Goretta Bonacorsi

The monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation.

Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B cells and the extent of infiltration is associated with adverse prognostic factors. We studied blood monocyte population by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate proliferation of T cells in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes cultured with CLL cells were also evaluated. The absolute number of monocytes increased in chronic lymphocytic leukemia patients compared to the number in normal controls (792±86 cells/μL versus 485±46 cells/μL, P=0.003). Higher numbers of non-classical CD14+CD16++ and Tie-2-expressing monocytes were also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment of phagocytosis. We also detected gene alterations such as down-regulation of PTGR2, a reductase able to inactivate prostaglandin E2, indicating immunosuppressive activity. Accordingly, the proliferation of T cells in contact with monocytes from patients was inhibited compared to that of cells in contact with monocytes from normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated “education” of immune elements may also include the establishment of a skewed phenotype in the monocyte/macrophage population.

Increased angiogenesis induced by chronic lymphocytic leukemia B cells is mediated by leukemia-derived Ang2 and VEGF

Emerging evidence suggests that angiogenic signalling pathways play important role in the patho-biology of chronic lymphocytic leukemia (CLL). Our goal was to investigate: (i) the spontaneous and hypoxia-induced production of pro-angiogenic factors, VEGF and Ang2, by Real-time PCR and ELISA, (ii) the degree of vascularization in CLL-infiltrated bone marrow (BM) compartment by CD34 immunohistochemical staining of microvessels and (iii) the direct angiogenic effect of CLL-derived VEGF and Ang2 by function-blocking experiments in Matrigel assays. The results demonstrated that CLL cells spontaneously express both VEGF and Ang2 and are able to secrete these factors in surrounding microenvironment. Full-length Ang2 mRNA and truncated form Ang2(443) were detectable. Moreover, CLL cells were shown to enhance secretion of both VEGF and Ang2 proteins when subjected to hypoxic condition. Furthermore, increased in vivo and in vitro angiogenesis was induced by CLL cells. Enhanced BM vascularity correlated with Ig-unmutated CLL subset and increased expression of Ang2. Then, we demonstrated that supernatants obtained from CLL cells significantly increase the HUVEC tube formation in Matrigel assays and that this enhanced angiogenic capacity is mediated by both CLL-derived VEGF and Ang2. Taken together, these results suggest that several simultaneous mechanisms may be involved in the CLL capacity to induce the disruption of pre-existing vessel structures to give rise to tumor neoangiogenesis. The preliminary studies in solid tumors, showing that the disruption of Ang2 function can inhibit tumor vessel density and growth, are encouraging and suggest the possibility of new future therapeutic options targeting CLL microenvironment.

Primary high-grade mucosa-associated lymphoid tissue-type lymphoma of the cervix presenting as a common endocervical polyp

Lymphomas of the uterine cervix are uncommon neoplasms and typically appear as diffuse cervical enlargement. We describe a rare case of primary high-grade lymphoma of mucosa-associated lymphoid tissue of the uterine cervix in a 46-year-old white woman. The tumor, incidentally disclosed at gynecological examination, appeared as a single common polyp. Immunohistochemical investigation found the lesion to consist of a monomorphic CD20-positive infiltrate of large blasts and rare intermingling centrocyte-like lymphoid cells. A dense area of monotypic (lambda light-chain restriction) plasma cells was found beneath the endocervical mucosa; only a few scattered lymphoepithelial lesions were present. The neoplastic cells did not stain for CD5, CD10, CD23, CD43, or cyclin D1. A bone marrow biopsy displayed a paratrabecular, centrocyte-like B-cell infiltration, but no lymphadenopathy was detected by instrumental examination (computed tomographic scan, magnetic resonance imaging). The tumor was successfully treated by multiagent chemotherapy followed by total hysterectomy. To our knowledge, this case represents the second reported example of mucosa-associated lymphoid tissue-type lymphoma occurring in the uterine cervix. We highlight the very unusual gross appearance of this case and emphasize the difficulty of interpreting lymphoid infiltrates in the lower genital tract by microscopy.

Endothelin-1 promotes survival and chemoresistance in chronic lymphocytic leukemia B cells through ETA receptor.

The endothelin axis, comprising endothelins (ET-1, ET-2 and ET-3) and their receptors (ETAR and ETBR), has emerged as relevant player in tumor growth and metastasis. Here, we investigated the involvement of ET-1/ETAR axis in chronic lymphocytic leukemia (CLL). CLL cells expressed higher levels of ET-1 and ETA receptor as compared to normal B cells. ET-1 peptide stimulated phosphoinositide-3-kinase and mitogen-activated protein kinase signaling pathways, improved survival and promoted proliferation of leukemic cells throughout ETAR triggering. Moreover, the blockade of ETAR by the selective antagonist BQ-123 inhibited the survival advantage acquired by CLL cells in contact with endothelial layers. We also found that blocking ETAR via BQ-123 interferes with ERK phosphorylation and CLL pro-survival effect mediated by B-cell receptor (BCR) activation. The pro-apoptotic effect of phosphoinositide-3-kinase δ inhibitor idelalisib and mitogen-activated protein kinase inhibitor PD98059 was decreased by the addition of ET-1 peptide. Then, ET-1 also reduced the cytotoxic effect of fludarabine on CLL cells cultured alone or co-cultured on endothelial layers. ETAR blockade by BQ-123 inhibited the ET-1-mediated protection against drug-induced apoptosis. Lastly, higher plasma levels of big ET-1 were detected in patients (n = 151) with unfavourable prognostic factors and shorter time to first treatment. In conclusion, our data describe for the first time a role of ET-1/ETAR signaling in CLL pathobiology. ET-1 mediates survival, drug-resistance, and growth signals in CLL cells that can be blocked by ETAR inhibition.

Physical contact with endothelial cells through β1- and β2- integrins rescues chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis and induces a peculiar gene expression profile in leukemic cells

Background Chronic lymphocytic leukemia B cells display prolonged survival in vivo, but when cultured in vitro rapidly undergo spontaneous apoptosis. We hypothesize that interactions with endothelial cells in infiltrated tissues and during recirculation may have a pathogenic role in chronic lymphocytic leukemia. Design and Methods We evaluated apoptosis of leukemic cells after co-culture on a monolayer of human umbilical vein endothelial cells with addition of fludarabine and antibodies that block adhesion. Then, we compared microarray-based gene expression profiles between leukemic cells at baseline and after co-culture. Results We found that the endothelial layer protected leukemic cells from apoptosis inducing a 2-fold mean decrement in apoptotic cells after 2 days of co-culture. Moreover, the endothelial layer decreased the sensitivity of chronic lymphocytic leukemia B cells to fludarabine-induced apoptosis. Physical contact with endothelium mediated by both β1- and β2- integrins is essential for the survival advantage of leukemic cells. In particular, blocking CD106 on endothelial cells or CD18 on leukemic B cells led to the almost complete abrogation of the survival advantage (>70% inhibition of viability). However, a reduction of apoptosis was also measured in leukemic cells cultured in conditioned medium collected after 2 days of co-culture, implying that survival is partially mediated by soluble factors. Overall, the contact with endothelial cells modulated 1,944 genes in chronic lymphocytic leukemia B cells, establishing a peculiar gene expression profile: up-regulation of angiogenesis-related genes, an increase of genes involved in TGFβ and Wnt signaling pathways, secretion of cytokines recruiting stromal cells and macrophages and up-regulation of anti-apoptotic molecules such as Bcl2 and Survivin. Conclusions Our study supports the notion that endothelial cells are major players in the chronic lymphocytic leukemia microenvironment. Adhesion to endothelium strongly supports survival, protects from drug-induced apoptosis and extensively modifies the gene expression profile of leukemic cells.

Lymphotropic herpes virus (EBV, HHV-6, HHV-8) DNA sequences in HIV negative Castleman's disease

Aim—To evaluate the possible involvement of lymphotropic herpes viruses in Castlemans disease. Methods—Archival formalin fixed, paraffin wax embedded biopsy specimens from 16 HIV negative patients (11 with localised and five of multicentric disease) were studied. Epstein-Barr virus (EBV), human herpes virus-6 (HHV-6) and human herpes virus-8 (HHV-8) DNA was detected using PCR. PCR was also used to characterise the EBV genomes and the clonal status of the lesions. Results—EBV sequences were identified in nine (56%) cases. The main EBV genotype detected was type 1. Two (12%) cases were positive for both HHV-6 and EBV sequences. HHV-8 sequences were detected in one case of localised Castlemans disease, the sequence of which differed from that of the HHV-8 prototype. No clonal immunoglobulin gene rearrangements were found. Conclusions—EBV DNA was detected in a substantial proportion of cases, suggesting that it may have a role in the pathogenesis of Castlemans disease, unlike HHV-6 which was detected rarely. This is the first report of HHV-8 specific sequences in the localised from of the disease.

Endothelium-mediated survival of leukemic cells and angiogenesis-related factors are affected by lenalidomide treatment in chronic lymphocytic leukemia

Lenalidomide is an IMID immunomodulatory agent clinically active in patients with chronic lymphocytic leukemia (CLL). We evaluated the activity of lenalidomide inside an in vitro coculture system of endothelial and CLL cells. Lenalidomide was able to inhibit CLL survival advantage mediated by endothelial contact. Moreover, the marked increase of in vitro angiogenesis determined by CLL-derived conditioned media was reduced by lenalidomide. We also analyzed peripheral blood collected from 27 patients with relapsed or refractory CLL being treated with lenalidomide within a phase II trial. Plasma levels of VEGF and THBS-1 decreased, whereas Ang2 and Ang increased during treatment. Patients who respond to lenalidomide showed a more pronounced decrease of VEGF and bFGF than did patients with stable or progressive disease (p = 0.007 and p = 0.005). Furthermore, lenalidomide reduced circulating endothelial cells and endothelial progenitors by increasing the percentage of apoptotic cells. Conversely, for six matched bone marrow biopsies available before and after treatment, we did not detect any modification in vessel density, suggesting a possible mechanism of vessel normalization rather than regression. In conclusion, our study provides further evidence that the anti-CLL effect of lenalidomide is mediated through the alteration of microenvironmental elements, implying the modulation of several angiogenesis-related factors and disruption of CLL crosstalk with endothelial cells.

Chronic eosinophilic leukaemia with ETV6-NTRK3 fusion transcript in an elderly patient affected with pancreatic carcinoma

To the Editor: An 82-yr-old woman with previous history of breast cancer, surgically treated in 1992, and arterial hypertension underwent distal pancreatectomy and splenectomy in July 2008 for a poorly differentiated adenocarcinoma of the pancreatic tail with regional lymph nodes and liver metastases. She was subsequently started on palliative chemotherapeutic treatment, consisting of gemcitabine 1000 mg ⁄m days 1 and 8 every 3 wk. On February 2009, having completed eight cycles, the chemotherapy was withdrawn because hepatic metastases increased in size and number. Until March 2009, the complete blood count was unrevealing (WBC count 7.2 · 10 ⁄L with a normal differential count, Hb 10.2 g ⁄dL, Plt count 300 · 10 ⁄L). One month later, the patient was admitted because of low-grade fever, bone pain and marked fatigue. The laboratory investigations revealed leukocytosis with marked eosinophilia and thrombocytopenia (WBC count 73 · 10 ⁄L with eosinophils 78%, Hb 11.4 g ⁄dL, Plt count 69 · 10 ⁄L). The diagnostic work-up excluded either allergic reactions or parasitic infestations or autoimmune disorders or T-cell populations with an abnormal phenotype (1, 2), raising the hypothesis of an eosinophilic leukaemoid reaction secondary to the metastatic pancreatic cancer (3). However, the morphological examination of peripheral blood (PB) smear showed eosinophilia accompanied with circulating myeloid immature precursors and blasts (3%) (Fig. 1A), while bone marrow (BM) trephine biopsy documented marked proliferation of eosinophil granulocytopoiesis, with a blast cell count < 20% (Fig. 1B). Moreover, moderate to severe BM fibrosis was documented on Gomori methenamine silver staining (not shown). These morphological pictures were consistent with a myeloproliferative neoplasm with eosinophilia (MNE) (4). Unfortunately, serum tryptase level was not measured. Extensive FISH and molecular studies on both PB and BM samples failed to detect PDGFRA, PDGFRB and FGFR1 rearrangements (4), but the clonality of the myeloid population was confirmed by further cytogenetic and FISH studies. In particular, conventional G-banding showed 46, XX, )7, +8 karyotype (Fig. 2A), while whole chromosome painting and metaphase FISH examinations performed with specific probes showed that ETV6 gene, located on 12p13, was cryptically rearranged with 15q25 (Fig. 2B,C). A submicroscopic t(12;15)(p13;q25) resulting in the fusion gene ETV6-NTRK3 on der(15) was thus documented. Moreover, RNA isolated from the BM sample was subjected to reverse transcriptase–PCR using primers designed to amplify either the 731-bp solid cancer ETV6-NTRK3 variant (5) or the 601-bp acute myeloid leukaemia (AML) ETV6-NTRK3 variant (6). These latter molecular analyses demonstrated the 731and 1265-bp products, respectively, as expected when the solid cancer-associated fusion transcript is detected (Fig. 2D). Both FISH and molecular examinations, performed as described (7) on formalin-fixed paraffinembedded pancreatic carcinoma sections, were negative for ETV6-NTRK3 fusion transcript, whereas amplification of ETV6 gene was documented (not shown).

Angiopoietin-2 expression in B-cell chronic lymphocytic leukemia: association with clinical outcome and immunoglobulin heavy-chain mutational status.

Angiopoietin-2 expression in B-cell chronic lymphocytic leukemia: association with clinical outcome and immunoglobulin heavy-chain mutational status

Distinct genomic events in the myeloid and lymphoid lineages in simultaneous presentation of chronic myeloid leukemia and B-chronic lymphocytic leukemia

Distinct genomic events in the myeloid and lymphoid lineages in simultaneous presentation of chronic myeloid leukemia and B-chronic lymphocytic leukemia