Marco Paulli

Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation

Next generation sequencing and copy number analysis provide insights into the complexity of the CLL coding genome, and reveal an association between NOTCH1 mutational activation and poor prognosis.

Differential Viral Protein Expression in Kaposi’s Sarcoma-Associated Herpesvirus-Infected Diseases : Kaposi’s Sarcoma, Primary Effusion Lymphoma, and Multicentric Castleman’s Disease

Kaposis sarcoma (KS)-associated herpesvirus (KSHV) is linked to KS, primary effusion lymphomas (PEL), and a subset of multicentric Castlemans disease (MCD). Transcript mapping studies using PEL cell lines have allowed preliminary classification of viral gene expression into constitutive (class I) and inducible (class II/III) categories. To determine whether viral gene expression differs in vivo, we examined tissue sections of KSHV-infected disorders, using specific antibodies against proteins that are representative of the different expression classes of KSHV genes. ORF73/LANA appears to be a surrogate marker for KSHV infection because it is constitutively expressed in vitro and in vivo in all KSHV-infected cells. Expression of vIRF1, vIL6, and PF-8 proteins in the infected B cells of MCD lymph nodes reproduces the expression pattern observed in TPA-stimulated KSHV-infected B-cell lines. In contrast, the protein expression of the inducible viral genes that we tested in KS and PEL biopsies is restricted to PF-8 and vIL6, respectively. The tightly restricted expression of KSHV proteins in vivo differs from the dysregulated expression of inducible KSHV genes in vitro and suggests that viral gene expression in KSHV-infected cell lines does not accurately reflect what occurs in diseased tissues. These differences may be related to either cell-specific or immune restriction of viral replication.

Primary cutaneous CD30‐positive large cell lymphoma: Definition of a new type of cutaneous lymphoma with a favorable prognosis. A European multicenter study of 47 patients

Background. CD30 (Ki‐1)‐positive anaplastic large cell lymphoma (LCL) has been described as a morphologically distinct group of LCL that generally are associated with a poor prognosis. Recent studies indicate that these lymphomas, when confined to the skin, have a favorable prognosis. However, there is no consensus regarding the definition of these primary cutaneous CD30‐positive LCL. Reported patients have been selected variously on the basis of morphologic (anaplastic cytology) or immunophenotypical (expression of CD30 antigen) criteria.

Marker Expression in Peripheral T-Cell Lymphoma: A Proposed Clinical-Pathologic Prognostic Score

PURPOSE Although peripheral T-cell lymphoma, unspecified (PTCL/U), is the most common T-cell tumor in Western countries, no study to date has been based on the application of a wide panel of markers to a large series of patients and assessed the impact of phenotype on survival. We evaluated the expression of 19 markers in 148 PTCLs/U and 45 PTCLs of the angioimmunoblastic type (AILD). PATIENTS AND METHODS The analysis was performed on tissue microarrays by immunohistochemistry and in situ hybridization. Clinical data were available in 93 PTCL/U patients, most of whom had been included in a previous study proposing a prognostic index (PIT). RESULTS An aberrant phenotype with frequent loss of CD5 and/or CD7 was typical for PTCLs, irrespective of whether they were U or AILD. Aberrantly expressed proteins rarely included CD20, CD15, and CD30. Positivity for Epstein-Barr virus-associated small RNAs and CD15 expression emerged as adverse prognostic factors. Among PTCLs/U, the proliferation-associated protein Ki-67 turned out to be prognostically relevant and was integrated in a new predictive score, incorporating age (> 60 years), high lactate dehydrogenase, poor performance status, and Ki-67 > or = 80%. This score was associated with the patient outcome (P < .0001) and was found to be more robust than PIT (P = .0043) in the present series. CONCLUSION Our retrospective analysis shows a wide range of protein expression in PTCLs and proposes a new prognostic index. The latter represents one of the first examples of mixed score (including patient- and tumor-specific factors) applied to malignant lymphomas and may be the basis for future prospective therapeutic trials.

CD30/Ki-1-positive lymphoproliferative disorders of the skin - Clinicopathologic correlation and statistical analysis of 86 cases: A multicentric study from the European organization for research and treatment of cancer cutaneous lymphoma project group

PURPOSE Recently, it has been shown that CD30 antigen expression is associated with a relatively favorable prognosis in primary cutaneous large-cell lymphomas (CLCLs). However, prognostic subsets within the CD30+ group have been difficult to identify due to lack of uniform clinicopathologic and immunophenotypic criteria, limited clinical information, and the inclusion of relatively few patients for statistical analysis in prior studies. To address these problems, we formed a multicentric study group of pathologists and dermatologists to classify and evaluate 92 cases of CD30+ cutaneous lymphoproliferative disorders. PATIENTS AND METHODS An expert panel established consensus diagnoses for 86 CD30+ cutaneous lymphomas. Cases, clinically and histologically classified as lymphomatoid papulosis (LyP), anaplastic large-cell lymphoma (ALCL), nonanaplastic lymphoma, and borderline histology between LyP and ALCL, were then analyzed statistically by univariate, multivariate, and Cox regression model analysis of potential prognostic features. RESULTS Spontaneous regression and age less than 60 years were associated with a favorable prognosis, while extracutaneous disease and age greater than 60 had a poor prognosis. Patients with LyP had the best prognosis, followed by those with primary CD30+ lymphomas, regardless of cytologic type (anaplastic or nonanaplastic). Borderline cases, morphologically indistinguishable from LyP and CD30+ ALCL, had a favorable prognosis, similar to LyP. CONCLUSION Our findings indicate that CD30+ cutaneous lymphoproliferative disorders comprise a spectrum of closely related skin lesions, which can be assigned a relatively favorable or unfavorable prognosis by a combined clinical and pathologic analysis.

Cytotoxic/natural killer cell cutaneous lymphomas. Report of EORTC Cutaneous Lymphoma Task Force Workshop.

Cutaneous lymphomas expressing a cytotoxic or natural killer (NK) cell phenotype represent a group of lymphoproliferative disorders for which there is currently much confusion and little consensus regarding the best nomenclature and classification.

Epithelium derived interleukin 15 regulates intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac disease

Background and aims: Epithelium derived interleukin (IL)-15 signalling via IL-15Rα is critical for the development, activation, and survival of intraepithelial lymphocytes (IEL). We aimed to better understand the IL-15 driven effects on IEL underlying mucosal damage and lymphomagenesis in coeliac disease (CD). Methods: Enterocytes, IEL, and lamina propria mononuclear cells (LPMC) were isolated from 46 patients with uncomplicated CD (25 untreated and 21 treated) and 22 controls. IL-15 and IL-15Rα expression were determined by immunoblotting. Secretion of IL-15, interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), and granzyme B into cell culture supernatants was assessed by ELISA. The ability of IL-15 to regulate IEL proliferation, perforin/granzyme dependent cytotoxicity, and apoptosis was tested by adding different combinations of IL-15, IL-15 blocking antibody, or chloroquine to IEL cultured alone or with Caco-2 cells as target. IL-15 mucosal levels were also determined by ELISA in five patients with complicated CD (two ulcerative jejunoileites, one refractory sprue, and two enteropathy associated T cell lymphomas) tested for T cell receptor γ chain clonality. Results: IL-15 was overexpressed in untreated CD enterocytes and LPMC, and in the mucosa of complicated CD patients and uncomplicated untreated CD patients, where its levels correlated with the degree of mucosal damage. Enterocytes from untreated, but not treated, CD patients and controls secreted IL-15. Untreated CD IEL, characterised by higher IL-15Rα expression, showed increased proliferation, production of IFN-γ and TNF-α, and perforin/granzyme dependent cytotoxicity, and a decreased propensity to apoptosis in response to IL-15. Conclusions: Our findings suggest that IL-15 plays a crucial role in the generation of epithelial damage in active CD. Its promotion of IEL survival in CD may predispose to the emergence of T cell clonal proliferations. Blocking IL-15, by suppressing uncontrolled IEL activation and survival, has the potential to provide new therapeutic tools to prevent tissue damage and lymphomagenesis in CD.

Preemptive Therapy of EBV-Related Lymphoproliferative Disease after Pediatric Haploidentical Stem Cell Transplantation

The treatment of Epstein‐Barr virus (EBV)‐related post‐transplant lymphoproliferative disease (PTLD) after hematopoietic stem cell transplantation (HSCT) is still unsatisfactory. We conducted a prospective trial to evaluate the impact of routine EBV surveillance and preemptive treatment with the anti‐CD20 monoclonal antibody rituximab on the development of PTLD in pediatric recipients of extensively T‐cell depleted HSCT from an HLA‐haploidentical relative. Twenty‐seven patients were included in the surveillance program, 12 developed EBV DNA positivity, with 8 of 12 presenting with sustained viral DNA levels requiring treatment with rituximab. Treatment was well tolerated, and induced clearance of EBV DNA in all patients. However, 4/8 patients showed a new increase in EBV load, coincident with the emergence of CD20−/CD19+ B cells in peripheral blood, accompanied by overt PTLD in 3 patients. The latter cleared PTLD after receiving donor EBV‐specific cytotoxic T‐lymphocytes (CTLs), and persist in remission at a median 30‐month follow‐up. EBV‐specific T‐cell frequency, undetectable at time of EBV DNA positivity, was restored by T‐cell therapy to levels comparable with controls. We conclude that preemptive therapy with rituximab is safe, but only partly effective in haplo‐HSCT recipients. Patients who progress to PTLD under rituximab treatment can be rescued permanently by infusion of EBV‐specific CTLs.

Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom's macroglobulinemia and related lymphoid neoplasms.

A study has shown that MYD88 (L265P) is a recurring somatic mutation in Waldenströms macroglobulinemia (WM). We developed an allele-specific polymerase chain reaction (PCR) for this mutation, and analyzed bone marrow or peripheral blood samples from 58 patients with WM, 77 with IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), 84 with splenic marginal zone lymphoma (SMZL), and 52 with B-cell chronic lymphoproliferative disorders (B-CLPD). MYD88 (L265P) was detected in 58/58 (100%) patients with WM, 36/77 (47%) with IgM-MGUS, 5/84 (6%) with SMZL, and 3/52 (4%) with B-CLPD. Compared to IgM-MGUS patients with wild-type MYD88, those carrying MYD88 (L265P) showed significantly higher levels of IgM (P < .0001) and presented Bence-Jones proteinuria more frequently at diagnosis (P = .002). During follow-up, 9 patients with IgM-MGUS progressed to WM or to marginal zone lymphoma. Using a case-control approach, the risk of evolution of patients carrying MYD88 (L265P) was significantly higher than that of patients with wild-type MYD88 (odds ratio 4.7, 95% confidence interval 0.8 to 48.7, P = .047). These findings indicate that the allele-specific PCR we developed is a useful diagnostic tool for patients with WM or IgM-MGUS. In this latter condition, MYD88 (L265P) is associated with greater disease burden and higher risk of disease progression, and the mutation may therefore also represent a useful prognostic marker.

The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation

Richter syndrome (RS) represents the development of diffuse large B-cell lymphoma in the context of chronic lymphocytic leukemia. The scarcity of biologic information about RS has hampered the identification of molecular predictors of RS outcome. We addressed this issue by performing a comprehensive molecular characterization of 86 pathologically proven RS. TP53 disruption (47.1%) and c-MYC abnormalities (26.2%) were the most frequent alterations, whereas common genetic lesions of de novo diffuse large B-cell lymphoma were rare or absent. By multivariate analysis, lack of TP53 disruption (hazard ratio, 0.43; P = .003) translated into significant survival advantage with 57% reduction in risk of death. An algorithm based on TP53 disruption, response to RS treatment, and Eastern Cooperative Oncology Group performance status had 80.9% probability of correctly discriminating RS survival (c-index = .809). RS that were clonally unrelated to the paired chronic lymphocytic leukemia phase were clinically and biologically different from clonally related RS because of significantly longer survival (median, 62.5 months vs 14.2 months; P = .017) and lower prevalence of TP53 disruption (23.1% vs 60.0%; P = .018) and B-cell receptor stereotypy (7.6% vs 50.0%; P = .009). The molecular dissection of RS into biologically distinct categories highlights the genetic heterogeneity of this disorder and provides clinically relevant information for refining the prognostic stratification of patients.