ALBERT

Description: Dysfunctional T-cells associated with human tumors can be identified utilizing multi-parameter flow cytometry and studied for intracellular signalling checkpoints using phospho-flow or time-of-flight mass spectrometry. Whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent has not yet been determined. Anergic T-cells are hypo-responsive with low cytokine production and induce tolerance to protect the host from autoimmune disease. T-cell exhaustion is usually associated with chronic viral infection such as CMV but also with some cancers. Both exhausted and anergic T-cells express PD-1, LAG-3, Tim-3, CD160 and CD28. Hypo-responsive T-cells associated with melanoma have the phenotype of exhausted T-cells which can be reactivated using targeted immune check point blockade, resulting in clinical benefit. Senescent T-cells accumulate in an oligoclonal manner with ageing and chronic antigen exposure. Senescence can be characterised by telomere shortening, the expression of CD57, KLRG-1, CD160 and the absence of CD28. However, not all senescent cells have shortened telomeres as telomere independent senescence involving the p21-p53 and p16-pRb pathways and senescence associated secretory phenotype (SASP) T-cells have been described. The aim of this study was to determine whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent and to identify targetable checkpoints to restore T-cell function. Cytotoxic T-cell clones (CD57+ CD28- TCRVβ restricted) determined by BetaMark TCRVβ analysis were present in 51% of patients with myeloma (n=264), are protective (OS χ2=6.2; p

Description: Abstract 1818 There is clinical evidence for the presence of a limited degree of host-tumor control in patients with multiple myeloma (MM) but the exact mechanisms involved are not known. Patients who survive for more than ten years are likely to have the most active immunological host-tumour control and are an ideal cohort to study. We now add to our preliminary observations using a range of immunological biomarkers in the 29 of these patients who attend our clinic. 51% of MM patients (n=264) had expanded CD3+CD8+ TCRVβ+ CD57+T cell clones detected by TCRVβ analysis (Beckman, BetaMark). Clonality was confirmed by IgH CDR3 sequencing. These clones accounted for 14.3% (median) of the CD3 cells (range 4–49%). CFSE tracking demonstrated the anergic nature of these clonal T cells (median 6% proliferation) compared with other CD8 cells (70% proliferation) while Geneset analysis of mRNA microarrays (Affymetrix U133) identified that anergy was caused by upregulated RAS, CSK, TOB and suppressed ERK pathways. Unlike recent reports for T-LGL, microarray analysis suggested that there was no evidence of STAT3 upregulation in the MM T cell clones. Functional studies suggested that these non-proliferating clones have split anergy as interferon-γ production was normal. In contrast, all ten year survivors had expanded T cell clones and serial studies demonstrated a 〉8 year persistence of the same clone in 8 out of 12 patients studied. More importantly, unlike the other MM patients, the T cell clones in 19 out of 21 of the ten year survivors studied were not anergic. The Treg/Th17 ratio in the ten year survivors was significantly lower than other MM patients (median 1.9 vs 12.0; p

Description: The transitional stage of B-cell development represents an important step where autoreactive cells are deleted, allowing the generation of a mature functional B-cell repertoire. In mice, 3 subsets of transitional B cells have been identified. In contrast, most studies of human transitional B cells have focused on a single subset defined as CD24hiCD38hi B cells. Here, we have identified 2 subsets of human transitional B cells based on the differential expression of CD21. CD21hi transitional cells displayed higher expression of CD23, CD44, and IgD, and exhibited greater proliferation and Ig secretion in vitro than CD21lo transitional B cells. In contrast, the CD21lo subset expressed elevated levels of LEF1, a transcription factor highly expressed by immature lymphocytes, and produced higher amounts of autoreactive Ab. These phenotypic, functional, and molecular features suggest that CD21lo transitional B cells are less mature than the CD21hi subset. This was confirmed by analyzing X-linked agammaglobulinemia patients and the kinetics of B-cell reconstitution after stem cell transplantation, which revealed that the development of CD21lo transitional B cells preceded that of CD21hi transitional cells. These findings provide important insights into the process of human B-cell development and have implications for understanding the processes underlying perturbed B-cell maturation in autoimmune and immunodeficient conditions.

Description: The transfer of membrane proteins between cells during contact, known as trogocytosis, can create novel cells with a unique phenotype and altered function. We demonstrate that trogocytosis is more common in multiple myeloma (MM) than chronic lymphocytic leukemia and Waldenstrom macroglobulinaemia; that T cells are more probable to be recipients than B or natural killer cells; that trogocytosis occurs independently of either the T-cell receptor or HLA compatibility; and that after trogocytosis, T cells with acquired antigens can become novel regulators of T-cell proliferation. We screened 168 patients with MM and found that CD86 and human leukocyte antigen G (HLA-G) were antigens commonly acquired by T cells from malignant plasma cells. CD3+CD86acq+ and CD3+ HLA-Gacq+ cells were more prevalent in bone marrow than peripheral blood samples. The presence of either CD86 or HLA-G on malignant plasma cells was associated with a poor prognosis. CD38++ side population cells expressed HLA-G, suggesting that these putative myeloma stem cells could generate immune tolerance. HLA-G+ T cells had a regulatory potency similar to natural Tregs, thus providing another novel mechanism for MM to avoid effective immune surveillance.

Description: Abstract 4957 Background Over 40% of patients with the most common lymphoid malignancy worldwide, DLBL, are over the age of 70. Although R-CHOP is inarguably the mainstay of therapy for DLBL patients, a significant number of elderly patients do not tolerate the regimen due to underlying frailty and/or co-morbidities. Most elderly patients with significant co-morbidities have limited treatment options and are not offered anthracycline-containing chemotherapy due to concerns regarding toxicity. Here we describe our single center experience with CEEP, a lower intensity regimen for elderly patients with newly diagnosed or relapsed DLBL whom are deemed inappropriate for CHOP-based chemotherapy. Method All patients 〉70 years old (median 78.5, range 71 – 85) with histologically proven DLBL treated with CEEP ± Rituximab (R) at Royal Prince Alfred Hospital from 2000 to 2010 were retrospectively reviewed. Modified CEEP, Cyclophosphamide 300mg/m2 Day 1 (D1) and D15, Epirubicin 50mg/m2 D1 and D15, Etoposide 100mg/m2 D1 and D15, and Prednisolone 50mg D1-D5 (reduced dose from original CEEP protocol) was administered every 2 weeks. Rituximab 375mg/m2 (when approved for use in Australia) was administered every 28 days. As per institutional protocol, all patients received Bactrim prophylaxis for Pneumocystis. Baseline characteristics, Charlson Comorbidity Index, Revised International Prognostic Index (RIPI), the number of CEEP cycles, treatment response and toxicity from treatment were identified and reviewed. Results A total of 22 patients were identified, 10 were male. 15 received CEEP as initial therapy, and 7 for relapsed disease. 23% (n=5) had an ECOG score ≥ 2. 55% (n=12) had RIPI ≥ 3. All patients had a Charlson Comorbidity Index ≥ 2, with 23% (n=5) ≥ 5, which was considered sufficient to preclude conventional CHOP-based chemotherapy. Median cardiac ejection fraction was 62% (range 55 – 85%). 73% (n=16) received Rituximab and 50% (n=11) received primary GCSF prophylaxis. The median number of CEEP ± R cycles was 6 (range 2 – 9 cycles). 5% (n=1) required dose reduction and 9% (n=2) required delays in treatment due to haematological toxicity. Median follow-up was 10.0 months (range 1 – 92.7 months). At completion of therapy, complete responses (CR) were demonstrated in 10 patients (45%), with partial responses (PR) seen in 32% (n=7). 18% (n=4) demonstrated progressive disease (PD) despite therapy. Of the 7 patients with relapsed disease prior to CEEP ± R, CR was seen in 2 cases, both of whom had previous exposure to R-CVP (cyclophosphamide, vincristine, prednisolone) chemotherapy. At most recent follow up, 32% (n=7) have remained in CR with a median follow up period of 28.1 months (range 13 – 92.7 months), 36% (n=8) had disease progression, 9% (n=2) demonstrated stable residual disease, while 23% (n=5) have died. Of the 5 deaths, 3 were attributed to progressive DLBL. The other deaths were a result of complications following further salvage chemotherapy. Grade 3 – 4 haematological toxicity was observed in 72% (n=16) of patients. Febrile neutropenia occurred in 41% (n=9). Overall, 50% (n=11) required at least one re-admission to hospital. Non-haematological grade 3 – 4 toxicity was detected in 2 patients, one of whom suffered unstable angina in the setting of anaemia, the other an acute cerebrovascular event in the setting of new atrial flutter post-chemotherapy. Discussion Although limited by a small sample size, our retrospective single center experience demonstrates that CEEP ± R chemotherapy can be administered to elderly patients with significant co-morbidities. Our cohort was all aged 〉70, with medical co-morbidities leading to the unsuitability of conventional CHOP-based therapy. Whilst an overall response rate of 77% (CR + PR) was observed, on prolonged follow up, 32% of patients remained in CR. Significant haematological toxicity (72%) and infectious complications (41%) were observed, however no deaths were directly attributed to the chemotherapy. Future prospective studies are required to further evaluate the safety and efficacy of R-CEEP in the elderly. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 983 HLA-G is a “non-classical” HLA class I molecule which is considered to be responsible for the immune tolerance of the fetus, allografts or tumors by inhibiting the antigen-specific cytotoxic T lymphocyte response and decreasing NK cell function. CD86 is a costimulatory molecule which binds to a T cell counter receptor (eg CD28) during antigen stimulation and T cell receptor engagement. T cells do not express CD86mRNA but can acquire CD86 and HLA-G by trogocytosis. We have studied the incidence and prognostic significance of HLA-G and CD86 on malignant plasma cells, the incidence of HLA-G and CD86 on T cells and the immunosuppressive function of HLA-G on malignant plasma cells and T cells. HLA-G and CD86 expression were determined on T cells (CD3+) and plasma cells (CD38++) of patients with myeloma (MM). Flow-sorted CD3+ HLA-G- cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with anti-CD3/CD2/CD28 beads in the presence and absence of either HLA-G+ T cells or HLA-G+ plasma cells. Suppression of the proliferation of CFSE-tracked HLA-G- T cells by HLA-G+ T cells or plasma cells was determined by flow cytometry. CD86+ plasma cells are present in 54% of patients and are associated with a poor prognosis (χ2=4.6;p5% CD38++ cells and expressing 〉12% HLA-G; χ2=12.4; p

Description: Abstract 3924 Prior to the introduction of novel therapies for patients with multiple myeloma (MM) few patients survived for more than 10 years. Several reports have suggested that 10 year survival is associated with a younger age at diagnosis and the duration of exposure to effective agents. Although the number of patients surviving 10 years is increasing, there have been no significant reports of immunological biomarkers in these patients. This is especially true in those for whom the prolonged survival has occurred without novel drugs. Previous studies have shown that the presence of expanded peripheral blood CD8 T-cell clones in MM patients is associated with a better prognosis; raising the possibility that these T-cells, confirmed as clones by TCR sequencing, mediate an anti-tumor effect. However microarray gene set enrichment analysis and proliferation tracking studies demonstrated that these cells are in an anergic state. In addition, there is evidence that Tregs inhibit and Th17 cells enhance autologous immune response in malignancy and that an imbalance in MM may impair disease control. Slan-DCs are a subset of myeloid dendritic cells and are of interest in MM because of their ability to stimulate cytotoxic T-cell responses and reverse anergy in tumor infiltrating lymphocytes. We have investigated the immune mechanisms of disease control which may contribute to long-term survival by analyzing Tregs, Th17 cells, Slan-DCs and the incidence and relative degree of anergy of T-cell clones in all current 〉10 year survivors. Peripheral blood samples were analyzed for the presence of CD3+ T-cell receptor Vβ restricted T-cell clones (BetaMark Kit), the number of CD3+CD4+CD25h+CD127- Tregs, CD4+IL-17+ Th17 cells and CD16+CD14low M-DC8+ Slan-DCs. Proliferation of T-cells was analyzed using CFSE tracked 4 day cultures stimulated with anti-CD3 and anti-CD28 beads. Results were compared with local data collected from 2 large MM cohorts and age-matched controls. Of 26 patients who had survived for 〉10 years, 22 were available for testing. At diagnosis the median age was 59y (median=57y in comparison cohort); 54% were female; median β2M was 2.2mg/L (range 1.1–7.3); 92% were ISS I; 73% of paraproteins were IgG, 23% IgA and 4% light chain only. 73% had end-organ effects with; 65% bone disease; 15% anemia and 8% renal impairment. 58% had received cytotoxic chemotherapy; 27% novel agents; 38% autologous transplants; 8% allogeneic transplants and 31% were untreated. Expanded T-cell clones were documented in all (100%) of the 10 year survivors, a significant increase compared with 54% (n=144) and 48% (n=120) in the previous MM cohorts (χ2=43.6;p

Description: The best conditioning regimen before allogeneic transplantation for high-risk diffuse large B-cell lymphoma (DLBCL) remains to be clarified. We analyzed data from 396 recipients of allotransplants for DLBCL receiving myeloablative (MAC; n = 165), reduced intensity (RIC; n = 143), or nonmyeloablative conditioning (NMAC; n = 88) regimens. Acute and chronic GVHD rates were similar across the groups. Five-year nonrelapse mortality (NRM) was higher in MAC than RIC and NMAC (56% vs 47% vs 36%; P = .007). Five-year relapse/progression was lower in MAC than in RIC/NMAC (26% vs 38% vs 40%; P = .031). Five-year progression-free survival (15%-25%) and overall survival (18%-26%) did not differ significantly between the cohorts. In multivariate analysis, NMAC and more recent transplant year were associated with lower NRM, whereas a lower Karnofsky performance score (〈 90), prior relapse resistant to therapy, and use of unrelated donors were associated with higher NRM. NMAC transplants, no prior use of rituximab, and prior relapse resistant to therapy were associated with a greater risk of relapse/progression. In conclusion, allotransplantation with RIC or NMAC induces long-term progression-free survival in selected DLBCL patients with a lower risk of NRM but with higher risk of lymphoma progression or relapse.

Description: Abstract 3950 Chemotherapy-resistant cancer stem cells are a major cause of relapse. It is considered that the small population of CD38++ cells which also exhibit “side population” (SP) characteristics after Hoescht 33342 staining contain chemotherapy resistant multiple myeloma (MM) stem cells. The hedgehog (Hh) signalling pathway is a key regulator of stem cell proliferation and differentiation. A range of Hh inhibitors are currently under development as therapeutics. Our aim was to determine the effect that NVP-LDE225, a novel Hh pathway inhibitor has on MM stem cells. NVP-LDE225 was kindly supplied by Novartis. Side population (SP) cells, considered to contain the MM stem cells, were detected after Hoescht 33342 staining using a BD ARIA II flow cytometer, Proliferation was monitored with CFSE tracking and viability with propidium iodide. MM cell lines RPMI8226, KMS-11, OPM2, U266 and primary MM cells were included. Expression of Hh pathway proteins PTCH1, Smo and Gli1 was determined by flow cytometry. Bone marrow samples were obtained from patients with myeloma after informed consent. CD38++ SP cells were identified in all 4 MM cell lines tested and 90% (26/29) of MM BM samples with the percentage of SP cells ranging from 0 to 9.5% of CD38++ cells. There was no correlation between %SP and %CD38++ and no significant difference in %SP during therapy. PTCH1 expression was higher on SP cells of KMS-11, OPM2 and U266 than non-SP cells (80.5% vs 51.7%) suggesting that the canonical Hh pathway was upregulated in CD38++ SP cells. PTCH1 expression varied from 0.1 – 87% on patient's CD38++ cells. In vitro dose response curves demonstrated increased killing of plasma cell lines above 1μM NVP-LDE225. The IC50 was 4.7uM for KMS-11 SP cells and 3.4uM for KMS-11 non-SP cells. During culture of flow-sorted CD38++ SP+ and non-SP cells (RPMI 8226 and KMS11), fluctuations in %SP suggest that SP cells differentiated to non-SP cells but also that the SP phenotype is unstable. This is supportive of the concept that the stem cell compartment is in a state of flux. The presence of NVP-LED225 caused a 90% inhibition of non-SP returning to SP and induced an additional 79% differentiation of SP to non-SP KMS11 cells on day 2. Proliferation studies using CFSE tracking of sorted SP cells demonstrated a 34% reduction in proliferation after exposure to NVP-LDE225. Studies with 9 primary human bone marrow samples from patients with MM confirmed the data from cell lines demonstrating that differentiation was significantly induced (t=2.78; p