Laura Pasqualucci

Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia.

The identification of new genetic lesions in chronic lymphocytic leukemia (CLL) prompts a comprehensive and dynamic prognostic algorithm including gene mutations and chromosomal abnormalities and their changes during clonal evolution. By integrating mutational and cytogenetic analysis in 1274 CLL samples and using both a training-validation and a time-dependent design, 4 CLL subgroups were hierarchically classified: (1) high-risk, harboring TP53 and/or BIRC3 abnormalities (10-year survival: 29%); (2) intermediate-risk, harboring NOTCH1 and/or SF3B1 mutations and/or del11q22-q23 (10-year survival: 37%); (3) low-risk, harboring +12 or a normal genetics (10-year survival: 57%); and (4) very low-risk, harboring del13q14 only, whose 10-year survival (69.3%) did not significantly differ from a matched general population. This integrated mutational and cytogenetic model independently predicted survival, improved CLL prognostication accuracy compared with FISH karyotype (P < .0001), and was externally validated in an independent CLL cohort. Clonal evolution from lower to higher risk implicated the emergence of NOTCH1, SF3B1, and BIRC3 abnormalities in addition to TP53 and 11q22-q23 lesions. By taking into account clonal evolution through time-dependent analysis, the genetic model maintained its prognostic relevance at any time from diagnosis. These findings may have relevant implications for the design of clinical trials aimed at assessing the use of mutational profiling to inform therapeutic decisions.

The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development

Notch2 mutations represent the most frequent lesion in splenic marginal zone lymphoma.

Alteration of BIRC3 and multiple other NF-κB pathway genes in splenic marginal zone lymphoma

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

Mediastinal large B‐cell lymphoma: clinical and immunohistological findings in 18 patients treated with different third‐generation regimens

We report on the immunophenotype, clinical findings and response to aggressive chemotherapy of 18 patients with mediastinal large B‐cell lymphoma (MLCL). Cases were collected from a series of 286 high‐grade non‐Hodgkins lymphomas (HG‐NHL) which, in the period September 1988 to August 1991, were enrolled in a prospective multicentre trial designed to compare the MACOP‐B and F‐MACHOP regimens. Immunostaining on frozen sections revealed a previously unrecognized phenotype, i.e. co‐expression of B‐cell (CD19, CD20, CD22, Ig‐associated dimer) and activation‐associated antigens (CD30 and CDw70) in about 60% of MLCL cases; in contrast, the activation‐associated antigens CD25 and Ki‐27 (unclustered) were consistently negative. This peculiar phenotype may reflect a derivation of the tumour from a subset of thymic activated B cells. Clinically, the patients (median age 31 years; F/M ratio 2.6) presented with bulky mediastinal mass (72%) associated with mediastinal syndrome in >50% cases; disease was stage IIA in most cases. All 18 patients received aggressive chemotherapy (F‐MACHOP 11; MACOP‐B 7). Complete response (CR) was achieved in 57.1% of cases treated with MACOP‐B. In contrast, the response of the 11 MLCL treated with F‐MACHOP was poor (CR 18.2%) as compared to that of the 135 HG‐NHL treated with the same regimen during the trial (CR 69.6%). This difference was still statistically significant after adjusting for negative prognostic factors (mediastinal mass > 10 cm plus increased LDH) and suggests that F‐MACHOP might not be the most appropriate regimen for this kind of lymphoma.

Genetic lesions associated with chronic lymphocytic leukemia chemo-refractoriness.

Fludarabine refractoriness (FR) represents an unsolved clinical problem of chronic lymphocytic leukemia (CLL) management. Although next-generation sequencing studies have led to the identification of a number of genes frequently mutated in FR-CLL, a comprehensive evaluation of the FR-CLL genome has not been reported. Toward this end, we studied 10 FR-CLLs by combining whole-exome sequencing and copy number aberration (CNA) analysis, which showed an average of 16.3 somatic mutations and 4 CNAs per sample. Screening of recurrently mutated genes in 48 additional FR-CLLs revealed that ~70% of FR-CLLs carry ≥1 mutation in genes previously associated with CLL clinical course, including TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (18.9%), and BIRC3 (15.5%). In addition, this analysis showed that 10.3% of FR-CLL cases display mutations of the FAT1 gene, which encodes for a cadherin-like protein that negatively regulates Wnt signaling, consistent with a tumor suppressor role. The frequency of FAT1-mutated cases was significantly higher in FR-CLL than in unselected CLLs at diagnosis (10.3% vs 1.1%, P = .004), suggesting a role in the development of a high-risk phenotype. These findings have general implications for the mechanisms leading to FR and point to Wnt signaling as a potential therapeutic target in FR-CLL.

Anti-CD30 (BER=H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful antitumour activity against CD30+ tumour cells in vitro and in SCID mice.

The anti‐CD30 immunotoxin (IT) Ber‐H2/saporin is effective in patients with refractory Hodgkin’s disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti‐CD30 ITs by covalently linking the mouse monoclonal antibody Ber‐H2 to the type 1 ribosome‐inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP‐S), which do not cross‐react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin’s disease and anaplastic large‐cell lymphoma (ALCL)‐derived CD30+ target cell lines with a very high efficiency (IC50 ranging from <u20035u2003×u200310−13u2003m to 2.75u2003×u200310−11m, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3u2003d treatment with non‐toxic doses of Ber‐H2/MOM (50% LD50), started 24u2003h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were a dose‐related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7u2003mg/kg for Ber‐H2/MOM and 0.45u2003mg/kg for Ber‐H2/PAP‐S. Sequential administration of two anti‐CD30 ITs (Ber‐H2/MOM and Ber‐H2/saporin) was well tolerated and did not result in formation of antibodies cross‐reacting with the two plant toxins. The results presented in this paper suggest that, in the future, sequential administration of anti‐CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP‐S) should be feasible.

Evaluation of immunotoxins containing single‐chain ribosome‐inactivating proteins and an anti‐CD22 monoclonal antibody (OM124): in vitro and in vivo studies

Immunotoxins were prepared with three ribosome‐inactivating proteins (RIP), momordin, pokeweed antiviral protein from seeds (PAP‐S) and saporin‐S6, linked to the anti‐CD22 monoclonal antibody OM124. These immunotoxins inhibited protein synthesis by CD22‐expressing cell lines Daudi, EHM, BJAB, Raji and BM21 with IC50 (concentration causing 50% inhibition) ranging from <u20035u2003×u200310−15 to 7.6u2003×u200310−11u2003M as RIP, and IC90 (concentration causing 90% inhibition) ranging from 5u2003×u200310−14 to 5u2003×u200310−8u2003M, with no effect on a CD22‐negative HL60 cell line at the highest concentration tested (5u2003×u200310−8u2003M). Apoptosis was induced in sensitive cells. The formation of bone marrow colonies was inhibited by no more than 40% by the immunotoxins at concentrations up to 10−9u2003M. Treatment with the immunotoxins, alone or in combination, significantly extended the survival time of mice bearing transplanted Daudi cells. A treatment with cyclophosphamide and OM124/saporin immunotoxin was particularly effective in SCID mice transplanted with a low number of cells (3u2003×u200310−6), when 60% of the animals remained tumour‐free.

Somatic CLL mutations occur at multiple distinct hematopoietic maturation stages: Documentation and cautionary note regarding cell fraction purity

Somatic CLL mutations occur at multiple distinct hematopoietic maturation stages: documentation and cautionary note regarding cell fraction purity