Frank Vari

Serum CD163 and TARC as Disease Response Biomarkers in Classical Hodgkin Lymphoma

Purpose: Candidate circulating disease response biomarkers for classical Hodgkin lymphoma (cHL) might arise from Hodgkin–Reed–Sternberg (HRS) cells or nonmalignant tumor-infiltrating cells. HRS cells are sparse within the diseased node, whereas benign CD163+ M2 tissue-associated macrophages (TAM) are prominent. CD163+ cells within the malignant node may be prognostic, but there is no data on serum CD163 (sCD163). The HRS-specific serum protein sTARC shows promise as a disease response biomarker. Tumor-specific and tumor-infiltrating circulating biomarkers have not been compared previously. Experimental Design: We prospectively measured sCD163 and sTARC in 221 samples from 47 patients with Hodgkin lymphoma and 21 healthy participants. Blood was taken at five fixed time-points prior, during, and after first-line therapy. Results were compared with radiological assessment and plasma Epstein-Barr virus DNA (EBV-DNA). Potential sources of circulating CD163 were investigated, along with immunosuppressive properties of CD163. Results: Pretherapy, both sCD163 and sTARC were markedly elevated compared with healthy and complete remission samples. sCD163 better reflected tumor burden during therapy, whereas sTARC had greater value upon completion of therapy. sCD163 correlated with plasma EBV-DNA, and associated with B symptoms, stage, and lymphopenia. Circulating CD163+ monocytes were elevated in patients, indicating that sCD163 are likely derived from circulating and intratumoral cells. Depletion of cHL CD163+ monocytes markedly enhanced T-cell proliferation, implicating monocytes and/or TAMs as potential novel targets for immunotherapeutic manipulation. Conclusion: The combination of circulating tumor-infiltrate (sCD163) and tumor-specific (sTARC) proteins is more informative than either marker alone as disease response biomarkers in early and advanced disease during first-line therapy for cHL. Clin Cancer Res; 19(3); 731–42. ©2012 AACR.

Restoration of tolerance to rat hepatic allografts by spleen-derived passenger leukocytes.

The tolerance induced by orthotopic liver transplantation (OLT) in certain combinations of rat strains can be prevented by total body irradiation (TBI) of the donor. We demonstrate here that the intravenous inoculation of splenic leukocytes into irradiated donors before OLT could re-establish tolerance in association with a state of microchimerism detected in the recipients. When donor DA (RT1a) strain rats were irradiated with 1000 rad 24 h before liver harvesting and subsequent liver implantation into PVG recipients, five out of six rats died from rejection in this normally tolerogenic OLT (DA-PVG) combination. Injection of 1.5x108 splenic leukocytes from naive DA rats into the irradiated DA donor rats 24 h before OLT restored the tolerogenic potential of the liver allografts. Immunofluorescence assay revealed an increased number of donor (DA) type cells in the PVG recipient bearing a repopulated DA liver, compared to the PVG recipient of an irradiated liver. These results suggest that passenger leukocytes reconstituted by splenic leukocytes have the capacity to protect liver allografts.

A phase I clinical trial of CD1c (BDCA-1)+ dendritic cells pulsed with HLA-A*0201 peptides for immunotherapy of metastatic hormone refractory prostate cancer

Preclinical studies have suggested that purified populations of CD1c (BDCA-1+) blood-derived dendritic cells (BDC) loaded with tumor-specific peptides may be a feasible option for prostate cancer immunotherapy. We performed an open-label dose-finding Phase I study to evaluate the safe use of CD1c+ BDC in patients with advanced metastatic hormone refractory prostate cancer. HLA-A*0201-positive patients with advanced metastatic prostate cancer were recruited and consented. The vaccine was manufactured by pulsing autologous CD1c+ BDC, prepared by magnetic bead immunoselection from apheresed peripheral blood mononuclear cells, with a cocktail of HLA-A*0201-restricted peptides (prostate-specific antigen, prostate acid phosphatase, prostate specific membrane antigen, and control influenza peptide) and keyhole limpet hemocyanin. The vaccine was administered intradermally or intravenously and peripheral blood was taken at predetermined intervals for clinical and immunologic monitoring. The vaccine was manufactured with a median purity of 82% CD1c+ BDC and administered successfully to 12 patients. Each patient received between 1 and 5×106 fresh CD1c+ BDC on day 0, followed by cryopreserved product in the same dose on days 28 and 56. The vaccine was well tolerated in all patients, with the most frequent adverse events being grade 1–2 fever, pain, or injection-site reactions. Vaccination with CD1c+ BDC is therefore feasible, safe, and well tolerated in patients with advanced-stage metastatic prostate cancer.

Allograft acceptance and rejection, mediated by a liver suppressor factor, LSF-1, purified from serum of liver transplanted rats.

In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart graft in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination, DA (RT1a) donor into PVG (RT1c) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosuppressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-1) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 micrograms of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RT1n) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-1), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In 5/6 cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the injection of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in vivo results, suggests a role for LSF-1 in the induction of tolerance.

CD4+ Tumor infiltrating lymphocytes are prognostic and independent of R‐IPI in patients with DLBCL receiving R‐CHOP chemo‐immunotherapy

Despite the Revised International Prognostic Indexs (R‐IPI) undoubted utility in diffuse large B‐cell lymphoma (DLBCL), significant clinical heterogeneity within R‐IPI categories persists. Emerging evidence indicates that circulating host immunity is a robust and R‐IPI independent prognosticator, most likely reflecting the immune status of the intratumoral microenvironment. We hypothesized that direct quantification of immunity within lymphomatous tissue would better permit stratification within R‐IPI categories. We analyzed 122 newly diagnosed consecutive DLBCL patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R‐CHOP) chemo‐immunotherapy. Median follow‐up was 4 years. As expected, the R‐IPI was a significant predictor of outcome with 5‐year overall survival (OS) 87% for very good, 87% for good, and 51% for poor‐risk R‐IPI scores (P < 0.001). Consistent with previous reports, systemic immunity also predicted outcome (86% OS for high lymphocyte to monocyte ratio [LMR], versus 63% with low LMR, P = 0.01). Multivariate analysis confirmed LMR as independently prognostic. Flow cytometry on fresh diagnostic lymphoma tissue, identified CD4+ T‐cell infiltration as the most significant predictor of outcome with ≥23% infiltration dividing the cohort into high and low risk groups with regard to event‐free survival (EFS, P = 0.007) and OS (P = 0.003). EFS and OS were independent of the R‐IPI and LMR. Importantly, within very good/good R‐IPI patients, CD4+ T‐cells still distinguished patients with different 5 year OS (high 96% versus low 63%, P = 0.02). These results illustrate the importance of circulating and local intratumoral immunity in DLBCL treated with R‐CHOP. Am. J. Hematol. 88:273–276, 2013.

Novel immunosuppressive proteins purified from the serum of liver-retransplanted rats.

Liver grafts between certain rat strain combinations, such as DA (RT1a)-into-PVG (RT1c), are accepted without the use of immunosuppressive agents. To explore the nature and role of serum proteins in liver-induced immunosuppression, we have developed a retransplantation model of rat liver grafting. In this procedure, orthotopic liver transplantation (OLT) is carried out in the DA-into-PVG combination; two days later the DA liver is removed and a new PVG liver implanted into the same recipient (re-OLT). Serum from re-OLT rats was immunosuppressive when tested in mixed lymphocyte reactions (MLR). Three novel proteins were detected in re-OLT serum by SDS-PAGE, with sizes of 180 kD, 87 kD, and 10 kD. The N-terminal sequences of these were distinct and did not match protein sequences in the computer databases, although there was some homology between the 10 kD sequence and the beta-chain of rat hemoglobin. Purified 87 kD and 10 kD proteins were immunosuppressive in MLR; in both cases suppression was dose-dependent and nonstimulator-specific. Production of the 180 kD and 87 kD molecules required the presence of the recipient spleen. We conclude that re-OLT serum contains novel immunosuppressive proteins, which may be products of immune recognition and associated with the immediate termination of graft rejection.

Ratios of T-cell immune effectors and checkpoint molecules as prognostic biomarkers in diffuse large B-cell lymphoma: a population-based study

BACKGROUND Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured, despite initial treatment with R-CHOP. The prognostic importance of the revised International Prognostic Index (R-IPI) and cell of origin of the malignant B cell are established in DLBCL. We aimed to develop a novel, easily applicable, tissue-based prognostic biomarker based on quantification of the tumour microenvironment that is independent of and additive to the R-IPI and cell of origin. METHODS We performed digital hybridisation on the NanoString platform to assess the relation between immune effector and inhibitory (checkpoint) genes in 252 formalin-fixed, paraffin-embedded DLBCL tissue specimens obtained from patients treated with R-CHOP. We used a tree-based survival model to quantify net antitumoral immunity (using ratios of immune effector to checkpoint genes) and to generate a cutoff as an outcome predictor in 158 of the 252 patients. We validated this model in tissue (n=233) and blood (n=140) samples from two independent cohorts treated with R-CHOP. FINDINGS T-cell and NK-cell immune effector molecule expression correlated with tumour-associated macrophage and PD-1/PD-L1 axis markers, consistent with malignant B cells triggering a dynamic checkpoint response to adapt to and evade immune surveillance. The ratio of CD4*CD8 to (CD163:CD68[M2])*PD-L1 was better able to stratify overall survival than was any one immune marker or combination, distinguishing groups with disparate 4-year overall survival. 94 (59%) of 158 patients had a score above the cutoff and 4-year overall survival of 92·1% (95% CI 82·9-96·7), and the remaining 64 (41%) patients had a score below the cutoff and 4-year overall survival of 47·0% (32·8-60·5; hazard ratio [HR] 8·3, 95% CI 4·3-17·3; p<0·0001). The CD4*CD8:M2*PD-L1 immune ratio was independent of and added to the R-IPI and cell of origin. Tissue findings in the independent tissue cohort accorded with those in our initial tissue cohort. 139 (60%) of 233 patients had a score above the cutoff and 4-year overall survival of 75·6% (95% CI 64·6-83·6), with the remaining 94 (40%) patients having a score below the cutoff (63·5% [52·5-72·7]; HR 1·9, 95% CI 1·1-3·3; p=0·0067). INTERPRETATION Ratios of immune effectors to checkpoints augment the cell of origin and R-IPI in DLBCL and are applicable to paraffin-embedded biopsy specimens. These findings might have potential implications for selection of patients for checkpoint blockade within clinical trials. FUNDING Leukaemia Foundation of Queensland, Kasey-Anne Oklobdzijato Memorial Fund, the Australasian Leukaemia and Lymphoma Group (Malcolm Broomhead Bequest), the Australian Cancer Research Foundation, and the Cancer Council of Queensland.

Expression profiling of Epstein-Barr virus-encoded microRNAs from paraffin-embedded formalin-fixed primary Epstein-Barr virus-positive B-cell lymphoma samples

Epstein-Barr virus (EBV) is implicated in a range of B-cell malignancies and expresses unique microRNAs (EBV-miRNAs). Due to the requirements for high-quality RNA, studies profiling EBV-miRNA in EBV-positive lymphomas have been restricted to cell-lines or frozen samples. However, the most commonly available archived patient material is paraffin-embedded formalin-fixed (FFPE) tissue. This has impeded the widespread profiling of EBV-miRNA expression in clinical samples. The requirements for accurate EBV-miRNA real-time RT-PCR quantitation in FFPE tissues representing a broad-spectrum of EBV-positive lymphomas were determined systematically, including where the neoplastic cells are sparse relative to the non-malignant infiltrate. The level of cellular EBV-load correlated strongly with the sum of EBV-miRNA expression and the number of EBV-miRNAs detectable. As calibrators for cellular EBV-load, the sum EBV-miRNA was optimal to EBV-genome copy number and EBER2 expression level, with the added advantage of not requiring additional assays. EBV-miRNA was profiled reliably within archival FFPE tissue in 14/23 patients, but not in tissues with low abundance EBV. This method enabled specific and simultaneous detection of numerous EBV-miRNAs in FFPE lymphoma samples that contain EBV at high to medium levels, making it as a useful tool for studies of EBV-miRNA in the majority of diagnostic biopsies.

Human kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+KLK4+ cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

A flow cytometry based assay for the enumeration of regulatory T cells in whole blood

The analysis of regulatory T cells (T-reg(s)) is becoming an increasingly important consideration in the development of novel immunotherapeutic strategies. Accurate quantification of T-regs during treatment protocols is crucial, particularly where the therapeutic strategy is targeting T-regs. The TruCOUNT™ method has utility for enumerating different immune cells but has not been used to detect T-regs. We have utilized this technology to develop an assay to enumerate human T-regs in whole blood, based on CD127 expression. The mean number of CD4(+)CD25(+)CD127(lo) T-regs per μl of whole blood was 48±16.9 with a range of 18 - 79 (n=22) and the average percentage was 6.1±1.9% (range 2.2-10.4%). The percentages of CD4(+)CD25(+)CD127(lo) T-regs were similar when detected in whole blood or density-gradient separated PBMC, and were comparable to those distinguished using the T-reg marker FoxP3. The assay was robust and reliable for enumeration of the lower frequency T-regs, with CVs for intra-assay repeatability and inter-assay precision of <9% and <35%, respectively. The CVs for the detection of total CD4(+) T lymphocytes using this assay were <2% for intra-assay repeatability and <18% for inter-assay precision, providing further evidence for reproducibility. This assay has a number of advantages over current methods, including small sample volume, the ability to determine absolute cell counts, and no need for hematology cell analyzers. This assay will simplify clinical trial immune monitoring and can be used to provide crucial data on patient T-reg numbers before, during, and after therapeutic interventions.