Bendamustine added to allogeneic conditioning improves long-term outcomes in patients with CLL.

Bendamustine has shown a favorable safety profile when included in chemotherapy regimens for several types of lymphoma, including CLL. This study investigated the long-term effect of adding bendamustine to a conditioning regimen on survival, rate of engraftment, immune recovery and GvHD after allogeneic stem cell transplantation (alloSCT) in CLL patients. These outcomes were compared with the fludarabine, cyclophosphamide and rituximab (FCR) conditioning regimen. We reviewed the data for 89 CLL patients treated on three trials at our institution. Twenty-six (29%) patients received bendamustine, fludarabine and rituximab (BFR) and 63 (71%) received FCR. Patient characteristics were similar in both groups. Ten (38%) BFR-treated patients vs only two (3%) FCR-treated patients did not experience severe neutropenia (P=<0.001). The 3-year overall survival estimates for the BFR and FCR groups were 82 and 51% (P=0.03), and the 3-year PFS estimates were 63% and 27% (P=0.001), respectively. The 2-year treatment-related mortality was 8 and 23% and the incidence of grade 3 or 4 GvHD was 4% and 10%, respectively. This study is the first to report that addition of bendamustine to alloSCT conditioning for CLL patients is associated with improved survival and lower mortality, myelosuppression, and GvHD.

Concomitant use of direct-acting antivirals and chemotherapy in hepatitis C virus-infected patients with cancer.

BACKGROUND: Antiviral therapy improves hepatic outcomes in hepatitis C virus (HCV)-infected cancer patients. However, such patients are not treated simultaneously with antivirals and chemotherapy, owing to overlapping toxicities with previous standard of care treatment of pegylated interferon and ribavirin. AIM: To examine the safety and clinically-significant drug-drug interactions observed in patients who received simultaneous treatment with direct-acting antivirals (DAAs) and chemotherapy. METHODS: Safety was determined by the presence of adverse events which were graded according to the division of AIDS Table (version 2.0). Adverse events were monitored throughout antiviral treatment and up to 3 months after its completion. Drug-drug interactions were assessed using current online databases. Sustained virological response (SVR) was defined as absence of serum HCV RNA 12 weeks after end of DAA treatment. Cirrhosis was diagnosed via imaging, biopsy or with the use of non-invasive fibrosis markers. RESULTS: Twenty-one patients received concomitant treatment with DAAs and chemotherapy between January 2013 and September 2016. Concomitant treatment was started either for virological (14; 67%) or oncologic (7; 33%) reasons. DAAs used were sofosbuvir, ledipasvir, simeprevir, daclatasvir ± ribavirin. The adverse events observed were mainly constitutional (12; 57%), hematological and gastrointestinal (7; 33% each). Physicians changed the DAA regimens in two patients (10%) in anticipation of drug-drug interactions with daclatasvir and dexamethasone. The overall SVR rate was 95% (20/21). CONCLUSIONS: Hepatitis C virus-targeted antiviral therapy can be used concomitantly with selected anti-neoplastic agents under close monitoring for drug-drug interactions. This therapeutic intervention may prevent delay in the administration of chemotherapy in HCV-infected cancer patients.

Patients with classical Hodgkin lymphoma experiencing disease progression after treatment with brentuximab vedotin have poor outcomes.

BACKGROUND: Brentuximab vedotin (BV) is a key therapeutic agent for patients with relapsed/refractory classical Hodgkin lymphoma (cHL). The outcomes of patients experiencing disease progression after BV are poorly described. PATIENTS AND METHODS: We reviewed our institutional database to identify patients with cHL treated with BV who were either refractory to treatment or experienced disease relapse. We collected clinicopathologic features, treatment details at progression and outcome. RESULTS: One hundred patients met inclusion criteria, with a median age of 32 years (range 18-84) at progression after BV. The median number of treatments before BV was 3 (range 0-9); 71 had prior autologous stem cell transplant. The overall response rate (ORR) to BV was 57%, and the median duration of BV therapy was 3 months (range 1-25). After disease progression post-BV, the most common treatment strategies were investigational agents (n = 30), gemcitabine (n = 15) and bendamustine (n = 12). The cumulative ORR to therapy was 33% (complete response 15%). After a median follow-up of 25 months (range 1-74), the median progression-free (PFS) and overall survival (OS) were 3.5 and 25.2 months, respectively. In multivariate analysis, no factors analyzed were predictive of PFS; age at progression >45 years and serum albumin <40 g/l at disease progression were associated with increased risk of death. Among patients who achieved response to therapy, allogeneic stem cell transplantation was associated with a non-significant trend toward superior OS (P = 0.11). CONCLUSIONS: Patients with BV-resistant cHL have poor outcomes. These data serve as a reference for newer agents active in BV-resistant disease.

Factors influencing outcome in advanced stage, low-grade follicular lymphoma treated at MD Anderson Cancer Center in the rituximab era.

BACKGROUND: The optimal initial therapy of follicular lymphoma (FL) remains unclear. The aims of this study were to compare primary treatment strategies and assess the impact of maintenance rituximab and patterns of treatment failure. PATIENTS AND METHODS: We retrospectively analyzed patients with treatment-naive advanced stage, grade 1-2 FL treated at our center from 2004 to 2014. We included 356 patients treated on clinical trials or standard of care with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP, n = 119); R-CHOP with maintenance (R-CHOP + M, n = 65); bendamustine/rituximab (BR, n = 45); BR with maintenance (BR + M, n = 35); R(2) (n = 94). We compared baseline characteristics, progression-free survival (PFS), overall survival (OS) and analyzed prognostic factors using univariate and multivariate analysis adjusted for treatment. RESULTS: After a median follow-up of 4 years (range 0.2-15.0), the 3-year PFS was 60% [95% confidence interval (CI) 51% to 69%] for R-CHOP, 72% (59% to 82%) for R-CHOP + M, 63% (42% to 78%) for BR, 97% (80% to 100%) for BR + M and 87% (78% to 93%) for R(2). Patients treated with R-chemotherapy had more high-risk features than patients treated with R(2) but, by adjusted multivariate analysis, treatment with R(2) [hazard ratio (HR) 0.39 (0.17-0.89), P = 0.02] was associated with a superior PFS. Eastern Cooperative Oncology Group Performance status of one or more predicted inferior OS. Among patients treated with R-chemotherapy, maintenance was associated with the superior PFS [HR 0.38 (95% CI 0.21-0.68)]. By adjusted multivariate analysis, disease progression within 2 years [HR 5.1 (95% CI 1.57-16.83)] and histologic transformation (HT) [HR 11.05 (95% CI 2.84-42.93)] increased risk of death. CONCLUSION: Induction therapy with R(2) may result in disease control which is comparable with R-chemotherapy. Early disease progression and HT are predictive of inferior survival.

Ehlers-Danlos syndrome: case report and an electron microscopy study.

Ehlers-Danlos syndrome (EDS) type III is a inherited connective tissue disorders characterized by extensibility of the skin, hypermobility of the joints, chronic pain, tissue fragility, easy bruising, and delayed wound healing with result of atrophic scars. The patients report commonly a history of recurrent dislocations of the shoulders and knees after low-impact trauma, chronic joint pain, and early osteoarthritis, which lead to diagnosis. The pathogenesis of this condition is unknown, and the diagnosis is generally made in adult age, based only on clinical criteria. In this report, we describe a case of a 50-year-old woman with a 30-year history of recurrent dislocations and atrophic scars. We performed diagnosis of EDS type III after a complete clinical and instrumental evaluation, comprising of histological and electron microscopic studies, that highlighted collagen abnormalities.

The emerging normal and disease-related roles of anaplastic lymphoma kinase.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, the normal role of which remains to be completely elucidated. Although work carried out in mammals suggests a function in neural development, results from studies in Drosophila indicate an additional role in visceral muscle differentiation. The aberrant expression of full-length ALK receptor proteins has been reported in neuroblastomas and glioblastomas, while the occurrence of ALK fusion proteins in anaplastic large cell lymphoma (ALCL) has resulted in the identification of the new tumor entity, ALK-positive ALCL. ALK represents one of few examples of a receptor tyrosine kinase implicated in oncogenesis in both haematopoietic and non-haematopoietic tumors, given that ALK fusions also occur in the mesenchymal tumor known as inflammatory myofibroblastic tumor (IMT). The study of ALK fusion proteins, besides demonstrating their importance in tumor development, has also raised the possibility of new therapeutic treatments for patients with ALK-positive malignancies.

Anaplastic lymphoma kinase proteins in growth control and cancer.

The normal functions of full-length anaplastic lymphoma kinase (ALK) remain to be completely elucidated. Although considered to be important in neural development, recent studies in Drosophila also highlight a role for ALK in gut muscle differentiation. Indeed, the Drosophila model offers a future arena for the study of ALK, its ligands and signalling cascades. The discovery of activated fusion forms of the ALK tyrosine kinase in anaplastic large cell lymphoma (ALCL) has dramatically improved our understanding of the pathogenesis of these lymphomas and enhanced the pathological diagnosis of this subtype of non-Hodgkin's lymphoma (NHL). Likewise, the realisation that a high percentage of inflammatory myofibroblastic tumours express activated-ALK fusion proteins has clarified the causation of these mesenchymal neoplasms and provided for their easier discrimination from other mesenchymal-derived inflammatory myofibroblastic tumour (IMT) mimics. Recent reports of ALK expression in a range of carcinoma-derived cell lines together with its apparent role as a receptor for PTN and MK, both of which have been implicated in tumourigenesis, raise the possibility that ALK-mediated signalling could play a role in the development and/or progression of a number of common solid tumours. The therapeutic targeting of ALK may prove to have efficacy in the treatment of many of these neoplasms.

Recombinant adenovirus-mediated cytotoxic gene therapy of lymphoproliferative disorders: is CAR important for the vector to ride?

The literature has seen an incredible booming of publications related to the use of recombinant adenoviruses as therapeutic tools for lymphoproliferative disorders over the last decade. Several approaches of adenovirus-mediated gene expression have been used to transfect cell lines that are derived from lymphoid tumors and would have otherwise been refractory to other transfection methods. The identification of high-affinity receptor for human adenoviruses serotype 2 and 5, the coxsackie-adenovirus receptor (CAR), has raised the question about its relevance for the efficacy of recombinant adenovirus-mediated gene therapy. We review published studies that have analyzed the use of recombinant adenovirus vectors expressing cytotoxic genes for gene therapy in lymphomas, chronic lymphocytic leukemia and multiple myeloma. For simplicity, we group all these diseases under the term lymphoproliferative disorders. We analyze the use of recombinant adenovirus-mediated cytotoxicity by assessing the importance of the biochemical and intrinsic signaling pathways interacting with the products of the exogenous viral-mediated expression. Ultimately, we discuss studies that have been finalized to by-pass the limitations of the biodistribution of CAR by modifying or targeting adenovirus to other membrane proteins in cells derived from lymphoproliferative disorders.

Comparison of the effects of recombinant adenovirus-mediated expression of wild-type p53 and p27Kip1 on cell cycle and apoptosis in SUDHL-1 cells derived from anaplastic large cell lymphoma.

Recombinant adenoviruses expressing wild-type p53 (AdWTp53) and p27KiP1 (Adp27) were used to compare the effects on cell cycle and apoptosis in SUDHL-1 cells derived from human anaplastic large cell lymphoma. Cells infected with AdWTp53 and Adp27 showed high level of wild-type p53 and p27KiP1 expression, respectively. The expression of these proteins resulted in G1 arrest after 24 h of infection. Although the cells persisted in G1 arrest in both cell populations after 48 and 72 h of infection, the level of apoptosis assessed by TUNEL analysis was higher in cells infected with AdWTp53. Interestingly, apoptosis was more pronounced in cells infected with Adp27 after the initial 24 h and reached a steady state at 48 and 72 h. A lower MOI of Adp27 resulted in G1 arrest associated with a low level of apoptosis in SUDHL-1 cells after 48 h of infection. This was correlated with lower expression of p27KiP1. We postulate that the time-lag and the different level of apoptosis occurring in SUDHL-1 cells infected with AdWTp53 and Adp27 are clearly related to the intrinsic biochemical pathways solicited. In this context our study provides a model to investigate these pathways and better understand the biology of this particular lymphoma. Our data also support a potential application of Adp27 for gene therapy of this lymphoma similarly to AdWTp53 as previously shown.

Biochemical differences between SUDHL-1 and KARPAS 299 cells derived from t(2;5)-positive anaplastic large cell lymphoma are responsible for the different sensitivity to the antiproliferative effect of p27(Kip1).

An inverse correlation between p27(Kip1) expression and proliferation has been recently established in tissues derived from human lymphomas. The nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)/phospholipase C-gamma (PLCgamma) complex also appears to play an important role in cell proliferation and malignant transformation of anaplastic large cell lymphoma (ALCL). In this study, we report that SUDHL-1 and KARPAS 299 ALCL-derived cell lines present different sensitivity to the antiproliferative effect of recombinant adenovirus-mediated p27(Kip1) expression or to serum-starvation in culture media. The results indicate that exogenous p27(Kip1) may interact with the NPM-ALK/PLCgamma pathway in SUDHL-1 but not in KARPAS 299 cells. This interaction correlates with changes in cell cycle and cell morphology observed mainly in SUDHL-1 cells. The percentage of SUDHL-1 cells in S phase declines, whereas it is almost unchanged in KARPAS 299 cells as compared to the controls after 96 h of infection with the recombinant adenovirus. Furthermore KARPAS 299 cells are resistant to serum-starvation due to deficient p27(Kip1)-upregulation and G1 arrest, whereas SUDHL-1 cells respond with increased G1 phase and p27(Kip1)-upregulation after 48 h of serum-starvation. Both cell lines express appropriate variation of levels of cyclins E and A, and Rb-phosphorylation as expected by growing them in culture media with different FBS content. Although both cell lines express cyclin D2, SUDHL-1 cells only present high level of cyclin D3. Moreover SUDHL-1 cells express high level of PTEN and the PKB/Akt pathway is constitutively activated in both cell lines. Lastly SUDHL-1 cells show higher levels of phosphotyrosine-containing proteins that is correlated with a higher NPM-ALK-associated autophosphorylation activity compared to KARPAS 299 cells. Our study clearly identifies some of the biochemical differences that may explain the difference in sensitivity to antiproliferative stimuli shown by two cell lines derived from the same type of lymphoma.

Adenovirus-p53-mediated gene therapy of anaplastic large cell lymphoma with t(2;5) in a nude mouse model.

Adenovirus-p53-mediated apoptosis has been extensively evaluated in animal xenografts derived from human epithelial tumors and recently began testing in phase I clinical trials, but has not been evaluated for lymphoid malignancies. Cell lines derived from anaplastic large cell lymphoma (ALCL) carrying the t(2;5) translocation are efficiently transduced by adenoviral vector expressing p53 and undergo apoptosis. To test the in vivo efficiency of adenovirus-mediated-p53 expression and apoptosis induction, SUDHL-1 cells (derived from human ALCL) were injected subcutaneously into athymic nude mice. Cells from the xenograft had typical morphology of human ALCL by standard hematoxylin-eosin staining, CD5+, CD45+ and CD30+ immunophenotype, the t(2;5) translocation by PCR. Six tumors from an initial set of mice were evaluated for apoptosis by TUNEL and for necrosis by hematoxylin-eosin staining 48-72 h after injection with 1 x 108 p.f.u. of AdWTp53 (adenoviral vector expressing p53), of AdNull (adenoviral vector backbone) and PBS (mock), respectively. TUNEL staining was positive only in tumors injected with AdWTp53 and was mainly localized around the needle track. Differences of the means of the counts of the necrotic cells were statistically significant at P = 0.02 between AdWTp53 and mock and only borderline between AdWTp53 and AdNull. Twenty-three tumors from a separate set of mice were subsequently injected with AdWTp53, AdNull and PBS and evaluated for in vivo tumor response. Three total injections of viral vectors (1 x 108 p.f.u.) and PBS were given every 48-72 h. Only tumors injected with AdWTp53 showed tumor growth inhibition with a mean final tumor volume that was statistically significantly smaller than AdNull (P = 0.007) and mock (P = 0.002). Based on these results we foresee a potential application of adenovirus-mediated p53 apoptosis as gene therapy of lymphomas.

In vitro adenoviral vector p53-mediated transduction and killing correlates with expression of coxsackie-adenovirus receptor and alpha(nu)beta5 integrin in SUDHL-1 cells derived from anaplastic large-cell lymphoma.

Adenoviral vector-mediated p53 expression induced apoptosis is a well established gene therapy approach that has been evaluated extensively in epithelial tumors but only recently in lymphoid malignancies mainly due to the known resistance of the lymphoid lineage to adenovirus infection. Recently, it was shown that this resistance is not absolute and that cell lines derived from anaplastic large cell lymphoma (ALCL) and some other lymphoid malignancies are efficiently transduced by adenoviral vectors. Normal circulating T lymphocytes do not express coxsackie-adenovirus receptor (CAR) and alpha(nu)beta integrins and are relatively resistant to infection by adenovirus. These molecules serve as receptors for adenovirus entry into the cells. ALCL-derived SUDHL-1 cells were evaluated for transduction efficiency and expression of p53 after infection with an adenoviral vector containing wild-type p53 (AdWTp53). Cells derived from ALCL and circulating mononucleated cells (MNCs) were also evaluated for expression of CAR and alpha(nu)beta integrins. AdWTp53-mediated expression of p53 resulted in p21/WAF1 induction, G1 arrest, and apoptosis in SUDHL-1 cells. The expression of CAR and alpha(nu)beta5 integrin was high in SUDHL-1 cells and comparable to levels observed with epithelial tumor cells, but it was absent in MNCs. The susceptibility to adenoviral vector transduction of the tumor-derived cells implies an important biological difference between them and circulating MNCs, possibly underlying the malignant transformation that ALCL cells undergo. Further studies will be required to evaluate this initial observation in more cell lines and tissue derived from ALCL.

Adenoviral vectors efficiently target cell lines derived from selected lymphocytic malignancies, including anaplastic large cell lymphoma and Hodgkin's disease.

We have hypothesized that adenoviral vectors might mediate gene transfer into cell lines derived from human lymphocytic malignancies, such as lymphoma, lymphocytic leukemia, and myeloma. A panel of 33 cell lines was studied for their ability to be transduced by an adenoviral (AD) vector encoding the Escherichia coli beta-galactosidase gene (AD-betagal). A cytochemical assay and a flow cytometry assay both demonstrated that a subset of lymphocytic cell lines can be efficiently transduced by adenoviral vectors. In particular, three of three anaplastic large cell lymphoma lines, two of two Hodgkin's disease cell lines, two of seven Burkitt's lymphoma cell lines, and three of five myeloma cell lines exhibited efficient gene transfer. The ability of an AD vector expressing the thymidine kinase (tk) gene from herpes simplex virus-1 (AD-tk) followed by ganciclovir (GCV) to kill 11 of these lymphocytic cell lines was studied. In eight of the cell lines tested, more than 68% of the cells were killed by AD-tk/GCV. Similar results were obtained using an adenoviral vector expressing the wild-type p53 tumor suppressor gene (AD-p53). Thus, AD-tk/GCV and AD-p53 both demonstrated efficient killing of these cell lines. These data document that adenoviral vectors are valuable reagents for the introduction of genes into selected lymphocytic cell lines. These data also suggest that adenoviral vectors might be useful for gene therapy of subsets of lymphocytic malignancy.

Ring appearance of Tc-99m MIBI thoracic SPECTs and increased uptake on Tc-99m HMDP thoracic SPECTs in a pulmonary mass of small cell carcinoma.

Tc-99m MIBI is taken up avidly by viable tumor tissue and does not accumulate in the necrotic carcinoma. We present a patient who underwent Tc-99m MIBI and Tc-99m HMDP thoracic SPECTs: a large area of increased MIBI uptake with central photopenia (ring appearance) in the right upper lung localizes bone imaging agent and does not localize multiple areas of intense uptake in the metastatic hilar mediastinum lymph nodes. Rapid growth of tumor cells in the lung leading to central necrosis/ischemia accounts for bone imaging agent localization in the tumor, as well as the ring-appearance of lung mass on Tc-99m MIBI imaging. These findings may reflect less viability of the lung tumor as compared with intense MIBI uptake in hilar/mediastinal lymph node uptake without bone agent localization.

Apo C-II deficiency type Bari.

We formerly studied an Italian family with apo C-II deficiency. Two probands were homozygous for the defect (unmeasurable circulating apolipoprotein C-II and absence of C-II bands on immunoelectrophoresis). We documented the synthesis of the protein at the intestinal level in the probands with immunohistological techniques. With the purpose of investigating the molecular basis of the defect, Southern analysis, polymerase chain reaction (PCR) amplification and sequence analysis were carried out on one of the two cases. We identified a point mutation C to G transversion in the third exon of the gene causing a premature stop codon. Our hypothesis is that the truncated protein of 36 aa., instead of 79 aa., lacks its functional domain. This causes inefficiency in the activation of lipoprotein lipase (LPL) and the instability of the circulating molecule, which could have an higher catabolic rate compared to a normal protein. The faster disappearance from the circulating compartment make it unmeasurable. The mutation destroys a Rsa I site, present in the normal gene sequence. We suggest the use of this site for a rapid Restriction Fragment Length Polymorphism (RFLP) on PCR amplification products to screen this defect in the Italian population.

Apolipoprotein C-II deficiency: detection of immunoreactive apolipoprotein C-II in the intestinal mucosa of two patients.

Recent data suggest that mutant immunoreactive forms of apolipoprotein C-II (apoC-II) can be detected in the plasma of patients with the apoC-II deficiency syndrome. We studied the possible presence of apoC-II mutants in the plasma of two patients with apoC-II deficiency by immunological means. The patients were hypertriglyceridemic, and apoC-II was undetectable in plasma as determined by radial immunodiffusion, electroimmunoassay, and immunonephelometry. Furthermore, apoC-II was undetectable either by electrophoresis or by immunoblotting in the plasma of the probands, while apoC-II was present in the plasma of their parents, although at less than half-normal concentration. Immunochemical localization of apoC-II, however, showed that the apoprotein could be detected within the enterocytes obtained from the intestinal mucosa of the patients. From these data we conclude that the patients synthesize apoC-II, at least in the intestine.