Citations

Abstract Objectives Distinguishing between T-cell and natural killer (NK)–cell neoplasms could be difficult given their overlapping immunophenotype. In this study, we investigated whether a flow cytometry assay with cytoplasmic staining for CD3 could be used for this purpose. Methods Flow cytometry immunophenotyping was performed on 19 surface CD3 (sCD3)–negative mature T-cell neoplasms, 10 sCD3-positive mature T-cell neoplasms, 13 mature NK-cell neoplasms, and 19 normal controls. In addition to routine antibody panels (CD2, sCD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A TCRγ/δ), cytoplasmic staining for a monoclonal CD3 antibody (clone SK7/Leu-4) was assessed in all cases. A molecular study for T-cell receptor (TCR) gene rearrangement and an immunohistochemical study for TCRβ were performed. Results Our data showed all T-cell neoplasms were uniformly positive for cytoplasmic CD3 (cCD3) regardless of sCD3 expression, whereas 85% of NK-cell neoplasms completely lacked cCD3 expression. The 2 cases with classic NK-cell immunophenotype but partial cCD3 expression showed no molecular genetic features of T-cell lineage by TCR gene rearrangement studies. Conclusions Uniform cCD3 positivity and homogeneous cCD3 negativity highly suggest T-cell and NK lineage, respectively. When partial cCD3 expression is encountered, additional confirmatory studies should be pursued for the most accurate lineage assignment.

Metabolites control immune cell functions, and delivery of these metabolites in a sustained manner modulate the function of the immune cells.

Abstract Background Flow cytometric detection of T‐cell clonality is challenging. The current available methodology for T‐cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T‐cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells. Methods Here, we used a single TRBC1 antibody, in conjunction with other T‐cell associated markers, to detect T‐cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T‐cell neoplasm and 97 patients with no T‐cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1. Results We examined TRBC1 expression on neoplastic T‐cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T‐cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T‐cell neoplasms, and arbitrarily dissected T‐cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets. Conclusions Single TRBC1 antibody detection of T‐cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.

Abstract Activation of NFkB has been noted in late B-cell malignancies, but no genetic basis for this activation has been shown except for rare mutations of the NFkB pathway in lymphoma and none have been described in MM. Bortezomib, highly tumoricidal in a subset of MM patients, inhibits NFkB suggesting it’s role as a therapeutic target. Here we show for the first time the critical importance of mutations that dysregulate this potential survival pathway in human myeloma cell lines (HMCL) and new diagnosis MM patients. We performed high-density oligonucleotide array CGH (Agilent 44k) on CD138+ selected MM cells from 68 patients and 42 unique HMCL. Initially we focused on small loci, bi-allelically deleted, as markers for inactivation of potential tumor suppressor genes. We next developed FISH probes for the identified loci to confirm the bi-allelic deletions seen by aCGH. We next sequenced all samples with monoallelic deletions, and additional unselected patients, and identified additional inactivating mutations. Multiple novel tumor suppressor genes (TSG) were identified and validated, whose loss of function (both in patients and HMCL) leads to activation of the non-canonical NFkB pathway including: TRAF2 (9q34), TRAF3 (14q32), BIRC2/BIRC3 (11q22) and CYLD (16q12). Functional studies showed that HMCL with deletion or mutations in these genes have markedly increased NFkB2 p52/p100 ratios relative to other HMCL (and for BIRC2/BIRC3 relative to an isogenic line that retains one copy of BIRC2/BIRC3), indicating genetic activation of the non-canonical NFkB pathway. Reintroduction of deleted TSG into HMCL inhibited cell growth and resulted in cell death (see data on abstract by J. Keats et al). We next focused our analysis (of combined aCGH and GEP data) on positive regulators of this pathway. Rare IgH translocations of NIK have recently been identified in MM. By aCGH we identified amplification and rearrangements of NIK. We identified dysregulated expression of TNF family receptors that activate the non-canonical pathway: CD40 (translocation), TACI (amplification) and LTBR. All of the HMCL with these mutations also showed increased NFkB2 p52/p100 ratios, consistent with activation of the non-canonical NFkB. These mutations correlate with a gene expression profile of NFkB activation that is detected in one half of patients. In summary, we have determined for the first time the genetic basis for constitutive NFkB activation - present in 19/42 HMCL, and we estimate in at least one quarter of newly diagnosed patients (approximately one half of those with evidence of NFkB activation by GEP). We propose a mechanism in which positive (TACI, CD40, LTBR) and negative regulators (TRAF2, TRAF3, BIRC2/BIRC3, CYLD) of the pathway converge on NIK to mediate the processing of NFKB2 p100 to p52. The preponderance of genetic evidence suggests that drugs that target the non-canonical NFkB pathway, will be most effective in treating the 50% of MM patients with NFkB activation.

Abstract We have conducted systematic small interfering RNA (siRNA) lethality screening of the kinome and “druggable genome” in human myeloma cell lines (HMCL) to functionally generate a comprehensive map of critical genes and vulnerabilities in human myeloma tumors. KMS11 human myeloma cells were screened with both an 1800 oligo (639 gene) siRNA library targeting the kinome and with a 13,984 oligo (6,791 gene) library targeting the druggable genome in duplicate using optimized conditions that resulted in >95% transfection efficiency and <5% background cytotoxicity. siRNA were used at low concentration (13nM) to minimise off-target cellular effects. Each gene was initially screened with at least 2 distinct siRNA. Universally lethal and non-silencing siRNA were tested in parallel as controls. Viability was measured at 96 hours by ATP-dependent luminescence and was normalized to the mean and distribution of controls by the B-score method. Approximately 14% of kinome siRNA and 5.8% of druggable genome siRNA caused important reductions in HMCL viability (greater than three standard deviations from control samples treated with non-silencing siRNA), compared with an anticipated rate due to chance of only 0.135%. Short-listed kinome vulnerabilities were validated by repeat targeting in a second lethality screening experiment in KMS11 using a total of four independent siRNA per gene. Forty-two kinases were confirmed as critical for myeloma cell survival. Unsupervised pathway analysis indicates that approximately half are concentrated within established pathways involved in cytokine (VEGFR, FGFR3, IGFR/IL6R) signaling and regulation of cell cycle, apoptosis or metabolism. The role of other survival kinases in myeloma are novel. The selectivity of kinome vulnerabilities identified in KMS11 for myeloma cells versus other tissues was examined by parallel silencing of these targets in KMS11 and JJN3 myeloma cells and in non-myeloma 293 and A549 epithelial cells. The most lethal kinase targets in myeloma that simultaneously show experimental evidence of tumor selectivity include aurora kinase A (AURKA), G-protein receptor coupled kinase 6 (GRK6) and protein kinase N1 (PKN1). By gene expression analyses, PKN1 and GRK6 are both preferentially expressed in plasma cells, compared with an analysis of >50 somatic tissues in which they are absent or only weakly expressed. The extent to which the kinome vulnerabilities identified in KMS11 occur in other HMCL was assessed by tertiary siRNA screening and by lentiviral shRNA techniques. More than 75% of kinases vulnerable in KMS11 cells are recurrently vulnerable in other myeloma tumor lines. Secondary validation studies of all genomic vulnerabilities identified from the much larger siRNA lethality screen of the druggable genome in myeloma cells is ongoing, however, to date 134 genes have been validated as essential myeloma survival factors during primary screening by concordant lethality of 2/2 independent siRNA (compared with an expected rate due to chance of n<1 genes). Significantly, proteosome subunits and specific ubiquitin ligases number amongst the most potently lethal genomic vulnerabilities in KMS11. Overall, this attempt to systematically and functionally document all genetic vulnerabilities in myeloma provides a strong rational impetus for the development of novel therapies that optimally target susceptibilities inherent within myeloma tumors.

Abstract HACS1 is a SH3 (Src homology 3) and SAM (sterile alpha motif) domain containing adaptor protein that is expressed in activated B and dendritic cells, is up-regulated by Interleukin 4 in the process of B cell activation and likely serves to dampen the immune response. To elucidate the function of HACS1, we generated HACS1 gene knockout mice by deletion of the SH3 and SAM domains. HACS1−/− mice were viable and fertile and had normal bone marrow B cell development and normal splenic T and B cell populations. However, adult HACS1−/− mice had increased numbers of peritoneal B1 cells (IgM+CD5+). Upon LPS plus BCR or TCR stimulations, splenic cells from HACS1−/− mice demonstrated an upregulation of activation markers with increased intensity of CD23 expression or increased population of CD69 positive cells. Purified B220+ splenic B cells from HACS1−/− mice showed increased cell proliferation upon BCR stimulation. Both T helper type 1 (Th1) and T helper type 2 (Th2) humoral responses were enhanced in HACS1−/− mice upon immunization with T cell-dependent antigen NP-KLH, which resulted in increased production of interferon-gamma (IFN-γ), anti-NP IgG2a and IL-4, as well as anti-NP IgG1 and IgE. Upon immunization with T cell-independent antigens such as TNP-LPS and TNP-Ficoll, HACS1−/− mice had increased production of anti-TNP IgM and IgG3 as compared to normal controls. The in vitro maturation of bone marrow-derived dentritic cells from HACS1−/− mice was similar to wild-type mice but HACS1−/− dentritic cells showed increased IL-12 production upon stimulation with anti-CD40 or LPS. There was no significant difference in antigen uptake by cultured dentritic cells from non-immunized wild-type or knockout mice. However, in immunized mice, an increase in antigen uptake in HACS1−/− dentritic cells was observed. We further demonstrate that the HACS1−/− B cells had increased tyrosine phosphorylation in the resting state. As deletion of the SH3 & SAM domains of HACS1 appears to generate a hypersensitive immune response, our results collectively support that HACS1 negatively regulates adaptive immunity.

Abstract Bortezomib and related proteasome inhibitors have profound pre-clinical and clinical activity in Multiple Myeloma (MM). However, the critical molecular target(s) modulated by proteosome inhibition remains unknown. The overall response rate to bortezomib in relapsed and refractory MM patients is consistently 35%–50%, suggesting specific molecular targets or resistance mechanisms are inherent in patients. None of the known effects of bortezomib has been shown to correlate with clinical responses in patients or to explain the unique sensitivity of MM (as opposed to other malignancies) to these reagents. Therefore, in this study, we have conducted a small interfering RNA (siRNA) screen of the druggable genome in human myeloma cells to identify genes that modulate sensitivity to bortezomib. KMS11 human myeloma cells were reverse transfected with 13,984 synthetic short interfering RNAs (siRNA) against each of 6992 druggable targets (2 siRNA/gene with a single-siRNA per well/384 well format). At 24 hours post transfection, bortezomib was added at IC10, 30, 60 and 90. Cell viability was measured by ATP-dependent luminescence assay 72 hours post drug treatment. The resulting data from primary screening was evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >99% global transfection efficiency, <0.25 CV values across the dataset with minimal plate-to-plate and set-to-set variation observed. From analysis of the primary screening data, a core panel of 320 (4.6% of druggable targets in library) bortezomib sensitizers were identified. 187 (58%) of these were further confirmed in a secondary screening experiment by multiple (2 to 4) independent siRNA. Among these 187 genes (2.6% of the genes investigated), some known targets, such as proteasome subunits are evident. Other genes belonging to the same functional gene families (e.g. TNF receptor family, Ras family, kinases, oncogenes and transporter proteins) or functioning within the same network of signal transduction (e.g. NF-kB, Wnt and estrogen receptor pathways) are evident. Our primary screen also identified genes that attenuate bortezomib-induced cytotoxicity. Members in this group include genes implicated in the negative regulation of cell proliferation and survival, such as NF-kB inhibitors, JNK pathway activators, tumor suppressors, apoptosis inducers and phosphatases. These studies enable dissection of the molecular mechanism of bortezomib in myeloma cells and may serve to point to biomarkers for response prediction and novel chemotherapeutic combination therapies.

AbstractRapid and functional bone‐tendon (B‐T) healing remains a difficulty in clinical practice. Tissue engineering has emerged as a promising strategy to address this problem. However, the majority of tissue engineering scaffolds are loaded with stem cells to enhance the regenerability in B‐T healing, which is complicated and inconvenient for clinical application. Accordingly, developing a cell‐free scaffold with chemotactic function and chondrogenic inducibility may be an effective approach. In this study, a collagen affinity peptide derived from the A3 domain of von Willebrand factor (a hemostasis factor) was fused into the C‐terminal of a stromal cell‐derived factor‐1α (SDF‐1α) to synthesize a recombinant SDF‐1α capable of binding collagen and chemotactic activity. The recombinant SDF‐1α was then tethered on the collagen fibers of a book‐shaped acellular fibrocartilage scaffold (BAFS), thus fabricating a novel scaffold (C‐SDF‐1α/BAFS) with chemotactic function and chondrogenic inducibility. In vitro tests determined that this scaffold was noncytotoxic and biomimetic, could attract stem cells migrating to the scaffold using sustainably released C‐SDF‐1α, and inducedthe interacting stem cells down the chondrogenic lineage. In vivo, the C‐SDF‐1α/BAFS significantly enhanced the B‐T healing in a rabbit partial patellectomy model, as shown by the larger cartilaginous metaplasia region, better fibrocartilage regeneration, additional bone formation, and improved biomechanical properties. Therefore, the findings of the study demonstrate that the C‐SDF‐1α/BAFS could potentially be applied for B‐T healing.

We examined the impact of a group-based self-empowerment intervention among diabetes patients, which uses multidisciplinary education, collaborative learning, peer support, and development of diabetes-specific social capital to improve glycemic control and weight management. Thirty-five patients who had primary care established at the Prisma Health Upstate, Internal Medicine Resident clinic and held the diagnosis of diabetes for longer than one year were recruited for our single-arm pilot intervention. Each group intervention session involved one to two internal medicine resident physician facilitators, a clinical diabetic educator, and 5–10 patients. Each session had a framework facilitated by the resident, with most of the discussion being patient-led, aiming to provide a collaborative learning environment and create a support group atmosphere to encourage self-empowerment. Patients’ hemoglobin A1c level and body mass index (BMI) before the intervention and 3 to 6 months after completion were collected from the laboratory results obtained in the participants’ routine clinic visits. All graduates from this three-week intervention were invited to attend monthly maintenance sessions, and we tracked the HgbA1c measures of 29 JUMP graduates one year after the intervention, even though 13 of the 29 chose not to participate in the monthly maintenance sessions. The pre-intervention HgbA1c level averaged 8.84%, whereas the post-intervention HgbA1c level averaged 7.81%. A paired t test showed that this pre–post difference of 1.03 percentage points was statistically significant (p = 0.0007). For BMI, there was an average decline of 0.78 from the pre-intervention mean value of 40.56 to the post-intervention mean value of 39.78 (p = 0.03). Among the 29 participants who agreed to participate in our follow-up measure of their HgbA1c status one year after the intervention, a paired t test showed that there was no significant difference between the post-JUMP measure and the follow-up measure (p = 0.808). There was no statistically significant difference between the HgbA1c level of those participating in the maintenance program and that of those not participating (post-intervention t test of between-group difference: p = 0.271; follow-up t test of between-group difference: p = 0.457). Our single-arm, pilot study of the three-week group intervention of self-empowerment shows promising results in glycemic control and weight loss. The short duration and small number of sessions expected could make it more feasible for implementation and dissemination as compared with popular intervention protocols that require much longer periods of attendance, if the effectiveness of this patient group-based self-empowerment approach can be further established by randomized controlled studies in the future.

Abstract Great heterogeneity in survival exists for patients newly diagnosed with diffuse large B-cell lymphoma (DLBCL). Three scoring systems incorporating simple clinical parameters (age, lactate dehydrogenase, number/sites of involvement, stage, performance status) are widely used: the International Prognostic Index (IPI), revised IPI (R-IPI), and National Comprehensive Cancer Network IPI (NCCN-IPI). We evaluated 2124 DLBCL patients treated from 1998 to 2009 with frontline rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP; or variant) across 7 multicenter randomized clinical trials to determine which scoring system best discriminates overall survival (OS). Median age was 63 years, and 56% of patients were male. Five-year OS estimates ranged from 54% to 88%, from 61% to 93%, and from 49% to 92% using the IPI, R-IPI, and NCCN-IPI, respectively. The NCCN-IPI had the greatest absolute difference in OS estimates between the highest- and lowest-risk groups and best discriminated OS (concordance index = 0.632 vs 0.626 [IPI] vs 0.590 [R-IPI]). For each given IPI risk category, NCCN-IPI risk categories were significantly associated with OS (P ≤ .01); the reverse was not true, and the IPI did not provide additional significant prognostic information within all NCCN-IPI risk categories. Collectively, the NCCN-IPI outperformed the IPI and R-IPI. Patients with low-risk NCCN-IPI had favorable survival outcomes with little room for further improvement. In the rituximab era, none of the clinical risk scores identified a patient subgroup with long-term survival clearly <50%. Integrating molecular features of the tumor and microenvironment into the NCCN-IPI or IPI might better characterize a high-risk group for which novel treatment approaches are most needed.