Immunological markers for prognostication in cytogenetically normal acute myeloid leukemia
Authors: Victoria Y. Ling, Denise Lee, Zoe Mcquilten, Sharon Avery, Michael Low, Stephen H. Cody, Trung Nguyen, Catriona Mclean, Malgorzata Gorniak, Andrew Wei, Stephen B. Ting
Journal: American Journal of Hematology (2015)

Cytogenetically normal acute myeloid leukemia (CN-AML) is heterogeneous and requires individualized prognostication. In spite of molecular characterization of CN-AML, many patients lack a clear indication for or against allogeneic stem cell transplant (SCT) in first complete remission (CR). Baseline immune response correlates with survival in solid organ malignancies, including melanoma, ovarian, breast, and especially, colorectal cancers, where a higher degree of tumor-infiltrating lymphocytes (TIL) predicts improved survival independent of TNM (tumor, node, metastases) staging 1. TIL have also shown prognostic value in various lymphoid hematological malignancies. In AML, evidence of immune modulation is seen in the "graft-versus-leukemia" effects post-allogeneic SCT together with promising preclinical activity of novel immune therapies. Furthermore, higher peripheral blood lymphocyte recovery after induction chemotherapy 2 and allogeneic SCT for AML 3 have predicted improved overall survival (OS). The prognostic significance of immune status at diagnosis has not been defined in AML. We therefore investigated whether degree of T lymphocyte infiltration within CN-AML diagnostic bone marrow trephines, which we have labeled the "leukemia-infiltrating lymphocyte" (LIL) index, had prognostic value. Patients diagnosed with CN-AML at an adult tertiary center between 2006 and 2013, treated with intensive chemotherapy and who had suitable stored, diagnostic bone marrow trephines were analyzed with institutional ethics approval. Trephines were formalin-fixed, decalcified in EDTA, and processed through to paraffin. Immunostaining for pan-T cell marker, CD3, and cytotoxic markers, CD8 and Granzyme B (GB), was performed at the time of analysis using routine methods. CD4 immunostaining was attempted, however high levels of non-specific staining precluded quantitative analysis. Positive cells were quantified using Fiji© image analysis software (v1.48o) from photographs taken at ×200 magnification of three representative areas of each trephine, expressed as a percentage of total cells and averaged over the three images. The cohort included 53 patients (51% male, median age 51 years (range 19–78)) followed-up over a median of 25.9 months (range 0.17–102.6 months). Most patients (96%) had de novo AML, 47 patients (89%) achieved CR (29 (62%) of whom relapsed), 21 (40%) underwent allogeneic SCT (10 in second CR) and 20 (38%) died. From a mean of 13,768 cells per trephine analyzed, median CD3% was 4.64% (range 0.56–37.24%) (Fig. 1A,B), CD8% was 5.08% (range 0.43–43.5%) and GB% was 0.34% (range 0–10.11%). In univariate Cox regression analyses, significant risk factors for death were higher aspirate blast% (P = 0.006), primary refractory disease (P = 0.03), and relapse (P = 0.04). Increasing age (P = 0.093), FLT3-ITDpositive(pos) (P = 0.099), and CD3% (P = 0.079) approached significance. Gender, preceding myelodysplastic syndrome, allograft, NPM1 mutation (NPM1mut), CD8%, and GB% were not significant. In Cox regression multivariate analyses, combining T cell numbers with the relevant univariate factors (i.e., P < 0.1), higher CD3 and CD8 were independent predictors of OS (CD3: hazard ratio (HR) 0.929 for death, 95% CI 0.870–0.992, P = 0.029; CD8: HR 0.920, 95% CI 0.869–0.973, P = 0.004). Patients were divided into quartiles to determine whether incremental differences in survival were present with increasing T cell number. Kaplan–Meier survival analyses demonstrated that CD3 and CD8 numbers were able to stratify patient survival, with significantly superior OS in patients with higher CD3% (Quartile 4 (>12.39%) vs. Quartile 1 (<1.63%), P = 0.042) (Fig. 1C, first panel) and a trend toward better OS with higher CD8% (Quartile 4 (>10.66%) vs. Quartile 1 (<2.2%), P = 0.057) (Fig. 1C, second panel). GB was not able to risk-stratify patients (Fig. 1C, third panel). Higher CD3% and CD8% measured by flow cytometry showed similar prognostic capability, however there was weak correlation between CD3 and CD8 numbers obtained by immunohistochemistry and flow cytometry (data not shown; correlation coefficient, r = 0.534 for CD3 and r = 0.613 for CD8). This may reflect inherent differences in the type of samples analyzed, particularly with sampling artifact and varying degrees of hemodilution in aspirates. A,B: Representative immunohistochemistry images of bone marrow trephines demonstrating low infiltration (Quartile 1) (A) compared with high infiltration (Quartile 4) (B) of CD3 T cells with percentage calculations based on Fiji© image analysis data displayed. Original magnification 200× for both panels. C–E: Kaplan–Meier overall survival analyses based on T cell numbers. C: Whole cohort (n = 53) separated into quartiles by CD3, CD8, and GB percentage. P-values denote analyses comparing Quartile 1 vs. 4. D: FLT3-ITDpos group (n = 18) separated by median CD3, CD8, and GB percentage; (E) NPM1wt/FLT3-ITDneg group (n = 17) separated by median CD3, CD8, and GB percentage. Further survival analyses incorporated known prognostic significance of FLT3-ITD and NPM1mut. Forty-four patients (83%) who were tested for both mutations formed three subgroups: FLT3-ITDpos (n = 18; 3 year OS 37%), NPM1mut without FLT3-ITD (NPM1mut/FLT3-ITDneg; n = 9; 3 year OS 70%), and "double-negative" (NPM1wt/FLT3-ITDneg; n = 17; 3 year OS 62%). As small numbers within each subgroup precluded analysis with quartiles, groups split by the median were compared. Although there were no statistically significant differences in OS for CD3, CD8, and GB within the FLT3-ITDpos (Fig. 1D) and NPM1mut/FLT3-ITDneg (data not shown) subgroups, there was a trend for better survival in the FLT3-ITDpos subgroup with higher T cells. This was in spite of older age in high (median age 63 years) compared with low T cell (median age 53 years) subgroups. In contrast, CD3 > median (16.06%) and CD8 > median (13.8%) in the NPM1wt/FLT3-ITDneg subgroup were associated with significantly superior OS (CD3: P < 0.001; CD8: P = 0.010) (Fig. 1E, first and second panels) with comparable patient characteristics between above and below median groups. GB > median (1.38%) was not related to survival (P = 0.247) (Fig. 1E, third panel). As the NPM1wt/FLT3-ITDneg group often lacks defining prognostic factors, T cell numbers may be most relevant for further risk-stratification of this group. The association of low T cell numbers with poor prognosis in our analyses may reflect the proliferative advantage of blasts gained by immune evasion mechanisms described in AML, including suppression of production and function of T lymphocytes 4. The corollary is that retained specific T cell immune responses against leukemia-associated antigens, including NPM1, may contribute to improved prognosis 5. In addition to prognostic implications, the baseline immune status may modify response to immune therapies. Patients with lower T cells generally had a dismal prognosis with cytotoxic therapy and may benefit from immune augmentation by immune-based therapies. Conversely, low T cells may limit response to immune therapies. In in vitro studies of chimeric antigen receptor T cells 6 and bispecific T cell engaging antibodies 7 in AML, less cytolysis was seen with lower T cells to blasts (i.e., effector to target (E:T)) ratios in a dose-dependent relationship. Whether this consideration applies in vivo requires further investigation. To the best of our knowledge, this study is the first to correlate higher baseline T cell infiltration in bone marrow trephines in CN-AML with improved survival. A noted strength of the study was the objective quantitation of large numbers of cells using image analysis software. Although our patient cohort was small, patient outcomes are consistent with larger cohort analyses. Notwithstanding the other limitations of a retrospective design and variability in post-remission therapies, our study introduces the LIL index as a novel prognostic marker in CN-AML, with greatest potential utility in the indeterminate-risk NPM1wt/FLT3-ITDneg group. This work provides rationale for further studies to explore the function of the infiltrating T cells, identify T cell subsets that portend the survival benefit, and assess the LIL index in other AML genetic subgroups. S.B.T is funded by an NHMRC Career Development Fellowship and NHMRC project grants 1006420 and 1047554. Victoria Y. Ling,1,2* Denise Lee,1 Zoe Mcquilten,3 Sharon Avery,1 Michael Low,1 Stephen H. Cody,4 Trung Nguyen,5 Catriona Mclean,5 Malgorzata Gorniak,6 Andrew Wei,1,2 Stephen B. Ting1,2 1Malignant Haematology & Stem Cell Transplantation Service, Department of Clinical Haematology, Alfred Health, Melbourne, Australia; 2Division of Blood Cancers, Leukaemia & Stem Cell Research, Australian Centre for Blood Diseases, Monash University, Melbourne, Australia; 3Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia; 4Monash Micro Imaging, Monash University, Melbourne, Australia; 5Department of Anatomical Pathology, Alfred Health, Melbourne, Australia; 6Department of Laboratory Haematology, Alfred Health, Melbourne, Australia