Gustafson, Michael P.

Abstract Abstract 1362 The role of anti-tumor immunity is not well understood in CLL but does have the potential to be manipulated to improve treatment responses. We studied the predictive values of 43 immune phenotypes in patients enrolled in a clinical trial of early treatment of high risk CLL. The study enrolled 33 patients with CLL diagnosed using standard NCI-WG96 criteria who had previously untreated early - intermediate stage disease and did not meet NCI-WG96 guidelines for conventional treatment. High risk disease was defined as at least one of the following: 17p13 deletion; 11q22 deletion or unmutated IGHV together with either expression of ZAP-70 (>20%) or CD38 (>30%). Patients were treated with GM-CSF 250 mcg sc 3 × week × 5 weeks, alemtuzumab sc with dose escalation over 3 days then 30 mg/d 3 × week × 4 weeks, and rituximab 375 mg/m2 weekly × 4 weeks for a total duration of therapy of 5 weeks. We measured immune phenotypes (representing Tregs, naïve, effector memory, central memory T cells, B cells, NK cells, monocytes, among others) at the time of enrollment, through treatment and at 1 year after enrollment in 28 patients. We identified pre-treatment immune phenotypes that correlated with disease burden (as measured by the number of circulating B cells). Increased disease burden was correlated with increased absolute numbers of CD3+, CD8+, CD4+ T cells, NK (CD56+CD16+), Dr+CD8+ T cells, and Tregs (CD4+CD25+CD127+); p<0.001. The disease burden was inversely related to the number of circulating monocytes (p<0.01) and unrelated to the circulating number of naïve T cells. We repeated the pre-treatment phenotype correlation studies using the CD19+ cell counts 12 months after treatment for 13 patients who have completed the final analysis. Interestingly, the number of naïve CD4 and CD8 cells (CD4+RA+62L+CD27+ and CD8+RA+62L+CD27+) of the patient prior to treatment correlated with CD19+ cells 12 months after treatment (p=0.024 and p=0.002 respectively; n=13). Finally, we correlated pre-treatment immune phenotypes with time to progression. Again, an elevated T cell phenotype (CD4+RA+62L+CD27- as a percent of CD4+ cells) correlated to increased time to progression (p=0.04; n=28). It is noteworthy that the naïve T cell phenotype did not correlate with tumor burden prior to therapy. Thus, while pre-treatment lymphocyte expansion is commensurate with tumor burden in CLL, the frequency and numbers of naïve T cells prior to treatment has some predictive value to response to treatment. These data suggest that CLL induces the expansion of lymphocytes, suppresses monocytes with an induction in naïve T cells acting as an indicator of higher B cell counts at 12 months in patients with durable remissions. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract Abstract 970 Despite advances in treatments for patients with non-Hodgkin's lymphoma (NHL), the relapse rates remain high and 40% of diffuse large B-cell NHL (DLBCL) patients die of disease. These patients have variable levels of immunodeficiency that may impact their prognosis. Previously we reported that monocytes in relapsed NHL patients suppress both autologous and allogeneic T cell proliferation and that these monocytes were unable to mature into dendritic cells in vitro. These suppressive functions were associated with increased frequency of CD14+HLA-DRneg monocytes in NHL patients (NHL: 29.4 ± 3.2%, n = 36; healthy donors 7.54 ± 0.97%, n = 27; p < 0.0001). This phenotype is distinct from other myeloid suppressors (Lin−CD33+HLA-DR−) or non-classical monocytes (CD16+), neither of which was different in numbers between NHL and healthy donors. We have now expanded our study cohort and found that the percentage of CD14+HLA-DRneg cells among circulating monocytes was increased in patients with more aggressive disease: DLBCL 37.5 ± 5.11%, n = 16 vs SLL and MALT lymphoma 15.8 ± 4.02%, n = 7; p = 0.01. Two newly diagnosed patients and three patients with localized disease amenable to resection all had frequencies of CD14+HLA-DRneg monocytes closer to that of healthy donors whereas 5 of the 6 patients with low grade NHL that transformed to DLBCL had significantly elevated percentage of these suppressive monocytes (49.1 ± 4.70%). To better understand the mechanism of immune suppression by CD14+HLA-DRneg monocytes, we investigated production of IFN-alpha induced by CpG oligodeoxynucleotide. Monocytes from NHL patients were less effective at IFN-alpha production (0.54 ± 0.22%, n = 4) compared to healthy donors (1.37 ± 0.25%, n = 4; p = 0.04). The corresponding culture supernatants also had a 4-fold reduction in IFN-alpha level by ELISA (p = 0.01). Reduced IFN-alpha production and subsequent decreased stimulatory response may contribute to diminished treatment response to CpG in NHL. Finally, we also found that the frequency of CD14+HLA-DRneg monocytes correlated with increasing plasma levels of arginase I, a known mediator of T cell suppression (n = 16, p = 0.06). Such correlation was not observed for circulating VEGF, IL-6 or IL-10. Taken together we have described a population of immunosuppressive CD14+HLA-DRneg monocytes in NHL patients that is more prevalent in aggressive disease and likely suppresses T cell function through multiple mechanisms including arginine metabolism. This work was supported through the University of Iowa and Mayo Clinic Lymphoma SPORE CA097274 and the Gerstner Family Foundation. Disclosures: Witzig: Novartis: Research Funding.

Abstract Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients. Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later. Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months. Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014–25. ©2018 AACR.

Abstract Purpose: Immunotherapeutic strategies to treat patients with renal cell carcinoma (RCC) offer new opportunities for disease management. Further improvements to immunotherapy will require additional understanding of the host response to RCC development. Experimental Design: Using a novel approach to understanding the immune status of cancer patients, we previously showed that patients with a certain immune profile had decreased overall survival. Here, we examine in more detail the phenotypic changes in peripheral blood and the potential consequences of these changes in RCC patients. Results: We found that CD14+HLA-DRlo/neg monocytes were the most predominant phenotypic change in peripheral blood of RCC patients, elevated nearly 5-fold above the average levels measured in healthy volunteers. Intratumoral and peritumoral presence of CD14 cells was an independent prognostic factor for decreased survival in a cohort of 375 RCC patients. The amount of peripheral blood CD14+HLA-DRlo/neg monocytes was found to correlate with the intensity of CD14 staining in tumors, suggesting that the measurement of these cells in blood may be a suitable surrogate for monitoring patient prognosis. The interaction of monocytes and tumor cells triggers changes in both cell types with a loss of HLA-DR expression in monocytes, increases of monocyte survival factors such as GM-CSF in tumors, and increased production of angiogenic factors, including FGF2. Conclusions: Our results suggest a model of mutually beneficial interactions between tumor cells and monocytes that adversely affect patient outcome. Clin Cancer Res; 21(18); 4224–33. ©2015 AACR.

Abstract Monocytes may contribute to tumor progression in part by mediating tumor-induced immunosuppression. Alterations to the monocyte populations and functions in untreated patients with late-stage melanoma are not fully understood. To characterize these alterations, we compared the frequency, phenotype, and functional capacity of peripheral blood monocytes and other myeloid cells in untreated, newly diagnosed stage IV melanoma patients (n = 18) with those in healthy volunteers. Stage IV untreated melanoma patients exhibited a sizeable decrease in the percentage of monocytes (P &lt; 0.0001) that included a drop in the percentage of the CD14+CD16− classical monocyte pool (P = 0.006). Although there was not a significant difference in the CD14+HLA-DRlow/− monocyte population between the patients with melanoma and the healthy volunteers, the HLA-DR levels were considerably lower in the patients' CD14+CD16+ intermediate (P &lt; 0.0001) and CD14lowCD16+ nonclassical monocyte populations (P = 0.001). Decreased surface expression of CD86 (P = 0.0006) and TNFRII (P = 0.0001) and increased expression of tissue factor and PD-L1 (P = 0.003) were identified on monocytes from patients with melanoma. Furthermore, these monocytes had decreased ability to upregulate CD80 expression and cytokine production following stimulation with agonist of Toll-like receptor 3 (TLR3). Peripheral blood dendritic cell subsets were decreased in untreated stage IV melanoma patients. Our study demonstrates that untreated late-stage melanoma patients exhibit monocytopenia in addition to phenotypic and functional deficiencies that may negatively affect their immune function. These findings open new avenues into examining the role of monocyte populations in melanoma development. Cancer Immunol Res; 2(3); 241–8. ©2013 AACR.

Case ReportA 50‐year‐old male with T‐cell lymphoma presented for autologous peripheral blood stem cell transplantation. After granulocyte–colony‐stimulating factor (G‐CSF) mobilization, his peripheral blood CD34+ cell count was 166 × 106/L on the day before collection, which predicted a high yield of CD34+ cells in the apheresis product. The first two collections had yields much lower than expected, triggering an investigation and changes to the apheresis collection methods since mobilization appeared adequate from the peripheral CD34+ values.ResultsChanges to the apheresis collection variables and instrumentation did not improve the yields in the next three collections. The laboratory investigation demonstrated that there was an interfering substance in the patient's plasma that was causing falsely high peripheral blood CD34+ cell values and that the low CD34+ cell yields in the collections were consistent with the actual peripheral blood CD34+ cell value. Based on this finding and after discussion with the clinical service, the patient then received plerixafor to increase the number of circulating CD34+ cells before Collections 6 and 7. The patient went on to achieve the target CD34+ cell dose and subsequently underwent a successful autologous transplant with full hematopoietic engraftment.ConclusionThis case demonstrates the importance of timely and critical review of laboratory results in the context of the specific patient. This case exemplifies how diligent review of laboratory results and open communication among the various teams can positively affect patient outcomes.

Abstract Pneumocystis carinii causes life-threatening pneumonia in immunocompromised patients. The inability to culture P. carinii has hampered basic investigations of the organism's life cycle, limiting the development of new therapies directed against it. Recent investigations indicate that P. carinii is a fungus phylogenetically related to other ascomycetes such as Schizosaccharomyces pombe. The cell cycles of S. pombe and homologous fungi are carefully regulated by cell-division-cycle molecules (cdc), particularly cell-division-cycle 2 (Cdc2), a serine–threonine kinase with essential activity at the G1 restriction point and for entry into mitosis. Antibodies to the proline–serine–threonine–alanine–isoleucine–arginine (PSTAIR) amino-acid sequence conserved in Cdc2 proteins specifically precipitated, from P. carinii extracts, a molecule with kinase activity consistent with a Cdc2-like protein. Cdc2 molecules exhibit differential activity throughout the life cycle of the organisms in which they occur. In accord with this, the P. carinii Cdc2 showed greater specific activity in P. carinii trophic forms (trophozoites) than in spore-case forms (cysts). In addition, complete genomic and complementary DNA (cDNA) sequences of P. carinii Cdc2 were cloned and found to be most closely homologus to the corresponding sequences of other pathogenic fungi. The function of P. carinii cdc2 cDNA was further documented through its ability to complement the DNA of mutant strains of S. pombe with temperature-sensitive deficiencies in Cdc2 activity. The P. carinii cdc2 cDNA restored normal Cdc2 function in these mutant strains of S. pombe, and promoted fungal proliferation. These studies represent the first molecular analysis of the cell-cycle-regulatory machinery in P. carinii. Further understanding of P. carinii's life cycle promises novel insights for preventing and treating the intractable infection it causes in immunocompromised patients.