Craig Y. Okada

Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles

We have developed a new procedure for introducing macromolecules into cultured mammalian cells based on osmotic lysis of pinocytic vesicles. Cells are first incubated in culture medium containing 0.5 M sucrose, 10% polyethylene glycol 1000 and the macromolecule to be transferred. Cells are then placed in medium diluted with 0.66 parts water. Most pinocytic vesicles formed in the presence of sucrose burst in hypotonic medium, thereby releasing the enclosed macromolecule. L929 cells remain fully viable after a single hypertonic sucrose treatment, and a majority survives four successive rounds of osmotic lysis. This procedure, termed osmotic lysis of pinosomes, has been used to transfer substantial amounts of horseradish peroxidase, antiricin antibodies and dextran 70,000 into the cytosol of L929 cells. Direct comparison of the degree of ricin resistance conferred by transfer of antiricin antibodies revealed pinosome lysis to be equal, if not superior, to injection mediated by red blood cells.

The murine T-cell receptor uses a limited repertoire of expressed V|[beta]| gene segments

Only 10 different Vβ gene segments were found when the sequences of 15 variable (Vβ) genes of the mouse T-cell receptor were examined. From this analysis we calculate that the total number of expressed Vβ gene segments may be 21 or fewer, which makes the expressed germline Vβ repertoire much smaller than that of the immunoglobulin heavy-chain or light-chain genes. We suggest that βchain somatic diversification is concentrated at the Vβ−Dβ−Jβ junctions.

Tumor Cell Lysate-Pulsed Dendritic Cells Are More Effective Than TCR Id Protein Vaccines for Active Immunotherapy of T Cell Lymphoma

TCR Id protein conjugated to keyhole limpet hemocyanin (KLH) (TCR Id:KLH) and injected with a chemical adjuvant (QS-21) induces a protective, Id-specific immune response against the murine T cell lymphoma, C6VL. However, Id-based immunotherapy of C6VL has not demonstrated therapeutic efficacy in tumor-bearing mice. We report here that C6VL lysate-pulsed dendritic cells (C6VL-DC) vaccines display enhanced efficacy in both the prevention and the therapy of T cell lymphoma compared with TCR Id:KLH with QS-21 vaccines. C6VL-DC vaccines stimulated potent tumor-specific immunity that protected mice against lethal challenge with C6VL and significantly enhanced the survival of tumor-bearing mice. Tumor-specific proliferation and secretion of IFN-γ indicative of a Th1-type immune response were observed upon ex vivo stimulation of vaccine-primed lymph node cells. Adoptive transfer of immune T cell-enriched lymphocytes was sufficient to protect naive recipients from lethal tumor challenge. Furthermore, CD8+ T cells were absolutely required for tumor protection. Although C6VL-DC and control vaccines stimulated low levels of tumor-specific Ab production in mice, Ab levels did not correlate with the protective ability of the vaccine. Thus, tumor cell lysate-pulsed DC vaccines appear to be an effective approach to generate potent T cell-mediated immune responses against T cell malignancies without requiring identification of tumor-specific Ags or patient-specific Id protein expression.

Cladribine plus rituximab is an effective therapy for newly diagnosed mantle cell lymphoma

Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma that is incurable with standard chemotherapy. There is no consensus on the best initial therapy, especially for elderly patients, who are not candidates for aggressive treatment approaches. Current National Comprehensive Cancer Network (NCCN) treatment guidelines include rituximab (R) plus cladribine for the initial treatment of MCL. However, few data are available to substantiate this recommendation. Therefore, to further define the role of R-cladribine for the initial treatment of MCL, we performed a retrospective chart review of 31 patients with MCL (median age, 67) treated with R-cladribine. The majority of responding patients also received R maintenance. The overall response rate was 87%, with 61% of patients achieving a complete remission (CR/CRu). The estimated median follow-up was 32.5 months, median PFS was 37.5 months, and median OS was 85.2 months. One of 19 (5.3%) subjects in CR/CRu relapsed (median follow-up of 23 months). CR/CRu was associated with improved survival (p < 0.0001), while a high mantle cell international prognostic index (MIPI) was associated with worse survival (p = 0.05). There was one toxic death (neutropenic pseudomonal sepsis) related to treatment. R-cladribine is an effective therapy for previously untreated MCL, and these results validate the use of R-cladribine for the initial treatment of MCL.

A rapid procedure for assaying nicotinamide phosphoribosyltransferase

Abstract A precipitation-filtration assay which measures the activity of nicotinamide phosphoribosyltransferase is described. The assay, based on the differential solubility of nicotinamide and nicotinamide mononucleotide in acetone, is rapid, convenient, and inexpensive when compared to chromatographic methods. It is sensitive enough to detect the formation of as little as 10 pmol of nicotinamide mononucleotide under the reaction conditions described. With minor modifications, the assay procedure can be adapted for measuring a variety of other enzymes.

TCR vaccines against a murine T cell lymphoma: a primary role for antibodies of the IgG2c class in tumor protection.

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8α knockout, μMT, and FcγR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1−/− splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-γ knockout mice demonstrated a requirement for IFN-γ, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-γ knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.

Galloping ophthalmoplegia and numb chin in Burkitt lymphoma.

A 57-year-old man developed complete bilateral ophthalmoplegia over a period of 10 days, together with bilateral facial pain and numbness of the chin. He had no other clinical manifestations. Findings on brain magnetic resonance imaging and spinal fluid formula from the first lumbar puncture were normal, but cerebrospinal fluid flow cytometry disclosed a kappa restriction monoclonal B-cell population, indicating malignant lymphoma. Computed tomography of the chest, abdomen, and pelvis then revealed multiple enlarged lymph nodes. Biopsy of an inguinal node showed findings consistent with Burkitt lymphoma. Within six weeks, intravenous and intrathecal chemotherapy resolved all neurologic findings except a partial right-side sixth nerve palsy and mild chin numbness. Eighteen months after disease onset, the patient remained in remission. Meningeal spread of Burkitt lymphoma is not commonly a presenting feature in immunocompetent adults. Chin numbness, a characteristic feature caused by infiltration of the mental nerve, should facilitate earlier recognition, which may be life saving.

T Cell Antigen Receptor Vaccines for Active Therapy of T Cell Malignancies

Abstract: T cell lymphoproliferative disorders continue to be serious management problems, and so alternative therapeutic modalities are continuously being explored. One such strategy involves immunotherapy using the T cell receptor (TCR) as a target. Specifically we are attempting to develop a T cell receptor idiotype (TCR‐Id) vaccine because the TCR‐Id can serve as a tumor‐specific antigen. In this article we will briefly review the rationale for TCR‐Id vaccines, the preclinical models as developed in our laboratory, and a discussion of our current plans for a vaccine trial in mycosis fungoides.

Diffuse large B-cell lymphoma in adults aged 75 years and older: a single institution analysis of cause-specific survival and prognostic factors

Background: Very elderly patients (75 years and older) with diffuse large B-cell lymphoma (DLBCL) will be increasingly considered for cancer treatment as the population ages, but are underrepresented in clinical trials. Here we report outcomes of very elderly DLBCL patients treated in the modern era at the Oregon Health and Science University (OHSU). Methods: We queried the OHSU Tumor Registry for DLBCL cases treated since 2002. A total of 73 patients aged 75 years or older were analyzed under Institutional Review Board approval. Results: With a median follow up of 31 months, cause-specific survival was 58% and overall survival 51% at 3 years. Incorporation of an anthracycline did not influence outcomes. More than one extranodal site or poor-risk disease by Revised International Prognostic Index score were adversely prognostic, but pathologic features studied were not. Conclusions: Very elderly patients with DLBCL require prospective studies, which employ novel risk stratification and therapeutic approaches.

Outcomes of DA-EPOCH-R induction plus autologous transplant consolidation for double hit lymphoma

Abstract High-grade B cell lymphoma with MYC and BCL2 rearrangements (double hit) has a poor prognosis with standard R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). We report here a treatment algorithm of DA-EPOCH-R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, rituximab) followed by BEAM (carmustine, etoposide, cytarabine, melphalan) autologous transplant in 36 cases of previously untreated double hit lymphoma (DHL) from 2010 to 2015. A high risk International Prognostic Index (IPI) was present in 42% of cases. At median follow-up of 38 months, the 2-year progression free survival (PFS) and overall survival (OS) were 69% (95% CI 54–84%) and 71% (95% CI 56–86%). Eight cases were refractory to induction with 1-year OS 20%, and no factors were predictive for primary refractory disease. Of 28 responders, 17 proceeded to transplant while 11 were observed, primarily due to age and co-morbidities. By 24-week landmark analysis after diagnosis, the 2-year PFS and OS were both 94% (95% CI 83–100%) vs 79% (95% CI 52–100%) for transplant vs observation (p = .59 for both PFS and OS). There was no significant benefit to consolidative transplant in our series, and primary refractory DHL needs novel approaches.