Devinder Gill

Loss of PKR activity in chronic lymphocytic leukemia

There are a number of observations that suggest the dsRNA‐activated protein kinase, PKR, may play an active role in formation and maintenance of leukemia, including nonrandom chromosomal deletions in acute leukemia as well as truncations and deletions of the PKR gene in some leukemia cell lines. However, there is little direct evidence from patient material that this is so. Here we show that full‐length PKR is present but not active in 21 of 28 patient samples from B‐cell chronic lymphocytic leukemia (B‐CLL). PKR from these patients was unable to auto‐activate or phosphorylate substrates but was able to bind dsRNA. Furthermore, the lack of PKR activation was not due to differing levels of the PKR activator, PACT nor of the PKR inhibitor, p58IPK. We compared PKR status with clinical parameters and disease staging. No differences were found between the 2 groups in terms of staging (modified Rai or Binet), age, CD38 status, p53 status, 11q23 deletion status or CEP12 deletion status. However, there was a significant correlation between deletion in 13q14.3 and lack of PKR activity. We show that B‐CLL cells appear to contain a soluble inhibitor of PKR, as lysates from cells lacking PKR activity were able to inhibit exogenous PKR in mixing experiments. Finally, we show suppression of PKR activity was still present following ultrafilitration through a 10,000 Da cutoff filter but was lost upon extraction with phenol/chloroform or by high salt washing. This data suggests loss of PKR activity may contribute to the formation and/or maintenance of CLL.

Effects of glycosylated recombinant human granulocyte colony-stimulating factor after high-dose cytarabine-based induction chemotherapy for adult acute myeloid leukaemia

The Australian Leukaemia Study Group (ALSG) investigated whether G-CSF would accelerate haemopoietic recovery after induction treatment for acute myeloid leukaemia (AML) intensified with high-dose cytarabine, and therefore improve response rates and survival. Patients were randomised to receive lenograstim (glycosylated recombinant human G-CSF) 5 μg per kg body weight subcutaneously daily from day 8 after starting chemotherapy, or no cytokine, following chemotherapy with cytarabine 3 g/m2 every 12 h on days 1, 3, 5, and 7, together with idarubicin 9 or 12 mg/m2 on days 1, 2, and 3, plus etoposide 75 mg/m2 on days 1 to 7 inclusive. Patients had untreated AML, and were aged 16 to 60 years. Overall, 54 evaluable patients were randomised to receive lenograstim and 58 to no cytokine. Patients in the lenograstim arm had a significantly shorter duration of neutropenia <0.5 × 109/l compared to patients in the no cytokine arm (median 18 vs 22 days; P = 0.0005), and also shorter duration of total leucopenia <1.0 × 109/l (17 vs 19 days; P = 0.0002), as well as a reduction in duration of treatment with therapeutic intravenous antibiotics (20 vs 24 days; P = 0.015) and a trend to reduced number of days with fever >38.0°C (9 vs 12 days; P = 0.18). There were no differences between the two groups in platelet recovery, red cell or platelet transfusions, or non-haematological toxicities. For patients achieving CR after their first induction course, a reduction in the time to the start of the next course of therapy was observed in the lenograstim arm, from a median of 40.5 days to a median of 36 days (P = 0.082). The overall complete response rates to chemotherapy were similar, 81% in the lenograstim arm vs 75% for the no cytokine arm (P = 0.5), and there was no significant difference in the survival durations. We conclude that the granulopoietic stimulating effect of G-CSF is observed after induction therapy for AML intensified by high-dose cytarabine, resulting in an improvement in a number of clinically important parameters with no major adverse effects.

CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro

Abstract Chronic lymphocytic leukemia (CLL) is predominantly a disease of accumulation rather than rapid proliferation. To date, no cell lines exist, as CLL cells undergo rapid apoptosis when cultured in vitro, suggesting that a favorable in vivo microenvironment is required. To identify survival signals we cultured primary CLL peripheral blood mononuclear cells (PBMCs) at high density, which has previously been shown to dramatically improve survival. Using antibody arrays we measured the level of 42 cytokines in culture supernatants and showed that inerleukin-6 (IL-6), IL-8, CXCL2 and CCL2 were highly up-regulated in culture. This is the first report to describe a role for CCL2 and CXCL2 in CLL cell survival. Importantly, CXCL2, IL-6 and IL-8 were significantly up-regulated in primary patient plasma. The addition of either CXCL2 or CCL2 enhanced CLL cell survival, while antibodies blocking these chemokines reduced survival. Co-culture of CLL cells and PBMC accessory cells separated by transwells provided a similar degree of survival protection compared to normal culture, whereas CLL cells cultured alone died rapidly. Interestingly, CCL2 and CXCL2 appeared to be produced by CLL cells but only when co-cultured with accessory cells. Thus, we speculate that accessory cells release soluble factors that promote the production of these pro-survival chemokines from CLL cells and physical interactions are not required. Our data support the concept that the CLL microenvironment is critical, and suggests that soluble factors are more important than physical interactions.

A ≥1 log rise in RQ-PCR transcript levels defines molecular relapse in core binding factor acute myeloid leukemia and predicts subsequent morphologic relapse

Core binding factor acute myeloid leukemia (CBF AML), with t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22) and the associated fusion gene transcripts AML1/ETO or CBFβ/MYH11, has a favourable clinical prognosis although significant numbers of patients still suffer relapse. We examined the prognostic utility of serial bone marrow minimal residual disease (MRD) monitoring by RQ-PCR in a cohort of patients with CBF AML with long term clinical follow-up. Twenty-nine patients were evaluated with a median follow of 34 months. Twelve relapses occurred at a median of 11 months (range 4 – 17) from diagnosis. RQ-PCR levels at diagnosis, post-induction chemotherapy and post-consolidation were not predictive of outcome. However, a ≥1 log10 rise at any stage in transcript level relative to the level from a remission bone marrow sample correlated with inferior leukemia free survival (LFS) and imminent morphologic relapse (hazard ratio 8.6). Relapses occurred a median of 60 days (range 45 – 272) after a log10 rise. A ≥1 log10 rise in transcript levels strongly predicts subsequent morphologic relapse in CBF AML and therefore defines molecular relapse. Our data support a simple RQ-PCR model for prediction of impending relapse which has the potential for widespread clinical applicability. Prospective identification of high risk patients will enable clinical trials to assess the efficacy of treatment initiated at molecular relapse.

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.

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.

Safety and efficacy of pegfilgrastim compared to granulocyte colony stimulating factor (G-CSF) supporting a dose-intensive, rapidly cycling anti-metabolite containing chemotherapy regimen (Hyper-CVAD) for lymphoid malignancy.

Pegfilgrastim (Neulasta®) has proven efficacy as supportive therapy in a variety of 21-day chemotherapy regimens, but has not been studied in dose intensive, rapidly cycling regimens utilising cell-cycle active drugs (e.g. anti-metabolites) such as hyper-CVAD. This study examined whether pegfilgrastim was safe and lead to similar kinetics of neutrophil recovery as daily granulocyte colony stimulating factor (G-CSF). Using retrospective analysis, patients receiving pegfilgrastim (6 mg) were matched with controls (G-CSF 5 μg kg−1 per day) for a cycle of chemotherapy, prior chemotherapy, dose of cytarabine received, age (<60 or >60 years), diagnosis and bone marrow involvement. The primary endpoint was duration of grade IV neutropenia (absolute neutrophil count, ANC < 500 μl−1). Secondary endpoints included time to neutrophil recovery, incidence of febrile neutropenia, positive blood cultures and delay in subsequent chemotherapy. This study identified 124 pegfilgrastim supported cycles in 43 patients and successfully matched them to 124 G-CSF supported cycles from 38 patients treated between January 1999 and July 2005. There were no significant differences between pegfilgrastim and G-CSF groups in baseline or treatment-related variables. The median duration of grade IV neutropenia was 4 days in both groups (P = 0.55). Time to neutrophil recovery, incidence of febrile neutropenia, positive blood cultures and delay in subsequent chemotherapy were similar in both groups. Once per cycle dosing of pegfilgrastim appears safe and as effective as daily G-CSF for supporting the hyper-CVAD chemotherapy regimen.

Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry

Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP‐70 protein expression in neoplastic B‐cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting.

Cessation of immunosuppression during chemotherapy for post-transplant lymphoproliferative disorders in renal transplant patients

BACKGROUND The optimal reduction of immunosuppressive therapy (IST) in renal transplant patients with post-transplant lymphoproliferative disorders (PTLDs) is uncertain. As chemotherapy is immunosuppressive, IST may be stopped during this time without compromising graft function. Subsequent long-term reduction of IST reduces relapse risk, but may increase risk of graft rejection. METHODS We performed a retrospective, matched cohort study of adult renal transplant patients in whom IST was ceased during chemotherapy and resumed at lower dose (calcineurin inhibitor at 50%, prednisolone ≤10 mg daily, no third agent) approximately 6 weeks after chemotherapy. Outcomes were compared with those of renal transplant patients without PTLD, matched for creatinine at equivalent time post-transplant that PTLD was diagnosed in cases, as well as for age, gender and year of transplant. RESULTS Twenty-four cases of PTLD occurring at a median of 9.2 years post-transplant were compared with 83 matched controls. PTLD cases were followed for a median of 11.9 years. Using competing risks analysis, time to 25% increase in serum creatinine was not significantly different between the two groups [adjusted hazard ratio (HR) 1.8, 95% confidence interval (CI) 0.89-3.6]. Similar results were obtained using multivariable Cox regression analysis (HR 1.19, 95% CI 0.44-3.23). Only one PTLD case experienced a ≥25% increase in creatinine <6 months after IST cessation in the setting of progressive PTLD and death. Three cases recommenced dialysis, compared with three controls (HR 2.5, 95% CI 0.47-13.00). Five-year patient survival rates for cases and controls were 70 and 94%, respectively (P = 0.01). CONCLUSIONS IST can be safely ceased during chemotherapy for PTLD in renal transplant patients. Furthermore, long-term reduction in IST is not associated with a significant difference in renal function deterioration. Prospective trials are needed to address the optimal reduction of IST in PTLDs.