Maria Paola Martelli

BRAF Mutations in Hairy-Cell Leukemia

BACKGROUND Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Full Haplotype-Mismatched Hematopoietic Stem-Cell Transplantation: A Phase II Study in Patients With Acute Leukemia at High Risk of Relapse

PURPOSE Establishment of hematopoietic stem-cell (HSC) transplantation from mismatched relatives is feasible for patients with acute leukemia. As our original method of graft processing was unsuitable for large-scale clinical studies, we use automated devices for CD34+ cell purification. PATIENTS AND METHODS Sixty-seven patients with acute myeloid leukemia (AML; 19 complete remission [CR] 1, 14 CR 2, nine CR > 2, 25 in relapse) and 37 with acute lymphoid leukemia (ALL; 14 CR 1, eight CR 2, two CR > 2, 13 in relapse) were conditioned with total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. Peripheral-blood progenitor cells were mobilized with recombinant human granulocyte colony-stimulating factor and depleted of T-cells using CD34+ cell immunoselection. No post-transplantation graft-versus-host disease (GvHD) prophylaxis was administered. RESULTS Primary engraftment was achieved in 94 of 101 assessable patients. Six of the seven patients who rejected the primary graft, engrafted after a second transplantation. Overall, 100 of 101 patients engrafted. Acute GvHD developed in eight of 100 patients, and chronic GvHD, in five of 70 assessable patients. Thirty-eight patients died of nonleukemic causes. Relapse occurred in nine of 66 patients receiving transplantation in remission and in 17 of 38 receiving transplantation in relapse. Median follow-up of the 40 patients who survived event-free was 22 months (range, 1 to 65 months). Event-free survival (+/- standard deviation) rate was 48% +/- 8% and 46% +/- 10%, respectively, for the 42 AML and 24 ALL patients receiving transplantation in remission. CONCLUSION Our transplantation procedure provides reliable, reproducible CD34+ cell purification, high engraftment rates, and prevention of GvHD. The mismatched-related transplant emerges as a viable, alternative source of stem cells for acute leukemia patients without matched donors and/or those who urgently need transplantation.

Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin

Acute myeloid leukemia (AML) carrying NPM1 mutations and cytoplasmic nucleophosmin (NPMc+ AML) accounts for about one-third of adult AML and shows distinct features, including a unique gene expression profile. MicroRNAs (miRNAs) are small noncoding RNAs of 19–25 nucleotides in length that have been linked to the development of cancer. Here, we investigated the role of miRNAs in the biology of NPMc+ AML. The miRNA expression was evaluated in 85 adult de novo AML patients characterized for subcellular localization/mutation status of NPM1 and FLT3 mutations using a custom microarray platform. Data were analyzed by using univariate t test within BRB tools. We identified a strong miRNA signature that distinguishes NPMc+ mutated (n = 55) from the cytoplasmic-negative (NPM1 unmutated) cases (n = 30) and includes the up-regulation of miR-10a, miR-10b, several let-7 and miR-29 family members. Many of the down-regulated miRNAs including miR-204 and miR-128a are predicted to target several HOX genes. Indeed, we confirmed that miR-204 targets HOXA10 and MEIS1, suggesting that the HOX up-regulation observed in NPMc+ AML may be due in part by loss of HOX regulators-miRNAs. FLT3-ITD+ samples were characterized by up-regulation of miR-155. Further experiments demonstrated that the up-regulation of miR-155 was independent from FLT3 signaling. Our results identify a unique miRNA signature associated with NPMc+ AML and provide evidence that support a role for miRNAs in the regulation of HOX genes in this leukemia subtype. Moreover, we found that miR-155 was strongly but independently associated with FLT3-ITD mutations.

EML4-ALK Rearrangement in Non-Small Cell Lung Cancer and Non-Tumor Lung Tissues

A fusion gene, echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK), with transforming activity has recently been identified in a subset of non-small cell lung cancer (NSCLC), but its pathogenetic, diagnostic, and therapeutic roles remain unclear. Both frequency and type of EML4-ALK transcripts were investigated by reverse transcription PCR in 120 frozen NSCLC specimens from Italy and Spain; non-neoplastic lung tissues taken far from the tumor were used as controls. In cases carrying the fusion transcript, we determined EML4-ALK gene and protein levels using fluorescence in situ hybridization, Western blotting, and immunoprecipitation. We also analyzed ALK protein levels in paraffin samples from 662 NSCLC specimens, including the 120 cases investigated in the molecular studies. EML4-ALK transcripts (variants 1 and 3) were detected in 9 of 120 NSCLC samples but were not specific for NSCLC since they were also found in non-cancerous lung tissues taken far from the tumor. Notably, no transcripts were detected in matching tumor samples from these patients. Fluorescence in situ hybridization analysis of cases expressing EML4-ALK transcripts showed that only a minority of cells harbored the EML4-ALK gene. None of these cases was found to express the EML4-ALK protein as examined by immunohistochemistry, Western blotting, and immunoprecipitation. The EML4-ALK transcript cannot be regarded as a specific diagnostic tool for NSCLC. Our results show therefore that the causal role and value of EML4-ALK as a therapeutic target remain to be defined.

Acute myeloid leukemia with mutated nucleophosmin (NPM1): is it a distinct entity?

After the discovery of NPM1-mutated acute myeloid leukemia (AML) in 2005 and its subsequent inclusion as a provisional entity in the 2008 World Health Organization classification of myeloid neoplasms, several controversial issues remained to be clarified. It was unclear whether the NPM1 mutation was a primary genetic lesion and whether additional chromosomal aberrations and multilineage dysplasia had any impact on the biologic and prognostic features of NPM1-mutated AML. Moreover, it was uncertain how to classify AML patients who were double-mutated for NPM1 and CEBPA. Recent studies have shown that: (1) the NPM1 mutant perturbs hemopoiesis in experimental models; (2) leukemic stem cells from NPM1-mutated AML patients carry the mutation; and (3) the NPM1 mutation is usually mutually exclusive of biallelic CEPBA mutations. Moreover, the biologic and clinical features of NPM1-mutated AML do not seem to be significantly influenced by concomitant chromosomal aberrations or multilineage dysplasia. Altogether, these pieces of evidence point to NPM1-mutated AML as a founder genetic event that defines a distinct leukemia entity accounting for approximately one-third of all AML.

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype.

Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.

AML with mutated NPM1 carrying a normal or aberrant karyotype show overlapping biologic, pathologic, immunophenotypic, and prognostic features

Acute myeloid leukemia (AML) with mutated NPM1 usually carries normal karyotype (NK), but it may harbor chromosomal aberrations whose significance remains unclear. We addressed this question in 631 AML patients with mutated/cytoplasmic NPM1. An abnormal karyotype (AK) was present in 93 of 631 cases (14.7%), the most frequent abnormalities being +8, +4, -Y, del(9q), +21. Chromosome aberrations in NPM1-mutated AML were similar to, but occurred less frequently than additional chromosome changes found in other AML with recurrent cytogenetic abnormalities according to WHO classification. Four of the 31 NPM1-mutated AML patients karyotyped at different time points had NK at diagnosis but AK at relapse: del(9q) (n = 2), t(2;11) (n = 1), inv(12) (n = 1). NPM1-mutated AML with NK or AK showed overlapping morphologic, immunophenotypic (CD34 negativity), and gene expression profile (down-regulation of CD34 and up-regulation of HOX genes). No difference in survival was observed among NPM1-mutated AML patients independently of whether they carried a NK or an AK, the NPM1-mutated/FLT3-ITD negative cases showing the better prognosis. Findings in our patients point to chromosomal aberrations as secondary events, reinforce the concept that NPM1 mutation is a founder genetic lesion, and indicate that NPM1-mutated AML should be clinically handled as one entity, irrespective of the karyotype.

Improved Outcome With T-Cell–Depleted Bone Marrow Transplantation for Acute Leukemia

PURPOSE To eliminate the risk of rejection and lower the risk of relapse after T-cell-depleted bone marrow transplants in acute leukemia patients, we enhanced pretransplant immunosuppression and myeloablation. PATIENTS AND METHODS Antithymocyte globulin and thiotepa were added to standard total-body irradiation/cyclophosphamide conditioning. Donor bone marrows were depleted ex vivo of T lymphocytes by soybean agglutination and E-rosetting. This approach was tested in 54 consecutive patients with acute leukemia who received transplants from HLA-identical sibling donors or, in two cases, from family donors mismatched at D-DR. No posttransplant immunosuppressive treatment was given as graft-versus-host disease (GVHD) prophylaxis. RESULTS Neither graft rejection nor GVHD occurred. Transplant-related deaths occurred in six (16.6%) of 36 patients in remission and in seven (38.8%) of 18 patients in relapse at the time of transplantation. The probability of relapse was .12 (95% confidence interval [CI], 0 to .19) for patients with acute myeloid leukemia and .28 (95% CI, .05 to .51) for patients with acute lymphoblastic leukemia who received transplants at the first or second remission. At a median follow-up of 6.9 years (minimum follow-up, 4.9 years), event-free survival for patients who received transplants while in remission was .74 (95% CI, .54 to .93) for acute myeloid leukemia patients and .59 (95% CI, .35 to .82) for acute lymphoblastic leukemia patients. All surviving patients have 100% performance status. CONCLUSION Adding antithymocyte globulin and thiotepa to the conditioning regimen prevents rejection of extensively T-cell-depleted bone marrow. Even in the complete absence of GVHD, the leukemia relapse rate is not higher than in unmanipulated transplants.

Targeting Mutant BRAF in Relapsed or Refractory Hairy-Cell Leukemia

BACKGROUND BRAF V600E is the genetic lesion underlying hairy-cell leukemia. We assessed the safety and activity of the oral BRAF inhibitor vemurafenib in patients with hairy-cell leukemia that had relapsed after treatment with a purine analogue or who had disease that was refractory to purine analogues. METHODS We conducted two phase 2, single-group, multicenter studies of vemurafenib (at a dose of 960 mg twice daily)--one in Italy and one in the United States. The therapy was administered for a median of 16 weeks in the Italian study and 18 weeks in the U.S. study. Primary end points were the complete response rate (in the Italian trial) and the overall response rate (in the U.S. trial). Enrollment was completed (28 patients) in the Italian trial in April 2013 and is still open (26 of 36 planned patients) in the U.S. trial. RESULTS The overall response rates were 96% (25 of 26 patients who could be evaluated) after a median of 8 weeks in the Italian study and 100% (24 of 24) after a median of 12 weeks in the U.S. study. The rates of complete response were 35% (9 of 26 patients) and 42% (10 of 24) in the two trials, respectively. In the Italian trial, after a median follow-up of 23 months, the median relapse-free survival was 19 months among patients with a complete response and 6 months among those with a partial response; the median treatment-free survival was 25 months and 18 months, respectively. In the U.S. trial, at 1 year, the progression-free survival rate was 73% and the overall survival rate was 91%. Drug-related adverse events were usually of grade 1 or 2, and the events most frequently leading to dose reductions were rash and arthralgia or arthritis. Secondary cutaneous tumors (treated with simple excision) developed in 7 of 50 patients. The frequent persistence of phosphorylated ERK-positive leukemic cells in bone marrow at the end of treatment suggests bypass reactivation of MEK and ERK as a resistance mechanism. CONCLUSIONS A short oral course of vemurafenib was highly effective in patients with relapsed or refractory hairy-cell leukemia. (Funded by the Associazione Italiana per la Ricerca sul Cancro and others; EudraCT number, 2011-005487-13; ClinicalTrials.gov number NCT01711632.).

CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice

Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34(+) cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34(+) cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34(+) leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34(+) subfraction exhibiting the phenotype (CD34(+)/CD38(-)/CD123(+)/CD33(+)/CD90(-)) of leukemic stem cells. When transplanted into immunocompromised mice, CD34(+) cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patients disease. These results indicate that the CD34(+) fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34(-) cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.