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We sought to evaluate the impact of cryopreservation of unrelated donor (URD) peripheral blood stem cell (PBSC) grafts on engraftment, chimerism, and immune reconstitution in the context of the COVID-19 pandemic. We reviewed stem cell product characteristics and clinical outcomes in 101 patients receiving cryopreserved PBSCs from URDs between January 1, 2019 and 31 December, 2020, compared with 203 patients receiving fresh URD PBSCs. We observed no differences in 6-month overall survival, progression-free survival, or nonrelapse mortality. Patients receiving cryopreserved PBSCs had delayed platelet engraftment and impaired reconstitution of white blood cells and T-cell subsets at day 30. Thirty-four percent of patients receiving cryopreserved grafts had CD3 chimerism 48 hours old at time of cryopreservation or infusion significantly increased the risk of graft failure (subdistribution hazard ratio = 4.57; 95% confidence interval, 1.71-12.3; P = .0025). Our data indicate that cryopreservation is associated with similar overall short-term clinical outcomes compared with fresh PBSC. However, patients must be monitored closely for increased risk of other potentially adverse outcomes, including graft failure and poor immune recovery, particularly for grafts with older overall age at infusion. Longer-term follow-up is needed to determine impact on relapse and survival.

PD-1 expression marks activated T cells susceptible to PD-1–mediated inhibition but not whether a PD-1–mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho–PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho–PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho–PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.

BackgroundResponses to conventional donor lymphocyte infusion (DLI) for post-allogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse.MethodsWe initiated this ongoing phase 1 trial of adoptively transferred cytokine induced memory-like (CIML) NK cells in patients with myeloid malignancies relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5-10 × million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High resolution profiling with mass cytometry and single cell RNA sequencing characterized the expanding and persistent NK cells subpopulations in a longitudinal manner after infusion. In vitro functional studies of infused CIML NK cells were complemented with in vivo evaluation of NK trafficking to disease sites using multiparameter immunofluorescence.ResultsIn the first 5 patients on the trial, infusion of CIML NK cells led to a rapid 10 to 50-fold in vivo expansion that was sustained over months. The infusion was well-tolerated, with fever and pancytopenia as the most common adverse events. Responses were attained in 4 of 5 patients at day +28. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires. AML relapse after initial response to CIML NK cell therapy was associated with low transcript expression of CD2 ligands, including CD48/SLAMF2 and CD58/LFA3.ConclusionGiven their rapid expansion and long-term persistence in an immune compatible environment, CIML NK cells serve as a promising platform for the treatment of post-transplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T-cells and tumor targets will lead to the development of novel cell-based immunotherapies.

Background : Autologous stem cell transplantation (ASCT) is a standard of care for patients with chemosensitive, relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL). While the clinical benefit of ASCT has traditionally been attributed solely to cytoreduction from intensive chemotherapy, ASCT has important immunogenic effects which may contribute to its anti-tumor efficacy and could provide a favorable immune environment for post-ASCT immune-based maintenance treatments. We previously reported clinical results of a phase II trial (NCT02362997) testing 8 doses of pembrolizumab maintenance therapy after ASCT for patients with R/R cHL or DLBCL. To clarify the impact of pembrolizumab on immune reconstitution, we compared the kinetics of peripheral blood immune cell recovery after ASCT for trial patients receiving pembrolizumab maintenance to those of a contemporaneous control cohort of similar patients undergoing ASCT without pembrolizumab maintenance. Objective : To characterize the impact of post-ASCT pembrolizumab maintenance therapy on immune reconstitution for patients with R/R DLBCL and cHL and to identify candidate biomarkers of efficacy and immune-related adverse events (irAEs). Study design : Peripheral blood mononuclear cell (PBMC) samples were prospectively collected 1-18 months after ASCT and analyzed by flow cytometry using a panel of fluorophore-conjugated monoclonal antibodies to identify B cells, natural killer (NK) cells, and various dendritic cell (DC) and T cell subsets. Results : A median of 5 (range 1-8) post-ASCT PB samples were collected from 144 patients (59 pembrolizumab, 85 control). Clinical characteristics of the two cohorts were similar. Compared to cHL patients, DLBCL patients (who all received anti-CD20 monoclonal antibody therapy before ASCT) had delayed CD19+ cell reconstitution which persisted for at least 18-month after ASCT. No other differences in immune reconstitution were observed based on lymphoma subtype. Post-ASCT pembrolizumab maintenance therapy was associated with 1) an elevation in circulating DCs (driven by higher levels of plasmacytoid and immature DCs) which persisted for the duration of pembrolizumab treatment, and 2) a significant reduction in PD-1+ T cells which persisted for 6-12 months after completion of pembrolizumab therapy. Despite the key role of T cells in mediating the effects of PD-1 blockade, pembrolizumab maintenance did not affect recovery of any T cell subsets. In an exploratory analysis, a higher baseline CD4+ TEMRA cell count (defined as CD3+ CD4+ CD45RA+ CD62L-) was associated with inferior PFS, but only among patients who received pembrolizumab maintenance (p=0.003). As continuous variables, lower absolute levels of NK cells (p=0.009), PD-1+ CD4+ T cells (p=0.005), and PD-1+ CD8+ T cells (p=0.005) before pembrolizumab initiation were each associated with a higher risk of grade 2+ irAEs. Conclusion : Post-ACST pembrolizumab maintenance therapy is associated with a persistent elevation in circulating DCs, but its impact on the reconstitution of other immune cells in peripheral blood appears limited. Our study suggests that early features of post-ASCT immune reconstitution could be associated with PFS and risk of irAE and warrant additional investigation.

Patients with chronic graft-versus-host disease (cGVHD) have a paucity of regulatory CD4 T cells (CD4Tregs) that mediate peripheral tolerance. In clinical trials, daily low-dose interleukin-2 (IL-2) has been administered safely for prolonged periods in patients with steroid-refractory cGVHD. Peripheral CD4Tregs expand dramatically in all patients during IL-2 therapy but clinical improvement was observed in ∼50% of patients. Here, we examined the impact of low-dose IL-2 therapy on functional T-cell markers and the T-cell repertoire within CD4Tregs, conventional CD4 T cells (CD4Tcons), and CD8+ T cells. IL-2 had profound effects on CD4Tregs homeostasis in both response groups including selective expansion of the naive subset, improved thymic output, and increased expression of Ki67, FOXP3, and B-cell lymphoma 2 within CD4Tregs. Similar changes were not seen in CD4Tcons or CD8 T cells. Functionally, low-dose IL-2 enhanced, in vitro, CD4Treg-suppressive activity in both response groups, and all patient CD4Tcons were similarly suppressed by healthy donor CD4Tregs. High-throughput sequencing of the T-cell receptor β (TCRβ) locus demonstrated that low-dose IL-2 therapy increased TCR repertoire diversity and decreased evenness within CD4Tregs without affecting CD4Tcons or CD8 T cells. Using clone-tracking analysis, we observed rapid turnover of highly prevalent clones in CD4Tregs as well as the conversion of CD4Tcons to CD4Tregs. After 12 weeks of daily IL-2, clinical responders had a greater influx of novel clones within the CD4Treg compartment compared with nonresponders. Further studies to define the function and specificity of these novel CD4Treg clones may help establish the mechanisms whereby low-dose IL-2 therapy promotes immune tolerance.

Vaccination using irradiated, adenovirus transduced autologous myeloblasts to secrete granulocyte–macrophage colony-stimulating factor (GVAX) early after allogeneic hematopoietic stem cell transplantation (HSCT) can induce potent immune responses. We conducted a randomized phase 2 trial of GVAX after HSCT for myelodysplastic syndrome with excess blasts or relapsed/refractory acute myeloid leukemia. Myeloblasts were harvested before HSCT to generate the vaccine. Randomization to GVAX vs placebo (1:1) was stratified according to disease, transplant center, and conditioning. Graft-versus-host disease (GVHD) prophylaxis included tacrolimus and methotrexate. GVAX or placebo vaccination was started between day 30 and 45 if there was engraftment and no GVHD. Vaccines were administered subcutaneously/intradermally weekly × 3, then every 2 weeks × 3. Tacrolimus taper began after vaccine completion. A total of 123 patients were enrolled, 92 proceeded to HSCT, and 57 (GVAX, n = 30; placebo, n = 27) received at least 1 vaccination. No Common Toxicity Criteria grade 3 or worse vaccine-related adverse events were reported, but injection site reactions were more common after GVAX (10 vs 1; P = .006). With a median follow-up of 39 months (range, 9-89 months), 18-month progression-free survival, overall survival, and relapse incidence were 53% vs 55% (P = .79), 63% vs 59% (P = .86), and 30% vs 37% (P = .51) for GVAX and placebo, respectively. Nonrelapse mortality at 18 months was 17% vs 7.7% (P = .18), grade II to IV acute GVHD at 12 months was 34% vs 12% (P = .13), and chronic GVHD at 3 years was 49% vs 57% for GVAX and placebo (P = .26). Reconstitution of T, B, and natural killer cells was not decreased or enhanced by GVAX. There were no differences in serum major histocompatibility chain-related protein A/B or other immune biomarkers between GVAX and placebo. GVAX does not improve survival after HSCT for myelodysplastic syndrome/acute myeloid leukemia. This trial was registered at www.clinicaltrials.gov as #NCT01773395.

Triple-negative/basal-like breast cancer (BC) is characterized by aggressive biological features, which allow relapse and metastatic spread to occur more frequently than in hormone receptor-positive (luminal) subtypes. The molecular complexity of triple-negative/basal-like BC poses major challenges for the implementation of targeted therapies, and chemotherapy remains the standard approach at all stages. The matricellular protein cysteine-rich angiogenic inducer 61 (CCN1/CYR61) is associated with aggressive metastatic phenotypes and poor prognosis in BC, but it is unclear whether anti-CCN1 approaches can be successfully applied in triple-negative/basal-like BC. Herein, we first characterized the prevalence of CNN1 expression in matched samples of primary tumors and metastatic relapse in a series of patients with BC. We then investigated the biological effect of CCN1 depletion on tumorigenic traits in vitro and in vivo using archetypal TNBC cell lines. Immunohistochemical analyses of tissue microarrays revealed a significant increase of the highest CCN1 score in recurrent tissues of triple-negative/basal-like BC tumors. Stable silencing of CCN1 in triple-negative/basal-like BC cells promoted a marked reduction in the expression of the CCN1 integrin receptor α(v)β(3), inhibited anchorage-dependent cell growth, reduced clonogenicity, and impaired migration capacity. In an orthotopic model of triple-negative/basal-like BC, silencing of CCN1 notably reduced tumor burden, which was accompanied by decreased microvessel density and concurrent induction of the luminal epithelial marker E-cadherin. Thus, CNN1/CYR61-targeting strategies might have therapeutic value in suppressing the biological aggressiveness of triple-negative/basal-like BC.

Administration of posttransplant cyclophosphamide (PTCy) has significantly expanded the number of patients undergoing HLA-haploidentical hematopoietic cell transplantation (haplo-HCT). To examine immune reconstitution in these patients, we monitored T- and natural killer (NK)-cell recovery in 60 patients receiving bone marrow or peripheral blood stem cell (PBSC) grafts after haplo-HCT with PTCy and 35 patients receiving HLA-matched donor PBSC grafts with standard graft-versus-host disease (GVHD) prophylaxis. Compared with HLA-matched recipients, early T-cell recovery was delayed in haplo-HCT patients and skewed toward effector memory T cells with markedly reduced naive T cells. We found higher regulatory T (Treg)-cell/conventional T (Tcon)-cell ratios early after HCT and increased PD-1 expression on memory T cells. Within the haplo-HCT, patients who did not develop chronic GVHD (cGVHD) had higher PD-1 expression on central and effector memory CD4+ Treg cells at 1 month after transplant. These findings suggest an immunologic milieu that promotes immune tolerance in haplo-HCT patients. NK cells were decreased early after haplo-HCT with preferential expansion of immature CD56brightCD16− NK cells compared with matched donor transplants. One month after transplant, mass cytometry revealed enrichment of immature NK-cell metaclusters with high NKG2A, low CD57, and low killer-cell immunoglobulin-like receptor expression after haplo-HCT, which partially recovered 3 months post-HCT. At 2 months, immature NK cells from both groups were functionally impaired, but interleukin-15 priming corrected these defects in vitro. Increased immature/mature NK-cell ratios were associated with cytomegalovirus reactivation and increased incidence of cGVHD after haplo-HCT. These homeostatic imbalances in T- and NK-cell reconstitution after haplo-HCT reveal opportunities for early immune-based interventions to optimize clinical outcomes.

Allogeneic hematopoietic cell transplantation (alloHCT) can cure previously treated high-risk chronic lymphocytic leukemia (CLL) patients if they are suitable for transplant through the graft-versus-leukemia effect. However, since the emergence of targeted therapies, the role of alloHCT for high-risk CLL is less clear. To address this question, we evaluated 108 high-risk CLL patients who underwent alloHCT from 2010 to 2018. Thirty patients from the period of 2013 to 2018 received targeted therapy prior to alloHCT. The median age for the targeted therapy cohort was 60 years (range, 30-71 years), and 20% and 73% had complete and partial remission, respectively: 76% had del(17p), 46.2% had 5 or more cytogenetic abnormalities, and 78.9% were IGHV unmutated. The median number of prior therapies was 4 (range, 1-9). With a median follow-up time of 36 months (range, 10-72 months), the 3-year overall (OS) and progression-free survival (PFS) were 87% and 69%, respectively. The 3-year cumulative incidence of nonrelapse mortality and relapse was 7% and 24%, respectively. For the control cohort of 78 patients who underwent alloHCT from 2010 to 2014 and received only chemoimmunotherapy prior to transplant, the 3-year OS and PFS were 69% and 58%, respectively. Patients treated with targeted therapy prior to alloHCT had a significantly higher number of circulating T and B cells and a lower ratio of CD4 regulatory T cells to CD4 conventional T cells early after transplant. In summary, despite multiple high-risk features, the clinical outcome of CLL patients who receive targeted therapy prior to transplant is excellent and alloHCT should be offered while the disease is under control.

We recently conducted a randomized double-blind study in which we demonstrated that moderate/severe chronic graft-versus-host disease (cGVHD) but not cGVHD-free survival was reduced in patients receiving anti-T lymphocyte globulin (ATLG) versus placebo. In a companion study we performed immunophenotypic analysis to determine the impact of ATLG on immune reconstitution (IR) and to correlate IR with clinical outcomes. The randomized study (n = 254) included patients (aged 18 to 65 years) who underwent myeloablative transplants for acute myeloid leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia from HLA-matched unrelated donors. Ninety-one patients consented for the companion IR study (ATLG = 44, placebo = 47). Blood samples were collected on days 30, 100, 180, and 360 after hematopoietic cell transplantation (HCT), and multiparameter flow cytometry was performed in a blinded fashion. Reconstitution of CD3+ and CD4+ T cells was delayed up to 6 months post-HCT in the ATLG arm, whereas absolute regulatory T cell (Treg) (CD4+25+127-) numbers were lower only in the first 100 days. Analysis of the CD4+ Treg and conventional T cells (Tconv) (CD4+25–127+) compartments showed a profound absence of naive Tregs and Tconv in the first 100 days post-HCT, with very slow recovery for 1 year. B cell and natural killer cell recovery were similar in each arm. Higher absolute counts of CD3+, CD4+, CD8+ T, Tregs, and Tconv were associated with improved overall survival, progression-free survival, and nonrelapse mortality but not moderate/severe cGVHD. Although ATLG delays CD3+ and CD4+ T cell recovery post-transplant, it has a relative Treg sparing effect after the early post-HCT period, with possible implications for protection from cGVHD. ATLG severely compromises the generation of naive CD4+ cells (Treg and Tconv), potentially affecting the diversity of the TCR repertoire and T cell responses against malignancy and infection.

Background Cytokine-release syndrome (CRS) is a life-threatening complication of haploidentical stem cell transplantation (haploSCT) occurring in the first few days after infusion of the stem cell graft prior to administration of post-transplant cyclophosphamide (PTCY). In the last few years, blockade of IL-6 receptor signaling with tocilizumab has emerged as an effective therapy for Grade 3-4 CRS. As IL-6 mediated signaling is a known regulator of the balance between regulatory T cells (Tregs) and other T cell subsets and has effects on NK cell function, questions remain regarding the effect of the blockade of IL-6 signaling on post-transplant immune cell recovery, engraftment, infection, acute graft-versus-host disease (aGVHD), and the graft-versus-tumor effect. We report on our experience with the use of tocilizumab to treat CRS, and we address the hypothesis that tocilizumab has no effect on the reconstitution of the post-haploSCT myeloid or lymphoid immune subsets. Methods A retrospective review of haploSCT patients with malignacies treated at the Dana-Farber Cancer Institute during the period of 2010-2019 was undertaken. Data included treatment with tocilizumab, cumulative incidence of NRM and relapse, day+30 and day+100 post-transplant chimerism, laboratory markers of viral and fungal infection, and immune reconstitution data at 1-month, 2-month, 3-month, and 6 months post-transplant. Kaplan-Meier analysis of overall survival (OS), including competing risk analysis of the cumulative incidence of relapse and non-relapse mortality (NRM) with the Fine-Gray model was preformed with EZR. Flow cytometry identification of relevant lymphoid populations was performed with a customized panel that included Treg (CD4+CD25+), Tcon (CD4+) and NK (CD3-CD56+) cells. Comparison of the lymphoid populations between the Tocilizumab-treated subgroup of haploSCT patients and the remaining patients was done with the Wilcoxon Mann-Whitney test. Results Out of 132 haploSCT patients, 19 received at least one dose of tocilizumab for the treatment of CRS out of whom one patient died from CRS. In tocilizumab-treated patients myeloid engraftment was 100% at day 30 post transplant. Tocilizumab use was not associated with any effect on lymphoid subset reconstitution at any time point post haploSCT (Figure 1A). Specifically, there was no difference in the reconstitution of NK cells (1-month NK: Mann-Whitney U = 120, p = 0.356; 3-month NK: Mann-Whitney U = 130, p = 0.528; 6-month NK: Mann-Whitney U = 110, p = 0.773), Treg (1-month Treg: Mann-Whitney U = 77, p = 0.412; 3-month Treg: Mann-Whitney U = 104, p = 0.203, 6-month Treg: Mann-Whitney U = 80, p = 0.186), or the Treg:Tcon ratio (1-month Treg:Tcon: Mann-Whitney U = 93, p = 0.835; 3-month Treg:Tcon: Mann-Whitney U = 97, p = 0.141; 6-month Treg:Tcon: Mann-Whitney U = 117, p = 0.959). The 1-year OS in the tocilizumab-treated patient subgroup was 62% (95% CI, 36%-80%) while in the rest it was 76% (95%CI, 67%-83%), with no significant difference between the two subgroups (p = 0.13). The 1-year cumulative incidence of relapse in the tocilizumab-treated subgroup was 19% (95% CI, 4%-42%) while in the rest it was 28% (95% CI, 20%-36%), with no difference between the subgroups (p=0.29). The 1-year NRM in the tocilizumab-treated subgroup was 33% (95% CI, 12%-55%) while in the rest it was 10% (95% CI, 5%-16%), and the difference between subgroups was statistically significant (p = 0.02). Fifty-five percent of patients treated with tocilizumab experienced reactivation of one or more of adenovirus, EBV, CMV or a fungal organism, with 50% of reactivations being CMV and 37% being fungal. Among the tocilizumab-treated patients, 42% experienced any aGVHD with only one patient experiencing Grade 3-4 aGVHD. Conclusions Tocilizumab use is not associated with any effect on post-transplant myeloid engraftment or reconstitution of the Treg, Tcon, and NK cell subsets. Although a significant proportion of tocilizumab-treated patients experienced reactivation of CMV or a fungal organism, the majority of these reactivations were not associated with any clinically significant symptoms. Treatment with tocilizumab was not associated with any significant effect on OS or disease relapse, but the tocilizumab-treated group had a higher NRM than the rest of the haploSCT patients. This association is consistent with prior studies correlating severe CRS with a high NRM, and merits further study. Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Biolojic Design Inc: Consultancy; Regeneron: Other: Research Support; BMS: Other: Research Support; Cugene: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Other: Research Support; Equillium: Consultancy; Moderna Therapeutics: Consultancy; Therakos: Membership on an entity's Board of Directors or advisory committees; EMD Serono: Consultancy; Clinigen: Other. Cutler:Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Be The Match/National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Celgene: Other; Juno: Other; Novartis: Consultancy; Alexion: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy; Gilead: Consultancy. Ritz:TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Rheos Medicines: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy.

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen-presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway modulates T cell activation, proliferation and trafficking playing a central role in inflammation. Early studies by Soiffer and colleagues demonstrated that ex-vivo depletion of CD6+ donor T cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized IgG1 anti-CD6 monoclonal antibody, has been shown to block CD6 signaling, leading to a reduction in T cell activation and proliferation. The aim of this study was to characterize expression of CD6 early after HCT and to examine the effects of Itolizumab on T cell responses in the setting of aGVHD. We analyzed immune reconstitution in 95 adult patients who underwent allogeneic HCT for hematological malignancies. Patients received myeloablative (50.5%) or reduced intensity (49.5%) conditioning. Almost all patients received peripheral blood stem cell grafts from HLA-matched related (MRD; 27.4%), matched unrelated (MUD; 66.3%) or mismatched unrelated donors (MMUD; 6.3%). Tacrolimus and methotrexate were used for GVHD prophylaxis. Forty-two of 95 patients developed grade I-IV aGVHD at a median of 50 days (range 20-294) after HCT and systemic immunosuppression was started in 81% of these cases. aGVHD grade severity was 38.1%, 40.5%, 7.1% and 14.3% of grade I, II, III and IV, respectively. Patient samples were collected at 1, 2, 3 and 6 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of Itolizumab was tested in vitro using peripheral blood mononuclear cells (PBMCs) obtained from HD and patients before (preGVHD) and after aGvHD onset (postGVHD) after 72 hour stimulation with antiCD3/CD2/CD28 coated beads. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation were monitored by expression of CD25 and CD45RO, respectively. To test if Itolizumab activity was dependent on the presence of the CD6 cognate ligand, ALCAM, HD CD3+ T cells were stimulated with anti-CD3 antibody with or without ALCAM-Fc for 96 hours. CD6+ T cells reconstituted early after transplant, accounting for 77.8% (range, 69.9-93.7) of CD4Treg, 98.1% (range, 96.5-99.5) of CD4Tcon, 84% (range, 77.1-92.1) of CD8 T cells at 1 month. This pattern remained unchanged at 2, 3 and 6 months and in HD. CD6 expression (MFI) was the highest in Tcon and lowest in Treg in patients as well as in HD. Compared to HD, CD6 MFI in CD4 Treg was significantly lower in patients at 1,2,3 and 6 months after HCT (p Prior to GVHD onset, Itolizumab inhibited CD4 and CD8 T cell proliferation in a similar fashion to HD control. This effect was less prominent in T cells collected after GVHD onset, when patients were already receiving immunosuppressive medications, especially for the CD8 T cells (Fig 1D). Similar results were observed for CD25 and CD45RO expression (Fig 1D). Addition of ALCAM-Fc to anti-CD3 antibody enhanced T cell proliferation, activation and maturation. Itolizumab efficiently inhibited T cell proliferation, activation and maturation in the presence of ALCAM-Fc and anti-CD3 antibody, while no effect was observed in the presence of anti-CD3 antibody alone (Fig 2A). Finally, Itolizumab alone did not induce significant CDC, ADC or ADCC in vitro (Fig 2B). In conclusion, we demonstrate that CD6 positive T cells reconstitute rapidly after HCT with higher levels of expression in Tcon compared to Treg and CD8 T cells. Percentages of CD6+ T cells do not change during aGVHD, but levels of CD6 expression decrease in Tcon and Treg after aGVHD. Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMCs from patients with aGvHD. Functional inhibition of the CD6-ALCAM pathway without T cell depletion may be a novel therapeutic strategy for treating aGvHD. A phase I/II study using Itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:EMD Serono: Consultancy; Biolojic Design Inc: Consultancy; Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; BMS: Other: Research Support; Miltenyi: Other: Research Support; Clinigen: Other; Therakos: Membership on an entity's Board of Directors or advisory committees; Equillium: Consultancy; Moderna Therapeutics: Consultancy; Amgen: Consultancy. Cutler:Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Cugene: Consultancy; alexion: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Consultancy; Mana Therapeutics: Consultancy; Rheos Therapeutics: Consultancy; Novartis: Consultancy; VOR Biopharma: Consultancy. Ampudia:Equillium: Current Employment, Current equity holder in publicly-traded company. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ritz:Rheos Medicines: Consultancy; Amgen: Research Funding; Avrobio: Consultancy; Talaris Therapeutics: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Equillium: Research Funding; Falcon Therapeutics: Consultancy; Kite Pharma: Research Funding; TScan Therapeutics: Consultancy.

Haploidentical hematopoietic cell transplantation (haplo-HCT) with post-transplant cyclophosphamide (PTCy) as graft versus host disease (GVHD) prophylaxis is frequently used for patients who do not have HLA-identical donors. However, the effect of PTCy on immune reconstitution (IR) has not been studied extensively in large prospective cohorts, despite a high incidence of early viral infections. We quantified IR in 60 patients after haplo-HCT with PTCy, mycophenolate mofetil and tacrolimus (TAC) and compared results to 34 patients with 8/8 HLA matched related or unrelated donors (MD) receiving TAC and methotrexate for GVHD prophylaxis. Both groups received reduced intensity conditioning for hematologic malignancies. Samples were prospectively collected at 1, 2, 3, 6, 9, 12, 18, 24 and 30 months after HCT and analyzed using multi-color flow cytometry to characterize distinct lymphocyte populations. Peripheral blood NK cells 1 month after PTCy were characterized using CyTOF and a panel of 35 metal-labeled monoclonal antibodies and compared to age-matched healthy donor NK cells. Recovery of all T cell subsets (CD4Tcon, CD4Treg, CD8) was significantly reduced in the PTCy cohort compared to MD early after HCT (Figure 1A, B, C). Despite delayed CD4Treg recovery, the Treg:Tcon ratio was significantly higher early after HCT in the PTCY cohort due to relatively deeper depression in CD4Tcon (0.18 vs 0.06, p Absolute NK cells were lower at 1 month after PTCy (Figure 1E), entirely due to reduced numbers of CD56dimCD16+ NK cells (Figure 1G). Subsequently, CD56dim NK cells remained significantly lower in the PTCy group until 3 months after HCT. In contrast, recovery of CD56brightCD16− NK cells was significantly increased in the PTCy cohort at 2, 3 and 6 months after HCT (p In summary, the effect of PTCy on IR was most pronounced at 1 month with significantly delayed recovery of CD3, CD4Tcon, CD4Treg, CD8 and CD56dim NK cells. After PTCy, NK cells display an immature phenotype and functional impairment that is at least partly corrected by priming with low dose IL-15. Impairment of NK and T cell recovery early after PTCy-based HCT underscores the need to adopt novel strategies to overcome immune defects induced by this platform.

Low-dose interleukin-2 (IL-2) therapy for chronic graft-versus-host disease (cGVHD) generates a rapid rise in plasma IL-2 levels and CD4+CD25+CD127−Foxp3+ regulatory T-cell (CD4Treg) proliferation, but both decrease over time despite continued daily administration. To test whether IL-2 dose escalation at the time of anticipated falls in plasma levels could circumvent tachyphylaxis and enhance CD4Treg expansion, we conducted a phase 1 trial in 10 adult and 11 pediatric patients with steroid-refractory cGVHD (www.clinicaltrials.gov: NCT02318082). Daily IL-2 was initiated in children and adults (0.33 × 106 and 0.67 × 106 IU/m2 per day, respectively). Dose escalations were scheduled at weeks 2 and 4 to a maximum dose of 1 × 106 IU/m2 per day in children and 2 × 106 IU/m2 per day in adults. Patients continued at their maximum tolerated dose (MTD) until week 8. Children tolerated IL-2 dose escalation with partial responses (PRs) in 9 of 11 patients (82%) at multiple cGVHD sites, including lung. Patient-reported outcome scores for skin and lung improved significantly in pediatric patients. In contrast, 5 of 10 adults required dose reduction, and only 2 of 7 evaluable patients (29%) had PRs at week 8. CD4Tregs and natural killer cells expanded in both cohorts without significant changes in conventional CD4+ T cells (Tcons) or CD8+ T cells. Children achieved a higher median CD4Treg/Tcon ratio at week 8 (0.4 vs 0.18, P = .02) despite lower IL-2 doses. We show for the first time that low-dose IL-2 is safe and effective in children with advanced cGVHD. In adults, escalation above the previously defined MTD did not improve CD4Treg expansion or clinical response.

Chronic graft-versus-host disease (cGVHD) affects >50% of hematopoietic stem cell transplant patients. Extracorporeal photopheresis (ECP), an immunomodulatory therapy, provides clinical benefit in steroid-refractory (SR) cGVHD, possibly via regulatory T (Treg) and natural killer (NK) cell expansion. We demonstrated that low-dose interleukin-2 (IL2) led to clinical improvement in SR-cGVHD and stimulated preferential Treg and NK-cell expansion with minimal effect on conventional T (Tcon) cells. We evaluated the effect of ECP (weeks 1-16) plus IL2 (1 × 106 IU/m2, weeks 9-16) in 25 adult patients with SR-cGVHD in a prospective phase 2 trial. Objective responses occurred in 29% and 62% of evaluable patients at weeks 8 (ECP alone) and 16 (ECP plus IL2), respectively. Eight weeks of ECP alone was associated with a marked decline in CD4+ Tcon (P = .03) and CD8+ T cells (P = .0002), with minimal change in Treg cells, Treg:Tcon cell ratio, or NK cells. Adding IL2 induced an increase in Treg cells (P < .05 at weeks 9-16 vs week 8), Treg:Tcon cell ratio (P < .0001 at weeks 9-16 vs week 8), and NK cells (P < .05 at weeks 9-16 vs week 8). Patients responding to ECP alone had significantly fewer CD4+ Tcon and CD8+ T cells at baseline compared with patients who responded after IL2 addition and patients who did not respond; neither Treg nor NK cells were associated with response to ECP alone. Altogether, ECP plus IL2 is safe and effective in patients with SR-cGVHD. ECP and IL2 have distinct immunologic effects, suggesting different therapeutic mechanisms of action. This trial was registered at www.clinicaltrials.gov as #NCT02340676.

Chronic graft-versus-host disease (cGVHD) is associated with inadequate reconstitution of tolerogenic CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs). Previous phase 1 studies identified a low daily dose of interleukin-2 (IL-2) that was well tolerated, did not exacerbate alloimmunity, augmented Treg in vivo, and was associated with improvement of active cGVHD. In the current phase 2 study, 35 adults with steroid-refractory cGVHD received daily IL-2 (1 × 10(6) IU/m(2)) for 12 weeks. Median time from transplantation and cGVHD onset was 616 days (range, 270-2145 days) and 317 days (range, 28-1880 days), respectively. Two patients withdrew and 5 required IL-2 dose reductions due to side effects. Twenty of 33 evaluable patients (61%) had clinical responses at multiple cGVHD sites (liver, skin, gastrointestinal tract, lung, joint/muscle/fascia). Three patients (9%) had progressive cGVHD. Compared with pretreatment levels, Treg and natural killer cell counts rosefivefold (P.001) andfourfold (P.001), respectively, without significant change in conventional CD4 T cells (Tcons) or CD8 T cells. The Treg:Tcon ratio rosefivefold (P.001). Clinical responders initiated IL-2 earlier (508 vs 917 days after transplantation, P = .005; 249 vs 461 days after cGVHD onset; P = .03). Treg:Tcon ratios ≥0.07 at baseline and ≥0.2 at week 1 also predicted clinical response (P = .003; P = .0003, respectively). After a 4-week treatment hiatus, clinical responders were eligible to continue IL-2 therapy indefinitely. During 2 years of extended IL-2 therapy, clinical and Treg immune responses persisted, while Tcon count and Treg:Tcon ratio gradually normalized. Low-dose IL-2 provides durable clinical improvement in active cGVHD and extended therapy is well-tolerated.

Donor lymphocyte infusions are used to treat relapse after allogeneic hematopoietic stem cell transplantation, but responses are inadequate. In addition to effector cells, infusions contain CD25+ regulatory T cells (Treg) that may suppress graft-versus-tumor responses. We undertook a phase I study of donor lymphocyte infusions depleted of CD25+ T cells in patients with hematologic malignancies who had relapsed after transplantation. Twenty-one subjects received CD25/Treg-depleted infusions following removal of CD25+ cells using antibody-conjugated magnetic beads. Sixteen subjects received prior cytoreductive therapy. Four were in complete remission at the time of infusion. Two dose levels were administered: 1×107 (n=6) and 3×107 CD3+ cells/kg (n=15). A median 2.3 log-depletion of CD4+CD25+FOXP3+ Treg was achieved. Seven subjects (33%) developed clinically significant graft-versus-host disease by 1 year, including one patient who died. At dose level 1, five subjects had progressive disease and one had stable disease. At dose level 2, nine subjects (60%) achieved or maintained responses (8 complete responses, 1 partial response), including seven with active disease at the time of infusion. A shorter period between relapse and infusion was associated with response at dose level 2 (P=0.016). The 1-year survival rate was 53% among patients treated with dose level 2. Four of eight subjects with acute myeloid leukemia remained in remission at 1 year. When compared to unmodified donor lymphocyte infusions in 14 contemporaneous patients meeting study eligibility, CD25/Treg depletion was associated with a better response rate and improved event-free survival. Circulating naive and central memory CD4+ T cells increased after CD25/Treg-depleted infusion, but no immunophenotypic signature for response was noted. CD25/Treg-depleted donor infusion appears feasible and capable of inducing graft-versus-tumor responses without excessive graft-versus-host disease. (ClinicalTrials.gov NCT#00675831).

Daily low-dose interleukin-2 (IL-2) at a fixed dose of 1 × 106 IU/m2/day leads to preferential expansion of CD4+CD25+CD127-Foxp3+ regulatory T cells (Treg) and clinical responses in 50-60% of adult patients with steroid-refractory chronic GVHD. During fixed-dose IL-2 therapy, plasma IL-2 levels rise rapidly, but decrease over time despite continued daily administration. Lower IL-2 levels correlate with decreased Treg proliferation, possibly due to IL-2 sequestration via binding to high-affinity IL-2 receptors on Treg. We reasoned that IL-2 dose escalation at the time of anticipated fall in plasma IL-2 levels would avoid tachyphylaxis and enhance Treg expansion. We conducted a phase 1 trial of individual patient IL-2 dose escalation over 8 weeks in adult and pediatric patients with steroid-refractory cGVHD. Daily SC IL-2 was initiated at 0.67 × 106 and 0.33 × 106 IU/m2/day in the adult and pediatric cohorts, respectively. Each participant had dose escalations at weeks 2 and 4 to a maximum dose of 2 × 106 IU/m2/day in adults and 1 × 106 IU/m2/day in children. We studied 21 participants (10 adult, 11 children) with median ages of 57 (range, 33-71) and 12 years (range, 3-22) in the adult and pediatric cohorts, respectively. The cohorts did not differ in terms of time since cGVHD onset, number of organ sites involved or failed therapies prior to enrollment. Individually dose-escalated IL-2 was well tolerated in children with 1 patient requiring dose reduction for electrolyte imbalances. In the adult cohort, 1 patient withdrew for cGVHD progression, 4 required dose reduction for injection site reactions and 2 were unevaluable. At week 8, objective cGVHD responses (PR) were seen in 8 of 11 pediatric patients (73%), but in only 1 of 8 evaluable adult patients (13%). Response sites included skin (n=4), joint/fascia/muscle (n=1), lung (n=5), and GI tract (n=2). 11 pediatric and 4 adult participants with clinical benefit (PR or SD with minor response) continued extended duration IL-2 therapy. 9 pediatric patients on IL-2 therapy were able to wean steroid therapy with a median dose reduction of 67% at 6 months (Fig. 1). Children had a superior immunologic response despite lower IL-2 doses. The median Treg:Tcon ratio at week 8 was significantly higher in the pediatric cohort (0.42 vs 0.21, p = 0.02). Both cohorts had increased NK cell numbers, with no significant changes in CD4Tcon, CD8 T cell or B cells (Fig. 2). Pediatric patients had greater thymic output of naive Treg as well as a larger fraction of PD-1-expressing effector memory Treg at baseline and during IL-2 therapy (Fig. 3), which has been correlated with improved Treg expansion and survival. We show for the first time that low-dose IL-2 is safe in children with advanced steroid-refractory cGVHD and results in a high clinical response rate. In adults, dose escalation above fixed-dose MTD did not improve Treg expansion or clinical response relative to fixed daily dosing.

Hematopoietic stem cell transplantation (HSCT) recipients lacking HLA-matched related donors have increased graft-versus-host disease (GVHD) and nonrelapse mortality (NRM). Bortezomib added to reduced-intensity conditioning can offer benefit in T cell–replete HLA-mismatched HSCT and may also benefit myeloablative conditioning (MAC) transplants. We conducted a phase II trial of short-course bortezomib plus standard tacrolimus/methotrexate after busulfan/fludarabine MAC in 34 patients with predominantly myeloid malignancies. Fourteen (41%) received 8/8 HLA-matched unrelated donor (MUD) and 20 (59%) received 7/8 HLA-mismatched related/unrelated donor peripheral blood stem cell grafts. Median age was 49 years (range, 21 to 60), and median follow-up was 25 months (range, 11 to 36). The regimen was well tolerated. No dose modifications were required. Neutrophil and platelet engraftment occurred at a median of 14 (range, 10 to 33) and 17 (range, 10 to 54) days, respectively. Median 30-day donor chimerism was 99% (range, 90 to 100), and 100-day grades II to IV and III to IV acute GVHD incidence was 32% and 12% respectively. One-year chronic GVHD incidence was 50%. Two-year cumulative incidence of both NRM and relapse was 16%. Two-year progression-free and overall survival rates were 70% and 71%, respectively. Outcomes were comparable to an 8/8 MUD MAC cohort (n = 45). Immune reconstitution was robust. Bortezomib-based MAC HSCT is well tolerated, with HLA-mismatched outcomes comparable with 8/8 MUD MAC HSCT, and is suitable for randomized evaluation. (clinicaltrials.gov: NCT01323920.)

We report a clinical trial testing vaccination of autologous myeloblasts admixed with granulocyte-macrophage colony-stimulating factor secreting K562 cells after allogeneic hematopoietic stem cell transplantation (HSCT). Patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with ≥5% marrow blasts underwent myeloblast collection before HSCT. At approximately day +30, 6 vaccines composed of irradiated autologous myeloblasts mixed with GM-K562 were administered. Tacrolimus-based graft-versus-host disease (GVHD) prophylaxis was not tapered until vaccine completion (∼day 100). Thirty-three patients with AML (25) and MDS (8) enrolled, 16 (48%) had ≥5% marrow blasts at transplantation. The most common vaccine toxicity was injection site reactions. One patient developed severe eosinophilia and died of eosinophilic myocarditis. With a median follow-up of 67 months, cumulative incidence of grade 2-4 acute and chronic GVHD were 24% and 33%, respectively. Relapse and nonrelapse mortality were 48% and 9%, respectively. Progression-free survival (PFS) and overall survival (OS) at 5 years were 39% and 39%. Vaccinated patients who were transplanted with active disease (≥5% marrow blasts) had similar OS and PFS at 5 years compared with vaccinated patients transplanted with

Aprior phase I/II trial of bortezomib/tacrolimus/methotrexate prophylaxis after human leukocyte antigen (HLA)-mismatched reduced intensity conditioning allogeneic hematopoietic stem cell transplantation documented low acute graft-versus-host disease incidence, with promising overall and progression-free survival. We performed an open-label three-arm 1:1:1 phase II randomized controlled trial comparing grade II-IV acute graft-versus-host disease between conventional tacrolimus/methotrexate (A) versus bortezomib/tacrolimus/methotrexate (B), and versus bortezomib/sirolimus/tacrolimus (C), in reduced intensity conditioning allogeneic transplantation recipients lacking HLA-matched related donors. The primary endpoint was grade II-IV acute graft-versus-host disease incidence rate by day +180. One hundred and thirty-eight patients (A 46, B 45, C 47) with a median age of 64 years (range: 24-75), varying malignant diagnoses and disease risk (low 14, intermediate 96, high/very high 28) received 7-8/8 HLA-mismatched (40) or matched unrelated donor (98) grafts. Median follow up in survivors was 30 months (range: 14-46). Despite early immune reconstitution differences, day +180 grade II-IV acute graft-versus-host disease rates were similar (A 32.6%, B 31.1%, C 21%; P=0.53 for A vs B, P=0.16 for A vs C). The 2-year non-relapse mortality incidence was similar (A 14%, B 16%, C 6.4%; P=0.62), as were relapse (A 32%, B 32%, C 38%; P=0.74), chronic graft-versus-host disease (A 59%, B 60% C 55%; P=0.66), progression-free survival (A 54%, B 52%, C 55%; P=0.95), and overall survival (A 61%, B 62%, C 62%; P=0.98). Overall, the bortezomib-based regimens evaluated did not improve outcomes compared with tacrolimus/methotrexate therapy. clinicaltrials.gov Identifier: 01754389.

International audience; Anti-programmed cell death protein 1 (PD-1) monoclonal antibodies are being increasingly tested in patients with advanced lymphoma. Following treatment, many of those patients are likely to be candidates for allogeneic hematopoietic stem cell transplant (HSCT). However, the safety and efficacy of HSCT may be affected by prior PD-1 blockade. We conducted an international retrospective analysis of 39 patients with lymphoma who received prior treatment with a PD-1 inhibitor, at a median time of 62 days (7-260) before HSCT. After a median follow-up of 12 months, the 1-year cumulative incidences of grade 2-4 and grade 3-4 acute graft-versus-host disease (GVHD) were 44% and 23%, respectively, whereas the 1-year incidence of chronic GVHD was 41%. There were 4 treatment-related deaths (1 from hepatic sinusoidal obstruction syndrome, 3 from early acute GVHD). In addition, 7 patients developed a noninfectious febrile syndrome shortly after transplant requiring prolonged courses of steroids. One-year overall and progression-free survival rates were 89% (95% confidence interval [CI], 74-96) and 76% (95% CI, 56-87), respectively. One-year cumulative incidences of relapse and nonrelapse mortality were 14% (95% CI, 4-29) and 11% (95% CI, 3-23), respectively. Circulating lymphocyte subsets were analyzed in 17 patients. Compared with controls, patients previously treated with PD-1 blockade had significantly decreased PD-1(+) T cells and decreased ratios of T-regulatory cells to conventional CD4 and CD8 T cells. In conclusion, HSCT after PD-1 blockade appears feasible with a low rate of relapse. However, there may be an increased risk of early immune toxicity, which could reflect long-lasting immune alterations triggered by prior PD-1 blockade.

Therapy for steroid-refractory acute graft-versus-host disease (SR-aGVHD) remains suboptimal. Preclinical data demonstrate increased CD30 expression on activated CD8+ T cells during aGVHD. Brentuximab vedotin (BV) is an antibody-drug conjugate targeting CD30. We conducted a multicenter phase 1 trial in 34 patients to establish the maximum tolerated dose (MTD) of BV for SR-aGVHD treatment. A 3+3 cohort design was conducted initially with BV given weekly × 3 doses followed by maintenance dosing (initial dose 0.6 mg/kg IV weekly). Six patients were treated with the initial weekly dosing scheme; 2 of these patients died of neutropenic sepsis complications. The trial was subsequently revised to escalating cohorts of 5 patients treated every 2 weeks × 4 doses with a 4-week dose-limiting toxicity (DLT) period. Twenty-eight patients were treated with every-2-week dosing (n = 10 at 0.6 mg/kg; n = 18 at 0.8 mg/kg). MTD was defined at 0.8 mg/kg with 1 DLT observed (sepsis). At day 28, the overall response rate was 38.2% with 5 complete responses (CRs; 14.7%) and 8 very-good-partial responses (23.5%). An additional 7 patients achieved CR by day 56. With 12 months' follow-up on all patients, overall survival was 41% (95% confidence interval [CI], 25%-57%) at 6 months and 38% (95% CI, 22%-54%) at 12 months. CD30 expression on central memory CD8+, central memory CD4+, and regulatory T-lymphocyte subsets at enrollment was not associated with clinical response. BV is tolerable and has activity in SR-aGVHD and merits further investigation. This trial was registered at www.clinicaltrials.gov as #NCT01940796.

Fibroepithelial lesions with cellular stroma are frequently termed cellular fibroadenomas although criteria for distinguishing them from a phyllodes tumor are vague and subjective. However, the clinical implications and surgical management for these 2 lesions may be different. We randomly selected 21 cases of fibroepithelial lesions sent in consultation to the senior author that were challenging to classify as cellular fibroadenoma or phyllodes tumor. One to 2 representative slides of each case along with patient age were sent to 10 pathologists who specialize in breast pathology. The World Health Organization criteria for phyllodes tumors and a diagnosis form were included with the study set. For the purposes of data reporting, fibroadenoma and cellular fibroadenoma are considered together. In only 2 cases was there uniform agreement as to whether the tumor represented a fibroadenoma or phyllodes tumor. Of the remaining 19 cases, if the diagnoses of fibroadenoma and benign phyllodes tumor were combined and separated from borderline and malignant phyllodes tumors, there was 100% agreement in 53% of cases and 90% agreement in 79% of cases. This study highlights the difficulty that exists in distinguishing some cellular fibroadenomas from phyllodes tumors even for pathologists who specialize in breast pathology. However, there appears to be considerable agreement when cellular fibroadenomas and benign phyllodes tumors are distinguished from borderline and malignant phyllodes tumors. Further studies are needed to determine if there is a clinically significant difference between cellular fibroadenomas and benign phyllodes tumors and how to better distinguish them from borderline and malignant phyllodes tumors.

Excessive levels of B cell activating factor (BAFF) are found in patients with active chronic graft-versus-host disease (cGVHD). In mice, BAFF has been shown to be essential for B cell recovery after myeloablation. To assess how BAFF levels relate to transplantation factors and subsequent development of cGVHD, we prospectively monitored 412 patients in the first year after allogeneic peripheral blood or bone marrow hematopoietic stem cell transplantation (HSCT) and censored data at time of cGVHD onset. In patients who did not develop cGVHD, we affirmed a temporal pattern of gradually decreasing BAFF levels as B cell numbers increase after myeloablative conditioning. In contrast, after reduced-intensity conditioning, BAFF levels remained high throughout the first post-HSCT year, suggesting that the degree of myeloablation resulted in delayed B cell recovery associated with persistence of higher BAFF levels. Given that high BAFF/B cell ratios have been associated with active cGVHD, we examined differences in early BAFF/B cell ratios and found significantly different BAFF/B cell ratios at 3 months post-HSCT only after myeloablative conditioning in patients who subsequently developed cGVHD. In addition to HSCT conditioning type, the use of sirolimus was significantly associated with higher BAFF levels after HSCT, and this also was potentially related to lower B cell numbers. Taken together, our results are important for interpreting BAFF measurements in cGVHD biomarker studies.

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation and trafficking and is central to inflammation. Early studies by Soiffer et al. demonstrated that ex vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized anti-CD6 monoclonal antibody, has been shown to modulate T cell activation and proliferation. The aim of this study was to characterize: (1) expression of CD6 and ALCAM, and (2) activity of itolizumab on T cell responses in peripheral blood from HCT patients pre- and post-aGvHD. We analyzed immune reconstitution in 31 adult patients who underwent HLA matched donor HCT for hematological malignancies. Patients received peripheral blood stem cell grafts and GVHD prophylaxis with tacrolimus and methotrexate. Twelve of 31 patients developed aGVHD at a median of 58 days, range 27-208, after HCT and systemic treatment was started in 83% of these cases. aGVHD grade severity was 25%, 58.3% and 16.7% of grade I, II and IV, respectively. Patient samples were collected at 1, 2 and 3 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of itolizumab was tested using peripheral blood mononuclear cells (PBMC) obtained from HD and patients before (preGVHD) and after (postGVHD) aGvHD onset (within 30 days). PBMC were stimulated with antiCD3/CD2/CD28 coated beads in the presence of itolizumab or isotype control (cetuximab) for 72 hours. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation was measured by expression of CD25 and CD45RO, respectively. For statistical analysis, non-parametric unpaired (Mann-Whitney) or paired (Wilcoxon matched-pairs signed rank) test were used. CD6+ T cells reconstituted early after transplant, accounting for 95% of positive CD3 T cells, range 57-100 at 1 month. Similar to HD PBMC, in the first 3 months after HCT, CD4 Tcon had the highest CD6 expression, while CD4 Treg had a lower CD6 expression compared to both CD4 Tcon and CD8 T cells (Fig 1A and 1B). To characterize the expression of CD6 on different T cell subsets, we used a t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and visualized the data using a viSNE map (Fig 1C). Within the Tcon compartment, there were no differences in expression of CD6 between HD and patients at all 3 time points. Within CD4 Treg and CD8 T cells, CD6 expression was reduced in naïve CD8 T cells and CM Treg after transplant compared to HD. In HD, ALCAM expression was detected in 35% of CD14+ monocytes, 23% of CD19+ B cells, 20% of myeloid (CD11c+ CD123-) DCs and 97% of plasmacytoid (CD11c-CD123+) DCs. After HCT, expression of ALCAM in DC compartments was similar to HD. In functional studies, itolizumab inhibited CD4 and CD8 T cell proliferation in preGVHD samples, similar to HD controls. This effect was less prominent in samples collected from patients who had developed GVHD and were already receiving immunosuppressive medications, potentially confounding the ability to assess the effect of itolizumab in this assay (Fig 2A). Similar results were observed for CD25 (Fig 2B) and CD45RO (Fig 2C) expression pre- and post-aGVHD. Finally, itolizumab did not increase rates of cell death in samples from HCT patients as assessed by Annexin V expression, suggesting that itolizumab-mediated T cell inhibition was not due to increased T cell apoptosis. There was a slight increase in Annexin V expression in HD vs isotype control (21%, range 10-43 vs 15%, range 11-31, p= 0.0273). In conclusion, we demonstrate for the first time that CD6+ T cells reconstitute rapidly in peripheral blood after HCT and that CD6 expression is highest in Tcon while lowest in Treg (Tcon>CD8>Treg). Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMC from aGvHD patients, thus representing a potential therapeutic for treating aGvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Cugene: Consultancy; Equillium: Consultancy. Cutler:Pharmacyclics: Consultancy; Omeros: Consultancy; Kadmon: Consultancy; BiolineRx: Other: DSMB; Cellect: Other: DSMB; Kalytera: Other: DSMB; ElsaLys: Consultancy; Genentech: Consultancy; BMS: Consultancy; Jazz: Consultancy; Incyte: Consultancy; Fate Therapeutics: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Ho:Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Soiffer:Jazz: Consultancy; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Mana therapeutic: Consultancy; Kiadis: Other: supervisory board. Ampudia:Equillium: Employment. Ng:Equillium: Employment, Equity Ownership. Connelly:Equillium: Employment, Equity Ownership. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy.

ACOSOG Z11 and other studies showing little benefit to axillary dissection (ALND) for early-stage breast cancers with limited nodal disease have led to questioning the value of preoperative axillary imaging ± ultrasound-guided needle biopsy (USNB). Data are lacking on the value of this approach in identifying cases that fall outside Z11 guidelines.We studied 988 consecutive patients with invasive breast cancers who underwent operation including axillary surgery in 2010-2011.Preoperative axillary ultrasonography (AUS) was performed in 92% and breast/axillary magnetic resonance imaging (MRI) in 51%; 82 (33.5%) of 245 patients with suspicious lymph nodes (LN) were USNB-positive. Regarding nodal status, AUS, MRI, and USNB had negative and positive predictive values of 78%, 76%, 70% and 54%, 58%, 100%, respectively. AUS/MRI visualization of one versus multiple abnormal LNs visualized predicted2LN+ on final pathology (13.5%/15.1% % vs 30.8%/32.6%, P.009). Among USNB-LN+ T1/T2 patients, 51.6% had 1-2 LN+ while 60% with multiple and 31% with one AUS-abnormal LN(s) had2LN+, P = .001.In our contemporary series, preoperative AUS±USNB streamlined surgical care for 29% of node-positive patients. Two-thirds of T1/T2 USNB-LN+ patients with multiple AUS-suspicious LNs had2LN+, suggesting they should undergo ALND without SLNB. AUS±USNB helps identify node-positive breast cancer patients who fall outside Z11 guidelines.

Purpose: Controversy exists about CYP2D6 genotype and tamoxifen efficacy. Experimental Design: A matched case–control study was conducted using the Austrian Breast and Colorectal Cancer Study Group Trial 8 (ABCSG8) that randomized postmenopausal women with estrogen receptor (ER)-positive breast cancer to tamoxifen for 5 years (arm A) or tamoxifen for 2 years followed by anastrozole for 3 years (arm B). Cases had disease recurrence, contralateral breast cancer, second non–breast cancer, or died. For each case, controls were identified from the same treatment arm of similar age, surgery/radiation, and tumor–node—metastasis (TNM) stage. Genotyping was conducted for alleles associated with no (PM; *3, *4, *6), reduced (IM; *10, and *41), and extensive (EM: absence of these alleles) CYP2D6 metabolism. Results: The common CYP2D6*4 allele was in Hardy–Weinberg equilibrium. In arm A during the first 5 years of therapy, women with two poor alleles [PM/PM: OR, 2.45; 95% confidence interval (CI), 1.05–5.73, P = 0.04] and women with one poor allele (PM/IM or PM/EM: OR, 1.67; 95% CI, 0.95–2.93; P = 0.07) had a higher likelihood of an event than women with two extensive alleles (EM/EM). In years 3 to 5 when patients remained on tamoxifen (arm A) or switched to anastrozole (arm B), PM/PM tended toward a higher likelihood of a disease event relative to EM/EM (OR, 2.40; 95% CI, 0.86–6.66; P = 0.09) among women on arm A but not among women on arm B (OR, 0.28; 95% CI, 0.03–2.30). Conclusion: In ABCSG8, the negative effects of reduced CYP2D6 metabolism were observed only during the period of tamoxifen administration and not after switching to anastrozole. Clin Cancer Res; 19(2); 500–7. ©2012 AACR.

© 2016 by The American Society of Hematology, The development and maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT) requires the balanced reconstitution of donor-derived CD4 regulatory T cells (CD4Tregs) as well as effector CD4 (conventional CD4 T cells [CD4Tcons]) and CD8 T cells. To characterize the complex mechanisms that lead to unbalanced recovery of these distinct T-cell populations, we studied 107 adult patients who received T-replete stem cell grafts after reduced-intensity conditioning. Immune reconstitution of CD4Treg, CD4Tcon, and CD8 T cells was monitored for a 2-year period. CD3 T-cell counts gradually recovered to normal levels during this period but CD8 T cells recovered more rapidly than either CD4Tregs or CD4Tcons. Reconstituting CD4Tregs and CD4Tcons were predominantly central memory (CM) and effector memory (EM) cells and CD8 T cells were predominantly terminal EM cells. Thymic generation of naive CD4Tcon and CD8 T cells was maintained but thymic production of CD4Tregs was markedly decreased with little recovery during the 2-year study. T-cell proliferation was skewed in favor of CM and EM CD4Tcon and CD8 T cells, especially 6 to 12 months after HSCT. Intracellular expression of BCL2 was increased in CD4Tcon and CD8 T cells in the first 3 to 6 months after HSCT. Early recovery of naive and CM fractions within each T-cell population 3 months after transplant was also strongly correlated with the subsequent development of chronic graft-versus-host disease (GVHD). These dynamic imbalances favor the production, expansion, and persistence of effector T cells over CD4Tregs and were associated with the development of chronic GVHD., This work was supported by a Collaborative Research Grant from the Harvard Medical School-Portugal Program in Translational Research HMSP-ICT/0001/201, and National Institutes of Health, National Cancer Institute grants CA183559, CA183560, and CA142106.

Lobular neoplasia (LN) includes atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS). LN often is an incidental finding on breast core needle biopsy (CNBx) and management remains controversial. Our objective was to define the incidence of malignancy in women diagnosed with pure LN on CNBx, and identify a subset of patients that may be observed. Patients diagnosed with LN on CNB between January 1993 and December 2010 were identified. Patients with an associated high-risk lesion or ipsilateral malignancy at time of diagnosis were excluded. All cases were reviewed by dedicated breast pathologists and breast imagers for pathologic classification and radiologic concordance, respectively. The study cohort was comprised of 184 (1.3 %) cases of pure LN (147 ALH, 37 LCIS) from 180 patients. Pathologic–radiologic concordance was achieved in 171 (93 %) cases. Excision was performed in 101 (55 %) cases and 83 (45 %) were observed. Mean follow-up was 50.3 (range, 6–212) months. Of cases excised, 1 of 81 (1.2 %) ALH and 1 of 20 (5 %) LCIS cases were upstaged to ductal carcinoma in situ (DCIS) and invasive lobular carcinoma (ILC), respectively. Only 1 of 101 (1 %) concordant lesions was upstaged on excision. Of the cases observed, 4 of 65 (6.2 %) developed ipsilateral cancer during follow-up: 1 of 51 (2 %) case of ALH and 3 of 14 (21.4 %) cases with LCIS (2 ILC, 2 DCIS). During follow-up, 2.9 % (4/138) patients with excised or observed LN developed a contralateral cancer. These data support that not all patients with LN diagnosed on CNB require surgical excision. Patients with pure ALH, demonstrating radiologic–pathologic concordance, may be safely observed.

The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERβ and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERβ and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERβ antibodies are insensitive for ERβ and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERβ antibody is shown to be highly specific and sensitive for detection of full-length ERβ and its variant forms. Strong and variable staining patterns for endogenous levels of ERβ protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERβ was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERβ antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERβ in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERβ, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.

Purpose. The Oncotype DX assay predicts likelihood of distant recurrence and improves patient selection for adjuvant chemotherapy in estrogen receptor–positive (ER-positive) early stage breast cancer. This study has two primary endpoints: to evaluate the impact of Oncotype DX recurrence scores (RS) on chemotherapy recommendations and to compare the estimated recurrence risk predicted by breast oncology specialists to RS. Methods. One hundred fifty-four patients with ER-positive early stage breast cancer and available RS results were selected. Clinicopathologic data were provided to four surgeons, four medical oncologists, and four pathologists. Participants were asked to estimate recurrence risk category and offer their chemotherapy recommendations initially without and later with knowledge of RS results. The three most important clinicopathologic features guiding their recommendations were requested. Results. Ninety-five (61.7%), 45 (29.2%), and 14 (9.1%) tumors were low, intermediate, and high risk by RS, respectively. RS significantly correlated with tumor grade, mitotic activity, lymphovascular invasion, hormone receptor, and HER2/neu status. Estimated recurrence risk by participants agreed with RS in 54.2% ± 2.3% of cases. Without and with knowledge of RS, 82.3% ± 1.3% and 69.0% ± 6.9% of patients may be overtreated, respectively (p = 0.0322). Inclusion of RS data resulted in a 24.9% change in treatment recommendations. There was no significant difference in recommendations between groups of participants. Conclusions. Breast oncology specialists tended to overestimate the risk of tumor recurrence compared with RS. RS provides useful information that improves patient selection for chemotherapy and changes treatment recommendations in approximately 25% of cases.