George Deeb

Immunomodulatory drugs stimulate natural killer‐cell function, alter cytokine production by dendritic cells, and inhibit angiogenesis enhancing the anti‐tumour activity of rituximab in vivo

The immunomodulatory drugs (IMiDs) lenalidomide and actimid (also known as CC‐4047) are thalidomide analogues which are more potent than their parental compound. In combination with rituximab, we have previously demonstrated that IMiDs have synergistic in vivo anti‐tumour activity in preclinical studies in a human lymphoma severe combined immunodeficiency mouse model. This report further explored the mechanisms by which IMiDs exert their anti‐lymphoma effects. Following exposure of subcutaneous lymphoma tumours in murine models to IMiDs, there was a significant increase in the recruitment of natural killer (NK) cells to tumour sites. This increase in NK cells was mediated via stimulation of dendritic cells and modification of the cytokine microenvironment associated with an increase in monocyte chemotactic protein‐1, tumour necrosis factor‐α and interferon‐γ and probably augmented rituximab‐associated antibody‐dependent cellular cytotoxicity. IMiDs also had significant anti‐angiogenic effects in our B‐cell lymphoma models. Thus, by modulation of the immune system mediated via dendritic cells and NK cells, changing the cytokine milieu, as well as by their anti‐angiogenic effects, IMiDs in combination with rituximab resulted in augmented in vivo anti‐tumour effects against B‐cell lymphoma. Our positive preclinical data adds additional support for the evaluation of IMiDs plus rituximab in patients with relapsed/refractory B‐cell lymphoma.

Higher response to lenalidomide in relapsed/refractory diffuse large B-cell lymphoma in nongerminal center B-cell-like than in germinal center B-cell-like phenotype.

There is a need to develop novel therapies for relapsed/refractory diffuse large B‐cell lymphoma (DLBCL) and to identify biomarkers predictive for therapeutic response. Lenalidomide was previously shown to induce an overall response rate (ORR) of 28% in patients with relapsed/refractory DLBCL. It is currently unknown if response rates differ between patients with different DLBCL subtypes.

Ofatumumab demonstrates activity against rituximab-sensitive and -resistant cell lines, lymphoma xenografts and primary tumour cells from patients with B-cell lymphoma

Ofatumumab is a new monoclonal antibody (mAb) targeting a novel membrane‐proximal epitope on CD20. To better define ofatumumab’s activity, we conducted pre‐clinical studies in rituximab‐sensitive cell lines (RSCL), rituximab‐resistant cell lines (RRCL), ofatumumab‐exposed cell lines (OECLs), primary lymphoma cells, and a lymphoma xenograft model. RRCL and OECL were generated by repeated exposure of sensitive cells to escalating doses of rituximab or ofatumumab ± human serum. Antibody‐dependent cellular cytotoxicity (ADCC) and complement‐mediated cytotoxicity (CMC) assays were performed to assess cellular sensitivity to rituximab or ofatumumab. Ofatumumab elicited a higher rate of CMC in RSCL, RRCL and primary tumour cells. The chronic exposure of lymphoma cells to ofatumumab resulted in rituximab resistance but less ofatumumab resistance. In an in vivo severe combined immunodeficiency mouse model of human lymphoma, ofatumumab prolonged median survival compared to rituximab. While rituximab CMC diminished with CD20 down‐regulation in RRCL passages, ofatumumab activity in vitro diminished to a lesser degree. Our data suggest that ofatumumab is more potent than rituximab in rituximab‐sensitive or rituximab‐resistant models and has the potential to decrease the development of biological resistance in patients with repeated exposure to anti‐CD20 mAbs.

Hypoxia-inducible factor-1α protein expression is associated with poor survival in normal karyotype adult acute myeloid leukemia

We examined the predictive impact of HIF-1α protein expression on clinical outcome of 84 normal karyotype acute myeloid leukemia (NK-AML) patients (median age 66.5 years) at our institute. Thirty percent of NK-AML cells expressed cytoplasmic HIF-1α. In univariate analysis, low HIF-1α (≤ 5%, n = 66) was associated with improved event-free survival (p = 0.0453, HR = 0.22). Multivariate analysis incorporating age, complete remission, FLT3-ITD mutation, and marrow blast percentage demonstrated that HIF-1α was independently associated with poorer overall and event-free survival. HIF-1α expression correlated with VEGF-C but not VEGF-A, marrow angiogenesis, FLT3 ITD or NPM1 mutations. These results support HIF-1α as an outcome marker for NK-AML.

Distinct cellular and therapeutic effects of obatoclax in rituximab-sensitive and -resistant lymphomas

Bcl‐2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3‐mimetic, has been designed to specifically target and counteract anti‐apoptotic Bcl‐2 proteins. We evaluated the biological effects of obatoclax on the anti‐tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy‐sensitive (RSCL), ‐resistant cell lines (RRCL) and primary tumour‐cells derived from patients with B‐cell lymphomas (N = 39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase‐independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient‐derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin‐1 knockdown. In summary, obatoclax is an active Bcl‐2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.

Rituximab-based therapy for gemcitabine-induced hemolytic uremic syndrome in a patient with metastatic pancreatic adenocarcinoma: a case report.

PurposeThe purpose of this report is to describe the management and outcome of an unusual complication of a commonly used chemotherapeutic agent. Gemcitabine is a known risk factor for hemolytic uremic syndrome (HUS), which can often have a rapidly fatal clinical course despite intervention with steroids, plasmapheresis and hemodialysis.MethodsA retrospective report of the first case of gemcitabine-related HUS, in a patient with metastatic pancreatic adenocarcinoma, treated with a variety of standard therapies in addition to rituximab is presented. The hematologic response parameters and clinical outcomes to each of the therapies given are described.ResultsChemotherapy-induced HUS was aggressively treated with plasmapheresis, high-dose steroids, vincristine and rituximab. Platelet recovery and clinical improvement coincided with administration of rituximab. In addition, aggressive supportive measures to manage renal failure (hemodialysis) and labile hypertension, allowed this patient to have an extended survival as a result of successful therapy for this complication despite an underlying rapidly fatal malignancy.ConclusionThis case highlights the importance of timely application of aggressive measures even in patients with known diagnosis of a fatal malignancy as these interventions can prolong life and be of palliative benefit.

Plasma cell leukaemia and other aggressive plasma cell malignancies.

Extramedullary plasma cell cancers, such as plasma cell leukaemia (PCL) and multiple extramedullary plasmacytomas (MEP) are very aggressive malignancies. These can be primary (de‐novo) or secondary due to progressive prior multiple myeloma (MM). Recent reports suggest an increase in incidence of these disorders. Compared to MM, organ invasion is common in PCL, while soft tissue tumors involving the head, neck or paraspinal area are common sites for MEP. Markers of poor prognosis are frequently observed in these extramedullary forms of plasma cell cancers, and survival is significantly inferior compared to patients with MM. Conventional chemotherapeutic and radiotherapy approaches have been employed with variable results. Even high dose chemotherapy with autologous stem cell rescue has not been able to demonstrate consistent improvement in survival outcome. Although not specifically evaluated, novel anti‐plasma cell agents, such as the proteasome inhibitor bortezomib, and immunomodulatory drugs, such as lenalidomide, appear to be active against these aggressive cancers. Clinical and translational research directed at improved understanding of disease biology and development of novel therapeutics is urgently needed.

CD52 over-expression affects rituximab-associated complement-mediated cytotoxicity but not antibody-dependent cellular cytotoxicity: Preclinical evidence that targeting CD52 with alemtuzumab may reverse acquired resistance to rituximab in non-Hodgkin lymphoma

In an attempt to define mechanisms by which B-cell non-Hodgkin lymphoma (NHL) may escape rituximab immunotherapy, we developed several rituximab-resistant cell lines (RRCL) generated from the rituximab-sensitive cell lines (RSCL) Raji and RL. Rituximab resistance was associated with CD20 downregulation and upregulation of CD52 and the complement inhibitory proteins (CIPs) CD55 and CD59. No significant alemtuzumab-associated complement-mediated cell lysis (CMC) or antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in RSCL. In contrast, in vitro exposure of RRCL to alemtuzumab resulted in a significant degree of CMC and ADCC. Of note, in vitro blocking of CD52 with anti-CD52 F(ab’)2 fractions in RRCL improved rituximab-associated CMC as compared to unblocked RRCL. Our current data provides a basis for further evaluation of alemtuzumab-based clinical trials for patients with rituximab-resistant NHL.

Genomic profiling of myeloid sarcoma by array comparative genomic hybridization.

Myeloid sarcoma (MS) is a tumor mass of myeloblasts or immature myeloid cells occurring in an extramedullary site. In this study, seven cases of MS [stomach (1), testis (1), skin (2), and lymph node (3)] and 3 synchronous and 1 follow‐up bone marrow (BM) samples were studied for genomic abnormalities using array comparative genomic hybridization (array‐CGH). Array‐CGH construction used ∼5,400 bacterial artificial chromosome clones from the RPCI‐11 library, spanning the human genome. Data were analyzed using the DNAcopy software and custom heuristics. All MS cases had genomic abnormalities detected by array‐CGH. Unbalanced genomic abnormalities in five MS cases were confirmed by conventional cytogenetics (CC) and/or fluorescence in situ hybridization (FISH); these abnormalities included loss of 4q32.1‐q35.2, 6q16.1‐q21, and 12p12.2‐p13.2 and gain of 8q21.2‐q24.3, 8, 11q21‐q25, 13q21.32‐q34, 19, and 21. Array‐CGH was also invaluable in identifying possible deletions, partner translocations, and breakpoints that were questionable by CC. The remaining two MS cases had genomic aberrations detected by array‐CGH, but were not studied further by CC/FISH. Chromosome 8 was most commonly abnormal (3/7 cases). Identical genomic abnormalities were demonstrated in MS and in synchronous BM in two cases. These results demonstrate that array‐CGH is a powerful tool to screen MS tissue for unbalanced genomic abnormalities, allowing identification of chromosome abnormalities when concurrent BM is nonanalyzable or nonleukemic.

Monoallelic and biallelic deletions of 13q14.3 in chronic lymphocytic leukemia FISH vs miRNA RT-qPCR detection

Deletion of 13q14.3 (del(13q)) is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL) and implies a favorable prognosis. We explored the feasibility of detecting del(13q) by real-time quantitative polymerase chain reaction (PCR) for miR-15a and miR-16-1, whose loci are located in the deleted region. We analyzed 23 cases of B-CLL with monoallelic (10 cases) or biallelic del(13q) (5 cases) and used trisomy 12-positive CLL samples (n = 8) as control samples. As expected, miR-15a was expressed at significantly lower levels in monoallelic del(13qx1) samples compared with trisomy 12 control samples (P = .001). Biallelic del(13q) (del(13qx2)) samples showed further reduction of miR-15a levels compared with monoallelic del(13q) (del(13qx1)) (P = .009). In contrast, miR-16-1 expression levels were generally much lower and variable, with the highest levels detected in del(13qx1). Analyzed retrospectively, miR-15a levels differ among the del(13q) groups. However, only del(13qx2) miR-15a levels are reduced enough to determine the allelic status of an individual sample prospectively by real-time quantitative PCR.