T cell repertoire diversity is generated in part by recombination of variable (V), diversity (D), and joining (J) segments in the T cell receptor β (TCR) locus. T cell clonal frequency distribution determined by high-throughput sequencing of TCR β in 10 stem cell transplantation (SCT) donors revealed a fractal, self-similar frequency distribution of unique TCR bearing clones with respect to V, D, and J segment usage in the T cell repertoire of these individuals. Further, ranking of T cell clones by frequency of gene segment usage in the observed sequences revealed an ordered distribution of dominant clones conforming to a power law, with a fractal dimension of 1.6 and 1.8 in TCR β DJ and VDJ containing clones in healthy stem cell donors. This self-similar distribution was perturbed in the recipients after SCT, with patients demonstrating a lower level of complexity in their TCR repertoire at day 100 followed by a modest improvement by 1 year post-SCT. A large shift was observed in the frequency distribution of the dominant T cell clones compared to the donor, with fewer than one third of the VDJ-containing clones shared in the top 4 ranks. In conclusion, the normal T cell repertoire is highly ordered with a TCR gene segment usage that results in a fractal self-similar motif of pattern repetition across levels of organization. Fractal analysis of high-throughput TCR β sequencing data provides a comprehensive measure of immune reconstitution after SCT.

Summary Patients with multiple myeloma (MM) undergoing high dose therapy and autologous stem cell transplantation (SCT) remain at risk for disease progression. Induction of the expression of highly immunogenic cancer testis antigens (CTA) in malignant plasma cells in MM patients may trigger a protective immune response following SCT. We initiated a phase II clinical trial of the DNA hypomethylating agent, azacitidine (Aza) administered sequentially with lenalidomide (Rev) in patients with MM. Three cycles of Aza and Rev were administered and autologous lymphocytes were collected following the 2nd and 3rd cycles of Aza‐Rev and cryopreserved. Subsequent stem cell mobilization was followed by high‐dose melphalan and SCT. Autologous lymphocyte infusion (ALI) was performed in the second month following transplantation. Fourteen patients have completed the investigational therapy; autologous lymphocytes were collected from all of the patients. Thirteen patients have successfully completed SCT and 11 have undergone ALI. Six patients tested have demonstrated CTA up‐regulation in either unfractionated bone marrow (n = 4) or CD138+ cells (n = 2). CTA (CTAG1B)‐specific T cell response has been observed in all three patients tested and persists following SCT. Epigenetic induction of an adaptive immune response to cancer testis antigens is safe and feasible in MM patients undergoing SCT.

4193 T cell repertoire diversity is generated by recombination of variable (V), diversity (D) and joining (J) segments in the T cell receptor (TCR) locus. Further variability and antigen recognition capacity is introduced by nucleotide insertion (NI) in the recombined sequences resulting in a complex repertoire, the organization of which is poorly understood. We postulate that TCR b D, J and V gene segment usage in an individual would result in a TCR repertoire with a fractal, self-similar frequency distribution of T cell clones with respect to gene segment usage. To determine this, the TCR repertoire of donors and recipients of HLA matched-related and unrelated allogeneic stem cell transplantation (SCT) was evaluated by high-throughput (HT) sequencing of the CDR3 region of TCR b. Ten SCT donor-recipient pairs were selected for HT-TCR b sequencing. cDNA was isolated from T cells obtained from the donors at baseline and recipients at day 100, 1 year post SCT or at the time of graft-versus-host disease (GVHD) diagnosis. HT-TCR β sequencing was performed and analyzed using the ImmunoSEQ analyzer tool (Adaptive Biotechnologies, Seattle, WA). TCR b clone frequencies were used to determine the TCR self-similarity score (SSS = log clone frequency × log scale), evaluating clonal frequency at each gene segment-scale (J, VJ and VJ+NI). This revealed a TCR SSS with a relatively narrow distribution of 1.64 ± 0.1 (mean ± SD) for J, 1.69 ± 0.2 for VJ, and 1.41 ± 0.01 for VJ+NI, which was consistent between all normal stem cell donors analyzed. Relative proportional distribution (RPD) graphs were then generated to allow for the depiction of the TCR b D, J, V distribution for simultaneous comparison at an individual level. Representative data in Figure 1 shows the relatedness across donors at the TCR b J segment level. Relative clonal frequency was determined for each unique TCR clone at the specified segment level and plotted according to frequency-rank. Slope of the resulting linear regression lines from log-log plots of rank-frequency was used to determine self-similarity and fractal dimension. These plots were comparable among donors with resulting slopes of 1.6 ± 0.01 and 1.8 ± 0.1, respectively for TCR J and VJ containing clones (Figure 2). The plots also revealed a hierarchy of T cell clones, with few dominant clones occupying the high ranks and a multitude of clones in the later ranks. For donor-recipient comparisons, we examined the dominant ranking clones which would be most likely to be involved in GVHD and response to infection. The ordered TCR clonal frequency distribution seen in donors was perturbed in recipients following SCT, with recipients demonstrating a lower level of complexity in their TCR repertoire, and a large shift in the frequency distribution of the dominant T cell clones compared to the donor. When dominant clones were compared between donors and recipients, recipients shared only a small proportion of TCR clonotypes with their donors despite full donor T cell chimerism. This difference did not change over time, suggesting an alternate T cell clonal hierarchy and repertoire develops in transplant recipients when compared with their donors. Using simple mathematical analysis, we demonstrate that the TCR b repertoire has a fractal, self-similar pattern with a hierarchy of dominant and minor clones. We note that the complexity of the TCR repertoire is diminished and TCR b hierarchy is altered following SCT. Restoration of this order may serve as a marker for post-SCT immune reconstitution. Further, by demonstrating shifts in TCR clonal dominance, fractal analysis comparing donor and recipient T cell repertoire may allow for more accurate monitoring of immunotherapy of malignancies in general, beyond allogeneic SCT. Figure 1. Graphical representation of self-similarity in TCR b J gene segment usage across segments and individuals. Relative-proportional-distribution (RPD) graph depicting TCR b J segment usage in ten hematopoietic stem cell donors. Each ring in the graph represents J segment usage frequency in a single donor, showing similarity in J segment frequency across ten different donors. Figure 1. Graphical representation of self-similarity in TCR b J gene segment usage across segments and individuals. Relative-proportional-distribution (RPD) graph depicting TCR b J segment usage in ten hematopoietic stem cell donors. Each ring in the graph represents J segment usage frequency in a single donor, showing similarity in J segment frequency across ten different donors. Figure 2. Log-Log plot depicting linear distribution of frequency ranked TCR b DJ (top panels) and VDJ (bottom panels) clones in two stem cell donors. Figure 2. Log-Log plot depicting linear distribution of frequency ranked TCR b DJ (top panels) and VDJ (bottom panels) clones in two stem cell donors. Disclosures: No relevant conflicts of interest to declare.

3123 Background: Multiple myeloma (MM) is a plasma cell malignancy with variable prognosis depending on disease features such as β2m level and cytogenetics. High dose therapy and stem cell transplantation (SCT) remains the current standard of care for MM, however the role of tandem SCT is controversial, particularly in the era of novel induction therapy. Methods: The Virginia Commonwealth University BMT program has a practice of risk-stratified transplant allocation in MM patients referred for SCT; those with high-risk (HR) disease (β2m >5.5, adverse cytogenetics, >1st remission) are preferentially assigned tandem SCT, and those with standard-risk (SR), a single SCT. Between 2008 and 2011, 146 MM patients underwent SCT, 64 (44%) SR patients received a single SCT (SRS), 32 (22%) HR patients received tandem SCT (HRT) & 50 (34%) a single SCT (HRS). Median age at SCT was 58 years. Maintenance therapy was administered in 48% SRS, 53% HRT & 63% HRS patients. Results: Disease status at day 100 post-transplant was complete response (CR) or very good partial response (VGPR) in 84%, 75% and 72% patients in the SRS, HRT, and HRS groups respectively. Patients in the HRT group (41%) were more likely to achieve CR than in the HRS group (18%) (P=0.04). At a median follow up of 23 months, there was no difference in the relapse incidence (Figure 1) between the HRT and SRS groups (Log Rank P=0.22) or between the HRT and HRS groups (Log Rank P=0.64), however the relapse incidence for SRS was lower than for HRS group (Log Rank P=0.01). Correspondingly, there is no difference in the estimated survival between the HRT and SRS (Log Rank P=0.12), or between the HRT and HRS groups (Log Rank P=0.57), though the survival for the SRS group is superior to HRS group (Log Rank P=0.025) (Figure 2). Estimated 2-year survival rates are 0.96 (95% CI: 0.88, 1.00), 0.87 (0.73, 1.0), and 0.79 (0.65, 0.94) in the SRS, HRT and HRS groups respectively, where the SRS survival is significantly higher than the HRS group (Log Rank P=0.047). This was attributable to a lower 1-year relapse rate of 0.10 (0.02, 0.19), 0.07 (0.00, 0.16) in the SRS, HRT groups compared to 0.29 (0.15, 0.43) in the HRS group (Log Rank P<0.03). Further analysis accounting for group status revealed that HR patients with IgA subtype, <CR/VGPR status, absence of bortezomib pre-SCT and absence of maintenance therapy post-SCT all experienced a higher rate of relapse (P <0.05, Cochran- Mantel-Haenszel test). Similarly, patients with <VGPR, and absence of maintenance therapy had an inferior survival. Conclusions: We report that HR MM patients undergoing tandem transplant have a higher response rate and thus a correspondingly lower relapse risk and improved survival comparable to SR. Therefore risk stratification in patients with MM using simple clinical and laboratory parameters may be used to guide single versus tandem SCT allocation. HR MM patients who cannot get tandem transplant for insurance or clinical reasons may benefit from receiving bortezomib as part of pre-transplant therapy with a goal to achieve maximal cytoreduction. Further benefit in these patients may be derived from post-transplant maintenance therapy. Disclosures: No relevant conflicts of interest to declare.

e18550 Background: Multiple myeloma (MM) is a plasma cell malignancy with variable prognosis depending on disease features such as β2m and cytogenetics. High dose therapy and stem cell transplantation (SCT) remains the current standard of care for MM, however the role of tandem SCT is controversial, particularly in the era of novel induction therapy. Methods: Our program has a practice of risk-stratified transplant allocation in MM patients referred for SCT, those with high-risk (HR) disease (β2m >5.5, adverse cytogenetics, >1st remission) are preferentially assigned tandem SCT, and those with standard risk (SR), a single SCT. Between 2008 and 2011, 129 MM patients underwent SCT, 43% SR patients received a single SCT (SRS), 22% HR received tandem SCT (HRT) & 36% HR a single SCT (HRS). Median age at SCT was 57 years. Maintenance therapy was administered in 51% SRS, 57% HRT & 67% HRS patients. Results: Complete response (CR) or very good partial response was achieved in 0.68, 0.72 and 0.80 for the HRT, HRS, and SRS groups. The HRT group (0.39) was more likely to achieve CR than HRS (0.20) (P=0.02). At a median follow up of 23.4 months, the overall survival for HRS was inferior to SRS (P=0.01) but there was no difference in the overall survival between HRT and SRS cohorts. Two-year survival rates were 0.81, 0.91 and 0.97 in the HRS, HRT and SRS cohorts (HRS vs. SRS P=0.02). This was attributable to a higher 1-year relapse rate in HRS (0.29) compared to HRT (0.07) and SRS (0.07) (P<0.01). Conclusions: Using a risk-stratified allocation system, we report that HR MM patients undergoing tandem SCT have outcomes comparable to SR patients. However, HR MM patients receiving a single SCT have inferior outcomes compared to those with SR. Notably, higher rate of CR and a lower relapse rate were observed in the HRT cohort when compared to HRS. This suggests that greater depth of remission achieved in HR patients undergoing tandem SCT may result in longer time to relapse and survival advantage compared to HR patients receiving a single SCT. In contrast, a single SCT may suffice for SR MM patients. These results demonstrate that risk-stratification based on disease prognostic features is an important treatment consideration when planning high dose therapy in MM patients.