (PROS) features. This finding provides insights into the pathophysiology of KHE, offering targeted therapeutic options by inhibition of the PI3K/Akt/mTOR pathway. We propose the inclusion of this mixed lymphatic and vascular anomaly within the PROS.

AimsNovel prognostic markers are warranted for gastrointestinal stromal tumours. Caveolin-1 is a multifunctional protein that proved to be associated with outcome in multiple tumour types. Aim of this study was to investigate Caveolin-1 expression and prognostic efficacy in a series of gastrointestinal stromal tumours.MethodsCaveolin-1 expression was assessed by immunohistochemistry in a retrospective series of 66 gastrointestinal stromal tumours representative of the different molecular subtypes. Correlations with clinical, histopathological and molecular features were investigated. Statistical analyses were performed as appropriate.ResultsThirty-five cases out of 66 (53.0%) expressed Caveolin-1. Presence of Caveolin-1 expression correlated with favourable histopathologic and clinical traits, including a lower mitotic count (p=0.003) and lower relapse rate (p=0.005). Caveolin-1 expression also resulted associated with the presence ofPDGFRAmutations (p=0.010). Outcome analyses showed a favourable prognostic significance of Caveolin-1 expression in terms of relapse-free survival (HR=0.14; 95% CI=0.03 to 0.63) and overall survival (HR=0.29; 95% CI=0.11 to 0.74), even after adjusting for the mutational subgroup (relapse-free survival: HR=0.14, 95% CI=0.04 to 0.44; overall survival: HR=0.29, 95% CI=0.11 to 0.51) and imatinib treatment (relapse-free survival: HR=0.14, 95% CI=0.02 to 0.81; overall survival: HR=0.29, 95% CI=0.17 to 0.48).ConclusionCaveolin-1 represents a novel prognostic marker in gastrointestinal stromal tumours. Further studies are warranted to validate these results and to explore the mechanisms linking Caveolin-1 expression with thePDGFRAoncogenic pathway.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified.

One of the most serious complications of advanced melanoma is the diffusion of cancer cells to the central nervous system. The diagnosis of leptomeningeal metastasis (LMM) is notoriously challenging and requires a combination of consistent MRI and cerebrospinal fluid (CSF) cytology. In ambiguous cases, mutations like BRAF V600E in CSF-cell-free (cf)DNA may help to clarify diagnosis of LMM. Here we present the case of a young woman who developed isolated LMM after the diagnosis of a node-positive primary melanoma with normal LDH. The CSF was negative for tumour cells by cytology but positive for cfDNA BRAF V600E mutation, thus allowing us to diagnose LMM. To our knowledge, this is the first case where CSF sampling for the detection of BRAF mutation was used to identify leptomeningeal disease in the presence of negative MRI and without involvement of any other distant sites.

Introduction Chronic myeloproliferative neoplasm (MPNs) are clonal malignant bone marrow (BM) diseases, arising from a hematopoietic stem cell. All therapies for these neoplasms have peculiar effects on the bone marrow, but little evidence has been described in the literature.Areas covered This review examines BM morphological changes following the main treatments in Philadelphia-negative MPNs. Hydroxyurea can reduce the cellularity of the erythroid and megakaryocyte lineages but has minimal impact on fibrotic evolution. There is general agreement on its dysplastic effects, with a high incidence of acute myeloid leukemia and myelodysplastic syndrome. Interferon treatment can reduce or normalize BM cellularity, improve erythropoiesis, and reduce the number and atypicality of megakaryocytes. Most data describe reduction or complete resolution of marrow fibrosis; dysplastic effects are not reported. Anagrelide may induce an increase in the number of BM megakaryocytes, especially immature megakaryocytes or precursors, and a worsening of marrow fibrosis or increased transformation of essential thrombocythemia into myelofibrosis. Ruxolitinib can improve or stabilize BM fibrosis and reduces the frequency and dense clustering of megakaryocytes.Expert opinion Since previous therapy can modify BM features, it is essential to obtain information on previous or current therapies and to collect complete clinical information.

Novel prognostic markers are warranted for gastrointestinal stromal tumours. Caveolin-1 is a multifunctional protein that proved to be associated with outcome in multiple tumour types. Aim of this study was to investigate Caveolin-1 expression and prognostic efficacy in a series of gastrointestinal stromal tumours.Caveolin-1 expression was assessed by immunohistochemistry in a retrospective series of 66 gastrointestinal stromal tumours representative of the different molecular subtypes. Correlations with clinical, histopathological and molecular features were investigated. Statistical analyses were performed as appropriate.Thirty-five cases out of 66 (53.0%) expressed Caveolin-1. Presence of Caveolin-1 expression correlated with favourable histopathologic and clinical traits, including a lower mitotic count (p=0.003) and lower relapse rate (p=0.005). Caveolin-1 expression also resulted associated with the presence ofCaveolin-1 represents a novel prognostic marker in gastrointestinal stromal tumours. Further studies are warranted to validate these results and to explore the mechanisms linking Caveolin-1 expression with the

The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.

Introduction: Mutations in the JAK2, CALR, and MPL genes have been shown to have prognostic value in essential thrombocythaemia (ET), but no clear association with morphological changes has been reported so far. We investigated the possible correlation between gene mutations and histopathological features in bone marrow (BM) biopsies of patients with ET. Methods: Marrow cellularity, fibrosis, and the number of total and dysmorphic megakaryocytes and clusters of megakaryocytes were compared to gene mutations in 90 cases of ET at diagnosis. Results: The JAK2V617F mutation was found in 58.9%, CALR in 28.9%, and MPL in 4.4% of the cases, and 7.8% were triple-negative. JAK2V617F-mutated ET showed a high BM cellularity, the lowest number of clusters of megakaryocytes and the highest number of dysmorphic megakaryocytes; CALR-mutated ET showed a reduced BM cellularity, many clusters of large megakaryocytes, and very few dysmorphic megakaryocytes; MPL-mutated ET showed the lowest BM cellularity, the highest number of clustered and large megakaryocytes, and the lowest number of dysmorphic megakaryocytes. Triple-negative ET cases had the highest BM cellularity. Conclusions: Distinct morphological patterns were associated with gene mutations in ET, supporting the classification of ET into different subtypes.

Background: Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies. Methods: We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs. Results: By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12). Conclusions: mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies

Background Technical biases due to PCR artefacts could represent an insidious obstacle for mutational analysis and precision medicine. Methods The authors report a retrospective analysis by fast COLD-PCR and sequencing of 31 suboptimal tumor DNA samples obtained from FFPE tissues and liquid biopsies. Results In FFPE tumor tissues and plasma liquid biopsies of patients with lung and colorectal adenocarcinoma, we observed a significant rate of artefactual KRAS mutations, unveiled by repeated analysis following UDG pretreatment as well as by simple repetition without UDG pretreatment step, thus suggesting a DNA damage different from cytosine deamination. UDG pretreatment was not only unnecessary to contrast artefacts occurrence, but also hampered the efficiency of mutational screening, reducing the analytical sensitivity. Taken individually or considered together, the reduced DNA input per reaction and UDG pretreatment limited the detection of 'real' mutated alleles, decreasing PCR sensitivity enough to hamper distinction between artefactual and true subclonal mutations of KRAS. Conclusions Careful validation of analytical sensitivities should always be carried out through standard controls, and strategies other than UDG pretreatment need to be identified to avoid both amplification of artefactual mutations and failure to identify real subclonal mutations.

Dear Editor, Myelodysplastic syndrome with isolated del (5q) and JAK2 mutation is a distinct entity within the World Health Organization (WHO) category myelodysplastic syndromes (MDS) [1]. JAK2 is a gain of function mutation, typically associated with myeloproliferative neoplasms (MPN) [2]. However, approximately 5 % of patients with isolated del (5q) also harbours the JAK2 mutation [3, 4]. No correlation was found between isolated del (5q) JAK2 mutated cases and clinical presentation, morphological features, disease transformation or patient outcome [4, 5], although a few cases presented with higher platelet and WBC count [3]. Lenalidomide was reported to be an effective treatment for the disease [6]. We have recently studied a patient with del (5q) and JAK2 mutation transformed to acute myeloid leukaemia (AML) with complex karyotype and increased JAK2 mutation load. A 66-year-old female presented with moderate anaemia and high platelet count (haemoglobin 100 g/l, WBC 7.2 × 10/l, platelets 800 × 10/l). Neutrophils were 4.95×10/l, eosinophils 0.21×10/l, monocytes 0.14×10/l and lymphocytes 1.9×10/l. No blast cell was present in the peripheral blood. There was no splenoor hepatomegaly. On marrow aspirate, only eight metaphases showing a normal karyotype (46,XX) could be analysed, due to the lack of growth of an adequate number of metaphases and the poor cellularity of the sample. FISH analysis was limited to the study of 5q and demonstrated 5q31 deletion in 12 % of the nuclei. Thus, additional aberrations could not be identified. BCR/ABL rearrangements (p210, p190) were absent, while JAK2 mutation displayed an allele burden of 4.28 %. Bone marrow (BM) biopsy showed a 70 % cellularity, slight dyserythropoiesis, moderate myeloid hyperplasia and marked proliferation of the megakaryocytic lineage with some dysplasia. A few megakaryocytes were large with hyperlobulated nuclei; others were medium-sized, with round and/or hypolobulated nuclei. There were 3 % CD34-positive blasts and moderate reticulin fibrosis (WHOMF-2). (Fig. 1a– c, e). A strong nuclear p53 immunostaining was detected in 2 % haematopoietic cells (Fig. 1d). We proposed a diagnosis of MDS with del (5q) and JAK2 mutation. The patient did not receive any therapy. No organomegaly nor leukoerythroblastosis was reported during the course of the disease. Twenty-seven months later, she was admitted to the hospital because of persistent fever, severe anaemia and neutropenia (Hb level 7.5 g/l, WBC 2.3×10/l, neutrophils 0.75×10/l, platelets 137×10/l). There were 5 % blast cells in the peripheral blood. Chromosome banding analysis showed the following: 46, XX [1] / 45,XX, −7, del(5)(q13q33), del(13)(q14q34) [7] / 45, idem, del(2)(p12) [2]. FISH analysis demonstrated −7, del(5)(q31), del(13)(q14) (80 %) without BCR/ABL, AML1/ETO, PML/RARA, inv(16), MLL(11q23) translocation and +8. Molecular analysis did not identify FLT3 (ITD, D835), NPM1 (exon 12) or MLL-PTD mutations. Real-time quantitative polymerase chain reaction (RQ-PCR) detected 4164 WT1 copies/10 ABL copies and 55.6 % JAK2 mutation load. p53 sequencing analysis of exons 2–11 showed * Achille Pich achille.pich@unito.it

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) is still under investigation. The aim of the present retrospective study was to assess the role of Wilms tumor gene 1 (WT1) overexpression in a large monocentric cohort of AML patients. Among 255 enrolled patients, MRD was investigated in those in complete remission (CR) with an available WT1 at baseline (>250 copies) and at two further time-points: after induction (n=117) and prior allogeneic hematopoietic cell transplantation (allo-HCT), n=65. Baseline BM WT1 overexpression was not associated with response to induction (p=0.244). Median overall survival (OS) and disease-free survival (DFS) were significantly shorter in patients with >350 WT1 copies after induction compared to those with ≤350 (HR for mortality 2.13; 95% CI 1.14-3.97, p=0.018 and HR for relapse 2.81; 95% CI 1.14-6.93, p=0.025). Patients with WT1>150 copies pre allo-HCT had a significantly higher 2-year cumulative incidence of relapse (CIR) compared to those with WT1≤150 (HR 4.61; 95% CI 1.72-12.31, p=0.002). The prognostic role of WT1 overexpression resulted independent from other well-established risk factors. According to these results, WT1 overexpression might represent an additional MRD tool for risk stratification in patients classified nowadays in CR.

We report the cellular and molecular characterization of two cases of Castleman's disease, plasma cell variant, that differed in their clinical presentation and course. Patient 1 had Castleman's disease in association with Kaposis's sarcoma unrelated to human immunodeficiency virus (HIV) infection and died while he was receiving an aggressive chemotherapeutic regimen for Kaposi's sarcoma. Patient 2 had an isolated retroperitoneal lymphoid mass with an adjacent enlarged limph nodes and his symptoms disappeared completely following the surgical removal of both. Pathologic and immunohistochemical analyses in both cases, revealed that there was a massive infiltration of polyclonal plasma cells in the interfollicular areas of the lymph nodes. Immunoglobulin gene rearrangement studies confirmed the polyclonal nature of B-lineage cells in the involved lymph nodes. Southern blot experiments failed to demonstrate the presence of EBV genome copies in the same lymph nodes. These paradigmatic cases lend further support to the notion that Castleman's disease is an extremely heterogeneous entity.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with an average survival of 4-6 months following diagnosis. Surgical resection is the only treatment with curative intent, but resectable PDAC patients are in the minority. Also, unlike other neoplasms, PDAC is resistant to conventional and targeted chemotherapy. Innovative treatments, such as immunotherapy, can be very important and the study of the immune response is fundamental. We previously demonstrated that PDAC patients show tumor-infiltrating T cells specific to α-enolase (ENO1), a glycolytic enzyme over-expressed by pancreatic tumor cells, which plays an important role in promoting cell migration and cancer metastasis. In the present study, we evaluate the functional anticancer proprieties of ENO1-specific T cells isolated from the peripheral blood of PDAC patients. Furthermore, comparing the T cell receptor repertoire of ENO1-specific peripheral and infiltrating tumor T cells from the same patient suggests that ENO1-specific T cells, despite having a different functional profile, can recirculate from the tumor to the periphery. Finally, of clinical relevance, the presence of peripheral ENO1-specific T cells has a prognostic value and significantly correlates with a longer survival.

Stacchini A, Barreca A, Demurtas A, Aliberti S, di Celle P F & Novero D (2012) Histopathology 60, 452–459 Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature Aim: To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). Methods and results: CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases. Conclusions: CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion.

Introduction: BCL6 is a key oncogene in lymphoma pathogenesis. Malignant lymphoid cells exploit several mechanisms to deregulate the expression of BCL6, including chromosomal translocations, somatic mutations in the promoter regulatory regions, or inactivation of the pathway that controls its degradation. FBXO11 was recently identified as a major ubiquitin ligase involved in the degradation of BCL6 and was found to be frequently inactivated by mutations or deletion in diffuse large B cell lymphoma (DLBCL). Thus, FBXO11 acts as an oncosuppressor in DLBCL by promoting the accumulation of BCL6. Given the prominent role of FBXO11 in regulating BCL6 stability, we searched for FBXO11 mutations in BCL6-positive lymphomas, other than DLBCL, and we investigated its role in lymphoma development in vivo. Methods: We sequenced the entire FBXO11 coding sequence by classical Sanger sequencing in 100 cases of follicular lymphoma (FL), 36 cases of Burkitt lymphoma (BL), 8 BL cell lines and 8 anaplastic large cell lymphoma cell lines, all BCL6-positive lymphomas. Moreover, we sequenced 50 cases of marginal zone B cell lymphoma (MZL), which show variable expression of BCL6. We functionally validated the FBXO11 mutations by developing mutant constructs and testing their ability to induce BCL6 and SNAIL degradation. We then applied the CRISPR/Cas9 system to disrupt the endogenous FBXO11 gene in BL cells and evaluated its effect on BCL6 stability. To dissect the in vivo role of FBXO11 in lymphomagenesis we generated conditional FBXO11 knock-out (KO) mice (FBXO11fl/fl) to delete protein expression in CD19-positive B cells. To investigate whether FBXO11 inactivation cooperates with c-myc in lymphomagenesis we crossed CD19/Cre-FBXO11fl/fl mice with Eμ-myc transgenic mice. Results: We identified FBXO11 mutations in BL cases and cell lines (10/44, 22.7%), one case of FL (1/100) and one case of MZL (1/50). In FL and MZL, the mutational analysis of tissue collected by microdissection showed that the mutation was specifically acquired by the high-grade component. The frequency of FBXO11 mutation in BL was further validated on an independent cohort of 51 BL cases (6/51, 11.7%), obtained from published data of a previous whole-exome sequencing study (Love et al. Nat Genet 2012). BL mutations were mostly missense mutations located in the functional CASH domains and also splice-site mutations that were never described before and that induced alternative splicing, as confirmed on the mRNA extracted from the tumor samples. All mutations produced a mutant FBXO11 with impaired ability to induce BCL6 and SNAIL degradation. CRISPR/Cas9 mediated KO of FBXO11 in BL cells resulted in an almost complete stabilization of BCL6, thus suggesting that FBXO11 is the main, if not unique, ubiquitin ligase that controls BCL6 stability in BL. Finally, we observed an acceleration of lymphoma development in the CD19/Cre-FBXO11fl/fl mice crossed with Eμ-myc transgenic mice. The lymphomas showed histologic features of high-grade disease with a more mature B-cell phenotype than the Eμ-myc tumors alone. Conclusions: Overall our results demonstrate that FBXO11 is frequently mutated in BL. All mutants identified impair the ability of FBXO11 to regulate the degradation of BCL6. Together with our previous findings (Duan et al. Nature 2012), this study shows that FBXO11 is mostly mutated in aggressive lymphomas such as DLBCL and BL, and suggests that FBXO11 mutations could contribute to their aggressiveness. In fact, we show that FBXO11 inactivation cooperates with c-myc in accelerating lymphomagenesis. We have established a novel murine lymphoma model that resembles more closely the human BL, providing a novel promising tool for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches with BCL6 and/or c-myc inhibitors. This abstract is also being presented as Poster 32. Citation Format: Chiara Pighi, Mara Compagno, Taek-Chin Cheong, Teresa Poggio, Qi Wang, Fernanda Langellotto, Anoop Sendamarai, Kyriacos Markianos, Paola Francia di Celle, Alberto Zamò, Roberto Chiarle. FBXO11 is recurrently mutated in Burkitt lymphoma and its inactivation accelerates lymphomagenesis in Eμ-myc mice [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR10.

Introduction Evaluation of WT1 expression is becoming an attractive marker in acute myeloid leukaemia (AML) for minimal residual disease (MRD) detection. Recent studies correlate therapy response with WT1 copy levels in the bone marrow (BM) offering an additional tool beside multiparameter flow-cytometry (MFC). No well-known data are available regarding its impact on the outcome after hematopoietic cell transplantation (HCT). Patients/Methods One hundred and two consecutive clinically and molecularly well characterized AML patients (pts) were transplanted in a single hematology center from 2004 to 2013. Indication for allogeneic HCT included high cytogenetic and molecular risk according to WHO criteria, high leukocytosis, extramedullary manifestations or secondary AML at diagnosis. Further, primary induction failure was considered as an indication for allogeneic HCT. WT1 expression was analyzed by real time polymerase chain reaction (RT-PCR), using the standardized European LeukemiaNet method on BM samples before HCT and during each follow up control. Only pts in first CR before HCT were included in the analysis. Cumulative incidence of relapse (CIR) was estimated considering death for other causes than relapse as a competing event. Univariate and multivariate Fine & Gray Regression models were used to test the association between CIR and pre-HCT BM WT1 levels. Linearity of the relationship between CIR and the WT1 level was investigated using a mathematical transformation (restricted cubic spline). Aim of this retrospective analysis was to investigate the impact of pre-HCT BM WT1 expression in first CR pts on predicting relapse after HCT. Results A BM WT1 evaluation pre-HCT was available in 89 out of 102 pts. Among them, 62 achieved a CR after induction treatment. Relapsed pts in CR after reinduction chemotherapy (n=16) or with disease persistence pre-HCT (n=2) and pts refractory to treatment (n=9) were excluded from analysis. The patient subgroup displaying a first CR before HCT had a median age at diagnosis of 49 years (range: 20-65). Patient/donor relationship involved 26 (42%) sibling, 32 (52%) matched unrelated and 4 (6%) haplo-identical donors. On the basis of standard cytogenetics, molecular biology and clinical criteria (global disease risk) pts were classified according to the following risk groups: 54 (87%) high, 5 (8%) intermediate and 3 (5%) low risk. Acute GVHD occurred in 24 (39%) whereas chronic GVHD was documented in 18 (28%) pts. Twenty-six (42%) deaths were documented after HCT, 21 (34%) due to relapse and 5 (8%) because of treatment related mortality (TRM). Pre-HCT BM WT1 expression was correlated with CIR. A cut point of 150 WT1 copies was applied according to the slope change of relapse hazard, and subsequently used for CIR analysis. Pts displaying a pre-HCT BM WT1 level > 150 copies (n=18) had a higher CIR (2-year CIR 52.5%, 95% CI: 27.1-77.8) compared to pts with a BM WT1 level ≤ 150 copies (n=44, 2-year CIR 27.7% (95% CI: 13.4-42.1). WT1 copy level > 150 showed a significantly higher risk of relapse in univariate analysis (HR 2.9, 95% CI 1.2-6.7, p=0.014). In multivariate analysis pre-HCT BM WT1 expression was confirmed as a significant independent risk factor for CIR when adjusted for patient/donor relationship, presence of GVHD, global disease risk and competitive risk of mortality due to TRM (HR 3.4, 95% CI 1.3-8.5, p=0,010), Figure. Conclusions In the present study, pre-HCT BM WT1 levels discriminated significantly for CIR in a cohort of AML in first CR. A cut off level of 150 BM WT1 copies pre-HCT had a powerful statistically significant discriminating value to predict the risk of relapse, independently from already established risk factors. The prognostic value of WT1 was confirmed also when TRM as competitive risk for mortality was added in the multivariable model. Based on these results, WT1 is a promising candidate as MRD tool. Further prospective studies are required to confirm these results. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of “CLL-biased” features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.

FBXO11 is identified as the F-box protein that normally targets BCL6 for degradation, and FBXO11 deletions or mutations that prevent this function and thus stabilize BCL6 are found in B-cell lymphomas. BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas1,2. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease3,4. In many DLBCL patients, BCL6 overexpression is achieved through translocation (∼40%) or hypermutation of its promoter (∼15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1–CUL1–F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene.

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (eg, splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.

Essential thrombocythaemia (ET) is a chronic myeloproliferative neoplasm (MPN) that primarily involves the megakaryocytic lineage.1 The JAK2V617F mutation has been detected in 50–60% of ETs, and homozygous mutation in only 5%.2 JAK2V617F mutation has been associated with a polycythaemia vera (PV)-like phenotype3 and increased risk of thrombosis,4 especially in homozygous patients.5 Increasing mutant allele load was correlated with older age, splenomegaly, microvessel symptoms and higher frequency of arterial thrombosis at diagnosis.2 Little is known on the association between JAK2V617F mutation and histological changes in bone marrow biopsy (BMB) of ET patients. One hundred and three consecutive patients with newly diagnosed ET, admitted to the Division of Haematology, S. Giovanni Hospital and University of Turin, Italy, from 2006 to 2010 were included in the study. Diagnosis of ET was performed according to WHO criteria.1 A general informed consent was obtained according to the local ethical committee guidelines. There were 56 women and 47 men; the mean age was 59.1 years (range, 25–86). Splenomegaly was detected in 38 patients (36.9%). Venous or arterial thrombosis (confirmed by ultrasound examination or CT scan and D-dimer assessment) were found in 21% and 7% of patients, respectively. …

INTRODUCTION: We recently described inactivating mutations of the FBXO11 gene in Diffuse Large B Cell Lymphoma (DLBCL). One major function of FBXO11 is to regulate BCL6 stability as well as other targets such as SNAIL. BCL6 acts as an oncogene in several human B-cell lymphomas and its expression levels can be increased by several mechanisms including chromosomal translocations, point mutations in the promoter region and reduced degradation by inactivation of FBXO11. Thus, FBXO11 acts as an oncosuppressor in DLBCL by promoting the accumulation of BCL6. However, a clear and complete picture of the distribution of FBXO11 mutations in different lymphoma subtypes is missing, and the in vivo functions of FBXO11 in normal tissue and lymphoma development are completely lacking. In the present work, we investigated the frequency and distribution of FBXO11 mutations in an extended panel of human BCL6 positive lymphoma and we functionally validated novel FBXO11 mutations for their ability to induce BCL6 degradation. METHODS: We sequenced the entire FBXO11 coding sequence by classical Sanger sequencing in 100 cases of Follicular Lymphoma (FL), 36 cases of Burkitt Lymphoma (BL), 8 BL cell lines and 8 Anaplastic Large cell lymphoma (ALCL) cell lines, which are typically BCL6-positive lymphomas. Moreover, we sequenced 50 cases of Marginal Zone B cell Lymphoma (MZL), which show variable expression of BCL6. To investigate whether the FBXO11 mutations interfere with FBXO11 activity we generated complementary DNA constructs containing these mutations. Wild-type FBXO11 or FBXO11 mutants were expressed in HEK-293T cells with constructs expressing BCL6 or SNAIL. Twenty-four hours after transfection, HEK-293T cells were treated with cycloheximide and harvested at different time points to check substrates expression and degradation by immunoblotting. RESULTS: BL carried the highest frequency of FBXO11 mutations (12/44 cases analyzed, 27%), whereas FL and MZL were very rarely affected by FBXO11 mutations (1/100 FL and 1/50 MZL). The analysis identified several mutations, either located in the coding sequence mostly in the CASH (functional) domains or located in intronic sequences controlling exon splicing. Sequence changes included missense mutations (n=8), deletions (n=2), a frameshift deletion introducing a premature stop codon (n=1) and nucleotide substitutions at consensus splice donor sites (n=2) and acceptor site (n=1). Remarkably, these last mutations represent the first report of splice site mutations affecting the FBXO11 gene. By cDNA amplification and sequencing, we demonstrated skipping of entire exons: the splice site mutations located at the donor site of exon 15 and 19 lead to the loss of the exon 15 and 19 respectively, while the splice site mutation affecting the acceptor site of exon 16 results in the deletion of the adjacent exon 16. Two of the point mutations (K631N and Y122C), affecting one case of FL and MZL respectively, were also found in the previous panel of DLBCLs (Duan et al, Nature 2012). Both cases consisted of a low grade component adjacent to an area of high grade transformation. By microdissection, we separately studied the two components of each of these cases to demonstrate that the mutation was present only in the high grade component. Finally, to validate all the mutations found in BL, we generated mutant constructs corresponding to each of them. By this approach, we showed that all the mutations identified are loss of function variants because they impaired the FBXO11 ability to promote BCL6 and SNAIL degradation. CONCLUSIONS: We identified several FBXO11 novel mutations in a large panel of BCL6-positive human lymphomas. All the mutations identified were inactivating the FBXO11 ability to induce BCL6 and SNAIL degradation. In contrast, mutations of FBXO11 are rare in low grade FL and MZL and, when present, are associated with high grade transformation. Together with our previous findings, this study showed that FBXO11 is mostly mutated in aggressive lymphomas such as DLBCL and BL, thus suggesting that FBXO11 mutations could contribute to the de-novo onset or transformation into high grade lymphoma. Disclosures No relevant conflicts of interest to declare.

Gain-of-function (GOF) mutations of Janus kinase 2 (JAK2) are frequently seen in myeloproliferative disorders (MPDs). Meanwhile, JAK3 activating substitutions have been found in a few megakaryocytic cell lines and in primary myeloid leukemia (AMKL). Here, we sought to discover novel leukemogenetic mutations in de novo acute myeloid leukemia of non-Down syndrome (N-DS) by DNA sequencing. A total of 191 normal Caucasian individuals were studied to define single nucleotide polymorphisms (SNPs) within the JH2 and JH6 domains. Although known activating substitutions were observed in rare cases of acute myeloid leukemia (AML) (V722I [2/134] or P132T [1/119]), all samples were wild-type (WT) for the oncogenic A572V (119/119). Interestingly, a novel homozygous mutation (P132A) was discovered in a patient with acute megakaryoblastic leukemia and in vivo studies demonstrated that its ectopic expression was oncogenic in a mouse xenotransplant model. This study defines a novel JAK3 mutation among patients with N-DS AML and demonstrates that normal individuals can also display germline JAK3 substitutions, previously proven to have oncogenic properties, in vitro and in vivo. The discovery of these substitutions in normal donors encourages future studies to define new risk factors among patients with MPDs.

Two cell lines were originated from the peripheral blood (PB-LAM) and bone-marrow (BM-LAM) of a patient with Burkitt-type acute lymphoblastic leukemia and AIDS. 26 and 7 clones were isolated from PB-LAM and BM-LAM respectively by limiting dilution. All of these had surface IgM lambda and the CD10 marker with low to absent CD23, CD30, CD39 and surface adhesion molecules. Furthermore, they shared the same chromosomal abnormalities (trisomy 7 and t(8;14) translocation) and the same rearrangements of immunoglobulin L and H chain and of c-myc gene loci. These features are those most frequently found in Burkitt's lymphoma (BL) cells and were different from those of the parental cell lines, which, besides cells identical to those of the malignant clones, also contained normal lymphoblastoid cells. Therefore, the cloning procedure used selected for the growth of cells with malignant features. EBV latent antigens were detected in all clones by Western blotting and their pattern of expression resembled that usually observed in BL cells. All the clones were positive for the EBV genome by Southern blotting and had monomorphic EBV-fused termini as determined by using cDNA probes specific for sequences at either end of the viral genome. However, the clones derived from PB-LAM had EBV fused termini of a different size from that of the clones derived from BM-LAM. The presence of different EBV-fused termini in otherwise monoclonal malignant cells indicate that EBV infection was possibly a late event in lymphomagenesis following rearrangement of the c-myc and the Ig gene loci.

GeneScan (GS) analysis is a highly sensitive method for the early detection of cutaneous T-cell lymphoma (CTCL) and allows the identification of clonal heterogeneity, defined as the coexistence of two or more different T-cell clones in multiple samples from the same patient. We analyzed by GS the incidence and the significance of long-lived oligoclonal expansions in multiple skin and blood samples from 24 Sézary syndrome (SS) patients, and tried to correlate them with the clinical outcome. A skin clonal heterogeneity with additional reproducible TCRgamma-gene rearrangements (TCRgamma-GRs) was detected at diagnosis in 19/24 patients, 13 of whom had a constant prevalence of pathological TCRgamma-GRs in both skin and blood (dominant clonal pattern). During follow-up, an increase in oligoclones that were present at diagnosis or the appearance of new oligoclones was observed in 10 patients; all of them achieved a clinical response to treatment with extracorporeal photochemotherapy (ECP). The TCRgamma pattern (homogeneity or heterogeneity) in the skin at diagnosis showed a relevant prognostic value, and patients with an oligoclonal pattern had a significantly longer survival than those with a homogeneous pattern. In conclusion, multiple-sample approach GS analysis allows the identification of clonal heterogeneity and could also help in identifying SS patients with a potential higher response to therapy.

Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell–specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell–specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor–related signaling molecules, including CD3ϵ, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALKK210R, downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling. [Cancer Res 2009;69(22):8611–9]

Interleukin-3 (IL-3) released by infiltrating inflammatory cells in different pathologic settings contributes to organ and tumor angiogenesis. Here we demonstrate that IL-3 expands a subset of CD45+ circulating angiogenic cells clonally derived from the hemopoietic progenitors. Moreover, CD45+ cells exposed to IL-3 acquire arterial specification and contribute to the formation of vessels in vivo. Depletion of signal transducer and activator of transcription 5 (STAT5) provides evidence that IL-3–mediated cell expansion and arterial morphogenesis rely on STAT5 activation. In addition, by means of Tie2-transgenic mice, we demonstrate that STAT5 also regulates IL-3–induced expansion and arterial specification of bonemarrow–derived CD45+ cells. Thus, our data provide the first evidence that, in inflammatory microenvironments containing IL-3, angiogenic cells derived from hemopoietic precursors can act as adult vasculogenic cells. Moreover, the characterization of the signaling pathway regulating these events provides the rationale for therapeutically targeting STAT5 in these pathologic settings.

In fourteen patients with hairy cell leukemia (HCL) the configuration of the immunoglobulin (Ig) heavy chain genes was used as a marker of clonality, to monitor the response of the neoplastic population to treatment with alpha-interferon (a-IFN). In agreement with the morphological, hematological and immunological data, twelve of them showed, after a variable length of therapy, a complete disappearance of rearranged bands in peripheral blood cells. In one patient, who was treated less intensively, the molecularly-defined neoplastic population was still present on two consecutive determinations, whilst in the last patient persistence of disease was repeatedly documented despite prolonged A-IFN treatment. Three further cases were analyzed sequentially: in two, no rearranged bands could be found at repeated determinations; the third, who was in complete remission whilst on 3 × 10(6) U of α-IFN every other day, showed recurrence of disease nine months later when on a maintenance protocol with 3 × 10(6) U/weekly. Nine bone marrow specimens were also analyzed following treatment with α-IFN. In four a monoclonally rearranged band could still be detected, while in another four, reversal of fibrosis and hemopoietic recovery wits coupled with the absence of a molecularly recognizable neoplastic clone. In the last (case, persistence of disease paralleled the findings in the peripheral blood cells. These data indicate that α-IFPJ is capable of producing a specific cytolytic effect on the leukemic population in HCL, which in some cases may lead to complete clonal remissions. Analysis at the DNA level may represent a valuable tool towards monitoring the clinical course of HCL patients and for optimal individual therapeutic scheduling.

We studied the histologic and multiparameter flow cytometry (MFC) features of 12 cases of angioimmunoblastic T-cell lymphoma (AITL), 13 of mature T-cell lymphoma, and 25 control cases of reactive lymphoid hyperplasia to evaluate the role of CD10 in the differential diagnosis of peripheral T-cell lymphomas (PTCLs). A characteristic immunophenotypic profile (CD2+/CD4+) with recurrent phenotypic aberrancies (eg, CD3 and CD7 loss) was identified in most AITL cases; MFC documented CD10 coexpression on T cells in 10 (83%). Mature T-cell lymphoma showed a more heterogeneous altered immunophenotypic pattern, and 2 cases of PTCL, unspecified, had clear evidence of aberrant CD10 expression on T cells. A small physiologic CD3+/CD4+/CD10+ T-cell population was detected by MFC in all control cases tested (range, 0.28%-4.71%), suggesting that a normal subset of peripheral CD10+ T cells exists. CD10 was a highly sensitive but incompletely specific phenotypic marker for diagnosing AITL; the differential diagnosis of PTCL, unspecified, must be related with traditional histologic features. A small number of CD10+ T cells in reactive lymph nodes suggests that this subpopulation may be the normal counterpart of neoplastic T cells in AITL. The biologic role of CD10+ T cells should be studied further.

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity.

In this study we investigated telomere restriction fragment (TRF) length in a panel of mature B-cell lymphoproliferative disorders (MBCLDs) and correlated this parameter with histology and histopathogenesis in relation to the germinal center (GC). We assessed 123 MBCLD samples containing 80% or more tumor cells. TRF length was evaluated by Southern blot analysis using a chemiluminescence-based assay. GC status was assessed through screening for stable and ongoing somatic mutations within the immunoglobulin heavy-chain genes. Median TRF length was 6170 bp (range, 1896-11 200 bp) and did not correlate with patient age or sex. TRF length was greater in diffuse large cell lymphoma, Burkitt lymphoma, and follicular lymphoma (medians: 7789 bp, 9471 bp, and 7383 bp, respectively) than in mantle cell lymphoma and chronic lymphocytic leukemia (medians: 3582 bp and 4346 bp, respectively). GC-derived MBCLDs had the longest telomeres, whereas those arising from GC-inexperienced cells had the shortest (P < 10-9). We conclude that (1) TRF length in MBCLD is highly heterogeneous; (2) GC-derived tumors have long telomeres, suggesting that minimal telomere erosion occurs during GC-derived lymphomagenesis; and (3) the short TRF lengths of GC-inexperienced MBCLDs indicates that these neoplasms are good candidates for treatment with telomerase inhibitors, a class of molecules currently the subject of extensive preclinical evaluation. (Blood. 2004;103:4644-4649)

2084 Background: Aim of this study is to describe clinical characteristics and outcome of CRC patients with CNS metastases undergone to surgical resection, and report preliminary data on the comparison between molecular aspects of primary tumor and metastasis. Methods: Thirty-two pts (17 males, 15 females) with a median age of 65 yrs (range 38-80 yrs) with brain metastases from colon (23 pts, 72%) or rectum (9 pts; 28% ) adenocarcinoma (G1 in 3 pts, 9%; G2 in 12 pts, 37%; G3 in 17 pts, 53%) underwent total surgical resection. Primary tumor TNM was: T2 in 1 pt (3%), T3 in 22 pts (69%) and T4 in 9 pts (28 %); no patient had N0 status , 10 pts had N1 lesions (31%) and 22 pts had N2 (69%) lesions; 25 pts were M0 (78%) while 7 (22%) were M1. Results: One patient had neurological symptoms as first manifestation of CRC; the other cases had metachronous metastases in the SNC after a median of 35.5 months (range 4-96). The CNS location was cerebellum in 15 pts (47 %), frontal lobe in 9 pts (28 %), spinal cord in 5 pts (16% ), parietal lobe in 2 pts (6%) and occipital lobe in 1 pt (3%). Perioperative mortality was 3%. Ten pts (31%) received post-operative whole-brain radiotherapy. Six patient (19%) had local recurrence. Primary cancers harbored a K-RAS mutation in 13 cases (41%) and a BRAF mutation in 1 case (3%). Preliminary data in 20 pts indicate a 100% mutation concordance between primary tumor and CNS metastases. Conclusions: Resectable CNS metastases from CRC adenocarcinoma were seen after a median of 3 yrs originating from T3-4 N1-2 M0-1 lesions. The preferential locations in the CNS were cerebellum and frontal lobe, being spinal metastases relatively frequent. The total resection was feasible with a good long-term local control ; the whole-brain irradiation given to one third of cases could have contributed to local control. Almost all pts died of extranervous metastases. There was a concordance of KRAS and BRAF mutation in primary and CNS lesions.

Atypical Chronic Myeloid Leukemia (aCML) is a heterogeneous disorder belonging to the group of myelodysplastic/myeloproliferative (MDS/MPN) syndromes. The molecular pathogenesis of this disease is still unclear and the outcome is poor with no improvement over the last 20 years. We applied whole exome sequencing approach in 9 aCML patient samples in order to identify possible recurrent alterations. The analysis revealed the presence of unique mutations in 70 genes with 3 cases of SETBP1 alterations. Some of the genes identified as mutated in the initial set of 9 patients (IDH2, MTA2, EPHB3, ETNK1, GATA2, IRAK4) and having a score higher than 1 in the oncogenic GeneRanker database were resequenced in a cohort of 40 aCML patients (15 with and 25 without SETBP1 mutations). With the exception of IDH2, no other gene was found mutated in any case apart from the index patient. Evaluation on a larger cohort of 70 aCML samples revealed recurrent SETBP1 mutations in 24.3% of cases (see designated abstract). To test the relationship between SETBP1 variants and mutations in oncogenes known to be involved in myeloid malignancies, mutations in ASXL1, CBL, CEBPA, DNMT3A, EED, EZH2, FLT3, IDH1/2, JAK2, JARID2, NPM1, N/KRAS, RBBP4, RUNX1, SF3B1, SUZ12, TET2 and WT1 were evaluated in a population of 61 aCML patients (14 with and 47 without SETBP1 mutations) by Sanger sequencing. Overall we identified 60 mutations in 14 genes: 28 were missense point mutations, 13 nonsense point mutations, 15 missense ins/del and 4 ins/del leading to a premature stop codon. No mutations were found in IDH1, RBBP4, NPM1, JAK2, FLT3, DNMT3A. Mutations in ASXL1 were present in 14 patients and appeared more frequent in patients with mutated SETBP1 (36% vs 19%) while the 15 TET2 mutations were more prevalent in patients with SETBP1 WT than in mutated samples(28% vs. 14%). Further associations will be presented at the meeting, although further analysis on larger cohorts of patients will be necessary to determine the significance of this differences. Additional data on epigenetic signature of aCML will clarify the role of epigenetic dysregulation in aCML and related diseases. Citation Format: Sara Redaelli, Simona Valletta, Rocco Piazza, Nils Winkelmann, Roberta Spinelli, Alessandra Pirola, Laura Antolini, Luca Mologni, Carla Donadoni, Elli Papaemmanuil, Susanne Schnittger, Kim Dong-Wook, Jacqueline Boultwood, Fabio Rossi, Giuseppe Gaipa, Greta De Martini, Paola Francia di Celle, Hyun Gyung Jang, Valeria Fantin, Graham R. Bignell, Vera Magistroni, Torsten Haferlach, Enrico Maria Pogliani, Peter Campbell, Andrew J. Chase, William J. Tapper, Nick C.P. Cross, Carlo Gambacorti Passerini. Patterns of recurrent mutations in SETBP1 mutated and wild-type atypical Chronic Myeloid Leukemia patients. [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 2993. doi:10.1158/1538-7445.AM2013-2993

Atypical Chronic Myeloid Leukemia (aCML) shares clinical and laboratory features with Chronic Myeloid Leukemia (CML), but it lacks the pathognomonic BCR-ABL1 fusion. The molecular pathogenesis of this disease has remained elusive and the outcome dismal. To investigate the molecular pathogenesis of aCML, we applied exome sequencing and RNA-SEQ to aCML, with the aim of identifying novel recurrent driver mutations. Whole-exome sequencing of 9 aCML patients(pts) revealed the presence of 70 unique mutations, including recurrent alterations of SETBP1 in 3 cases: 2 cases with a G870S and a case with a D868N alteration were found. Targeted resequencing in 70 aCMLs, 574 pts with different hematological malignancies and 344 cell lines, identified SETBP1 mutations in 17/70 aCML (24.3%; 95% CI: 16-35%), 4/30 (13%) MDS/MPN-u, 3/82 (3.6%) CMML and 0/100 MDS. aCML pts with SETBP1 mutations had higher white blood cell counts (p=0.008) and worse prognosis (p=0.01) when tested in multivariate analysis. The SETBP1 gene encodes for a predominantly nuclear protein with a predicted MW of 170kDa. Germline mutations of SETBP1 were previously described in pts affected by the Schinzel-Giedion syndrome (SGS), a rare disease characterized by bone, muscle and cardiac abnormalities and neuroepithelial neoplasms. The vast majority of aCML SETBP1 mutations (85%) was located between residues 858 and 871 and were identical to the germline changes seen in pts with SGS. This region may be critical for ubiquitin binding and for subsequent protein degradation, since the Eukaryotic Linear Motif identified a putative functional site (aa. 868-873) for beta-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase. The prediction was experimentally validated using biotinylated, phosphorylated peptides encompassing this region (aa 859-879): while the wild type (wt) peptide could efficiently bind beta-TrCP, a peptide presenting G870S was incapable of binding this subunit, indicating a possible alteration in SETBP1 protein stability caused by the mutation. In line with the known binding model of beta-TrCP, dephosphorylated peptides failed to bind beta-TrCP, therefore confirming the specificity of the interaction. In agreement with these findings, TF1 cells transduced with SETBP1 G870S showed increased SETBP1 and SET protein levels, decreased PP2A activity and increased proliferation rates when compared to cells expressing the wt SETBP1 gene. Mutated SETBP1 represents a novel oncogene specifically present in aCML and closely related diseases. These data allow for a better understanding of the molecular pathogenesis of this disease; they provide evidence that SETBP1 mutations might be a new biomarker for future diagnosis and classification of aCML and related diseases, and indicate a potential strategy to develop new treatment modalities for malignancies caused by mutated SETBP1. Citation Format: Rocco Piazza, Simona Valletta, Nils Winkelmann, Sara Redaelli, Roberta Spinelli, Alessandra Pirola, Laura Antolini, Luca Mologni, Carla Donadoni, Elli Papaemmanuil, Susanne Schnittger, Kim Dong-Wook, Jacqueline Boultwood, Fabio Rossi, Giuseppe Gaipa, Greta De Martini, Paola Francia di Celle, Hyun Gyung Jang, Valeria Fantin, Graham R. Bignell, Vera Magistroni, Torsten Haferlach, Enrico Maria Pogliani, Peter Campbell, Andrew J. Chase, William J. Tapper, Nick C.P. Cross, Carlo Gambacorti Passerini. Recurrent SETBP1 mutations in atypical chronic myeloid leukemia abrogate an ubiquitination site and dysregulate SETBP1 protein levels. [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 3176. doi:10.1158/1538-7445.AM2013-3176

The application of molecular technologies to the study of lymphoid cells has led to the characterization of several molecular events which are associated with the normal differentiation pathway (i.e., immunoglobulin (Ig) and T-cell receptor (TCR) chain gene rearrangements), and to the identification of genes that may be activated during the process of malignant lymphoid transformation (i.e., oncogenes and tumour suppressor genes). Ig and TCR studies may help in the definition of clonality and lineage affiliation, as well as in the detection of minimal residual disease. Oncogene deregulation promotes cell growth and/or rescues the tumour population from programmed cell death. Tumour suppressor gene inactivation contributes to the abnormal growth typical of neoplastic cells. These molecular lesions are characteristically associated with a defined type of lymphoid leukaemia or lymphoma and represent useful tumour markers for early diagnosis, for monitoring the course of the disease or for the detection of the transformation of lymphoid neoplasms. In this review, we will summarize the most important results obtained with the utilization of DNA analysis in the study of malignant lymphoid leukaemias and lymphomas. In addition to the important clinico-diagnostic implications, we will underline the contribution of these studies to our understanding of the biological mechanisms of lymphomagenesis.