Minimal Residual Disease Status in Multiple Myeloma 1 Year After Autologous Hematopoietic Cell Transplantation and Lenalidomide Maintenance Are Associated With Long-Term Overall Survival.

PURPOSE: Prognostic Immunophenotyping in Myeloma Response (PRIMeR) is an ancillary study of minimal residual disease (MRD) assessment for multiple myeloma by next-generation multiparameter flow cytometry (MFC). Patients were enrolled on a three-arm randomized control trial (Blood and Marrow Transplants Clinical Trials Network 0702 Stem Cell Transplant for Myeloma in Combination of Novel Agents [STaMINA]; ClinicalTrials.gov identifier: NCT01109004). METHODS: Four hundred and thirty-five patients consented to the MRD panel, which included 10 monoclonal antibodies measured via six-color MFC. MRD was measured at baseline/preautologous hematopoietic cell transplant (BL/preAutoHCT), premaintenance (PM), and 1 year (Y1) after AutoHCT with a sensitivity of 10-5 to 10-6. The primary objective was to assess MRD-negative (MRDneg) at 1 year after AutoHCT and progression-free survival and overall survival (PFS/OS). RESULTS: Similar to the STaMINA results, at a median follow-up of 70 months, there was no significant difference in PFS/OS by treatment arm in the PRIMeR patients. MRDneg at all three time points was associated with significantly improved PFS, and MRDneg at Y1 had significantly longer OS. Multivariate analysis of PFS, adjusting for disease risk and treatment arm, demonstrated hazard ratios (HRs) in MRD-positive patients compared with MRDneg patients at BL, PM, and Y1 of 1.55 (P = .0074), 1.83 (P = .0007), and 3.61 (P < .0001), respectively. Corresponding HRs for OS were 1.19 (P = .48), 0.88 (P = .68), and 3.36 (P < .001). Patients with sustained MRDneg or who converted to MRDneg by Y1 had similar PFS/OS. CONCLUSION: To our knowledge, this first, prospective US cooperative group, multicenter study demonstrates that MRDneg at Y1 after AutoHCT with lenalidomide maintenance is prognostic for improved 6-year PFS and OS. Serial MRD measurements may direct trials to test how further therapy may improve long-term PFS and OS.

Outcomes of Human Leukocyte Antigen-Matched Related Donor and Haploidentical Allogeneic Hematopoietic Cell Transplantation Recipients by Immune Profiles of Recipients and Donors.

Haploidentical (Haplo) allogeneic HCTs (alloHCT) have been used more frequently over the last decade as survival is similar to HLA-matched related donor (MRD) alloHCTs. We aimed to identify donor and recipient immune signatures before alloHCT that are associated with clinically meaningful outcomes in MRD vs Haplo alloHCT recipients. This retrospective cohort study of 165 MRD (n = 132) and Haplo (n = 33) alloHCT recipients and their related donors between 2007-2019 with paired peripheral blood samples immunophenotyped for T-cell, B-cell, NK cell and dendritic cell (DC) subsets. Immune cells were quantified before alloHCT in donors and recipients; calculations of immune cell ratios were classified as high, intermediate, and low and analyzed with alloHCT outcomes. Haplo donors were younger than MRD donors (median: 35 vs 51 years), whereas Haplo recipients were older than MRD recipients (median: 68 vs 54 years), were more likely to have a Karnofsky Performance Score ≤ 70 (76% vs 57%), 3+ comorbidities (54% vs 47%), and were in complete remission prior to alloHCT (58% vs 42%). In MRD alloHCT, a lower ratio of CD4+ to CD8+ effector memory cells in the donor was associated with lower 4-yr overall survival (OS; 25% vs 61%; P = .009), lower 4-yr progression free survival (PFS; 25% vs 58%; P = .014) and higher incidence of 1-yr transplant-related mortality (TRM; 39% vs 7%; P = .009) in recipients. A higher ratio of CD8+ effector memory to total NK cells measured in MRD recipients was associated with a higher incidence of grade II-IV aGvHD (63% vs 37%; P = .004) but was not statistically significant for III-IV aGvHD (23% vs 12%). In Haplo alloHCT, a lower ratio of total T-regulatory to CD4+ central memory cells in the donor was associated with lower 4-yr PFS (22% vs 60%; P = .0091). A higher ratio of CD4+ effector memory to CD8+ effector memory cells measured in Haplo recipients pre-alloHCT was associated with lower 4-yr OS (25% vs 88%; P = .0039). In both MRD and Haplo recipients, a higher ratio of CD4+ naïve to CD4+ central memory cells was associated with a higher incidence of grade II-IV aGvHD (64% vs 38%; P = .04). Evaluation of pre-alloHCT immune signatures of the donor and recipient may influence clinically meaningful patient outcomes in both MRD and Haplo transplants.

T Cell Exhaustion Markers in Multiple Myeloma Patients are Lower After Physical Activity Intervention.

PURPOSE: There is compelling evidence that CD4+ and CD8+T cells are dysfunctional in multiple myeloma, compromising their ability to control disease progression. Pre-clinical models suggest that exercise represents a non-pharmacologic means to reduce immune exhaustion, but no studies to date have examined the relationship between an exercise intervention and biomarkers of immune exhaustion in multiple myeloma patients. PATIENTS AND METHODS: The current study includes 24 multiple myeloma patients who participated in a six-month physical activity intervention, consisting of supervised strength training (n = 12) and unsupervised home-based walking arms (n = 12). Comprehensive flow cytometry was utilized to assess the frequency of CD4+ and CD8+T cells and subpopulations expressing the markers of exhaustion PD-1, TIGIT, TIM3 and/or LAG3. Ratios of exhausted to non-exhausted cell populations, and percentages of exhausted to total populations of the same lineage, were calculated for the baseline and final timepoints. RESULTS: Eighteen of 20 exhaustion measures were lower at the end of the intervention than at baseline, and several were significantly or borderline significantly reduced in the entire sample or in one of the arms. The entire sample saw improvements in the ratios of CD4+ TIGIT+ to non-exhausted CD4+ (0.7 [0.6] to 0.6 [0.4], P = .04) and CD8+ PD1+ to non-exhausted CD8+ (1.8 [2.6] to 1.5 [2.0], P = .06), and in total exhausted CD8+ as a percent of total CD8+ (72.9 [21.9] to 68.3 [19.6], P < .01). CONCLUSIONS: This pilot study suggests that physical activity induces changes in MM patients' immune systems, potentially rendering a less exhausted T cell state.

Probing cell proliferation: Considerations for dye selection.

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

Monitoring Cell Proliferation by Dye Dilution: Considerations for Panel Design.

High dimensional studies that include proliferation dyes face two inherent challenges in panel design. First, the more rounds of cell division to be monitored based on dye dilution, the greater the starting intensity of the labeled parent cells must be in order to distinguish highly divided daughter cells from background autofluorescence. Second, the greater their starting intensity, the more difficult it becomes to avoid spillover of proliferation dye signal into adjacent spectral channels, with resulting limitations on the use of other fluorochromes and ability to resolve dim signals of interest. In the third and fourth editions of this series, we described the similarities and differences between protein-reactive and membrane-intercalating dyes used for general cell tracking, provided detailed protocols for optimized labeling with each dye type, and summarized characteristics to be tested by the supplier and/or user when validating either dye type for use as a proliferation dye. In this fifth edition, we review: (a) Fundamental assumptions and critical controls for dye dilution proliferation assays; (b) Methods to evaluate the effect of labeling on cell growth rate and test the fidelity with which dye dilution reports cell division; and. (c) Factors that determine how many daughter generations can be accurately included in proliferation modeling. We also provide an expanded section on spectral characterization, using data collected for three protein-reactive dyes (CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far Red) and three membrane-intercalating dyes (PKH67, PKH26, and CellVue® Claret) on three different cytometers to illustrate typical decisions and trade-offs required during multicolor panel design. Lastly, we include methods and controls for assessing regulatory T cell potency, a functional assay that incorporates the "know your dye" and "know your cytometer" principles described herein.

Camelid-derived CD38 antibody successfully circumvents epitope blockade by the therapeutic anti-CD38 antibody daratumumab.

OBJECTIVES: To evaluate the efficacy of an anti-CD38 nanobody to detect plasma cells in a flow cytometry myeloma minimal residual disease (MRD) panel in patients treated with daratumumab and other immunotherapies. METHODS: Twenty-three bone marrow samples from as many patients were collected during or at the end of daratumumab treatment cycles. A 5-tube, 8-color flow cytometry MRD panel was performed. Dotplots were reviewed, and the median fluorescence intensity (MFI) was calculated. RESULTS: Patients' ages ranged from 45 to 77 years, and the cohort was made up of 13 men and 10 women who had undergone 2 to 24 cycles of daratumumab therapy at the time of myeloma MRD testing. In all 23 cases, therapeutic use of daratumumab impaired pathologists' ability to measure CD38 on plasma cells when using a conventional murine monoclonal antibody (anti-CD38 fluorescein isothiocyanate [FITC], clone T16; Beckman Coulter). In 21 of the 23 cases, the measurement of CD38 was restored when the anti-CD38 nanobody was employed. Compared with anti-CD38 FITC, the anti-CD38 Alexa Fluor 488 nanobody (Beckman Coulter) produced higher MFI and allowed measurement of a higher frequency of discernable plasma cells. CONCLUSIONS: The camelid-derived CD38 antibody successfully circumvents the steric inhibition of CD38 that the therapeutic use of daratumumab imparts and facilitates myeloma MRD plasma cell detection.

Short-Term Storage of Mobilized Peripheral Blood Stem Cells in a Closed System Changes the Microenvironment and May Affect the Quantity of CD34+ and CD34+CD38-CD45RA-CD90+ Cells.

Hypoxic conditions preserve the multipotency and self-renewing capacity of murine bone marrow and human cord blood stem cells. Blood samples stored in sealed blood gas tubes become hypoxic as leukocytes metabolize and consume oxygen. Taken together, these observations suggest that peripheral blood stem cell (PBSC) samples stored under airtight conditions become hypoxic, and that the stem cells contained may undergo qualitative or quantitative changes. This study aimed to determine the effect of storage for 8 hours in a sealed system on PBSC samples. Granulocyte colony-stimulating factor-mobilized PBSC samples were collected prospectively from 9 patients with myeloma or amyloidosis prior to apheresis, followed by measurement of CO2, O2, hydrogen ion (pH), lactate, and glucose concentrations in the blood and immunophenotyping of stem cell and multipotent progenitor cell populations before and after 8 hours of storage in sealed blood collection tubes. Blood concentrations of O2 and glucose and pH measurements were significantly decreased, whereas concentrations of CO2 and lactate were significantly increased after storage. Significantly higher concentrations of CD34+ cells (552 ± 84 cells/106 total nucleated cells [TNCs] versus 985 ± 143 cells/106 TNCs; P = .03), CD34+CD38- cells (98 ± 32 cells/106 TNCs versus 158 ± 52 cells/106 TNCs; P = .03), CD34+CD38+ cells (444 ± 92 cells/106 TNCs versus 789 ± 153 cells/106 TNCs; P = .03), and CD34+CD38-CD45RA-CD90+ cells (55 ± 17 cells/106 TNCs versus 89 ± 25 cells/106 TNCs; P = .02) were detected after 8 hours of storage. The changes in concentrations of CD34+CD38+ cells and CD34+ cells were inversely associated with the change in glucose concentration (P = .003 and P < .001, respectively) and positively associated with the change in lactate concentration (P = .01 and P <.001, respectively) after 8 hours of airtight storage. Storage of PBSC samples in a sealed, airtight environment is associated with microenvironmental changes consistent with hypoxia and increased concentrations of immunophenotypically defined stem cells. These results may have clinical implications with regard to the collection and processing of stem cell products and warrant confirmation with functional and mechanistic studies.

Spatiotemporal assessment of immunogenomic heterogeneity in multiple myeloma.

Spatial heterogeneity is a common phenomenon in metastatic solid tumors and an evolving concept in multiple myeloma (MM). The interplay between malignant plasma cells (PCs) and the microenvironment has not yet been analyzed in MM. For this purpose, we performed bone marrow aspirates and imaging-guided biopsies of corresponding lesions in newly diagnosed MM (NDMM) and relapsed/refractory MM (RRMM) patients. PCs were isolated and subjected to whole-exome sequencing (WES). Non-PCs were studied with next-generation flow (NGF) and T-cell receptor sequencing (TCRseq) to analyze the connection between malignant and nonmalignant cells in the bone marrow and in lesions. Although we observed a strong overlap from WES, NGF, and TCRseq in patients with intramedullary disease, WES revealed significant spatial heterogeneity in patients with extramedullary disease. NGF showed significant immunosuppression in RRMM compared with NDMM as indicated by fewer myeloid dendritic cells, unswitched memory B cells, Th9 cells, and CD8 effector memory T cells but more natural killer and regulatory T cells. Additionally, fewer T-cell receptor (TCR) sequences were detected in RRMM compared with NDMM and healthy individuals. After induction therapy, TCR repertoire richness increased to levels of healthy individuals, and NGF showed more regulatory T cells and myeloid-derived suppressor cells, regardless of depth of response. Clinical significance of imaging-guided biopsies of lesions was demonstrated by detection of monoclonal PCs in patients without measurable residual disease (MRD) in aspirates from the iliac crest as well as identification of secondary primary malignancies in MRD- patients. Furthermore, site-specific clones with different drug susceptibilities and genetically defined high-risk features were detected by our workflow.

Initial and follow-up evaluations on cerebrospinal fluid involvement by hematologic malignancy.

Central nervous system (CNS) involvement is a serious complication in hematologic malignancy, and early detection and management of CNS involvement in these cases significantly impact the prognosis. Currently, there is no consensus on the use of multiparametric flow cytometry (MFC) and conventional cytology (CC) testing for initial and follow-up cerebrospinal fluid (CSF) specimens to diagnose CNS involvement by hematologic malignancy. In our institution, after initial MFC and CC, two subsequent negative MFCs are required before discontinuing MFC. The aim of this study is to evaluate the outcome of this approach. CSF cytology and MFC reports were retrieved from Laboratory Information System, and data was reviewed. Between January 2020 and December 2021, 1789 CSF samples from 280 patients were submitted for CSF analysis. For those 517 CSF samples tested by both MFC and CC, 97 cases tested positive by both MFC and CC with 95% concordance. Eighteen cases were MFC + /CC - and 7 were MFC - /CC + . Thirty-six cases had initially positive MFCs followed by more than one MFC evaluation. Among those 36 cases, 22 cases (61.1%) converted to negative after the second follow-up sample, 9 cases (25%) were continuously positive for at least three samples, and 5 cases (13.9%) exhibited negative to positive conversion. Compared to negative CSF cases, positive CSFs had higher total nucleated cell count and higher total protein levels while red blood cells, glucose, and lactate dehydrogenase levels remained at comparable levels. The concordance between MFC and CC was excellent. The high incidence of positive MFCs on two or more follow-up samples and the high frequency of negative MFC to positive conversion indicate the necessity of repeated negative MFCs before discontinuing MFC. The fact that more than half of the positive cases converted to negative after the second CSF specimen and most follow-up positive cases can be detected by CC alone suggests it is adequate to use CC alone for follow-up CSF study after two consecutive negative MFCs.

Multiparameter flow cytometry and ClonoSEQ correlation to evaluate precursor B-lymphoblastic leukemia measurable residual disease.

Measurable residual disease (MRD) detection for precursor B-lymphoblastic leukemia (B-ALL) has become the standard of care. However, the testing methodology has not been standardized. We aim to correlate COG multiparameter flow cytometry (MFC) and ClonoSEQ techniques to assess the test characteristics, to study abnormal immunophenotype for B-ALL MRD, and to observe B-ALL clonal evolution and the impact of blinatumomab therapy on MFC testing. MFC and molecular reports were retrieved from electronic medical records and data was reviewed. Included in this study were 74 bone marrow samples collected from 31 B-ALL patients at our institution between January 2021 and March 2022. COG MFC and ClonoSEQ results were concordant in 59/74 samples (80%) with positive concordant results in 12 samples (16%) and negative concordant results in 47 samples (64%). Discordant results were seen in 15/74 samples (20%), with 14 samples (19%) showing ClonoSEQ + /MFC- results and only 1 sample (1%) showing MFC + /ClonoSEQ- result. ClonoSEQ + /MFC- cases had MRD values ranging from 1 to 1400 cells/million nucleated cells with 86% of cases showing MRD values of < 100 cells/million nucleated cells. Newly identified dominant sequences were detected using ClonoSEQ in 2/31 patients (6%) during follow-up. All 14 bone marrow samples from 8 patients, who had gone through blinatumomab immunotherapy, were MRD negative by MFC, but 3 cases were MRD positive by ClonoSEQ. Our results show strong correlation between COG MFC and ClonoSEQ (r = 0.96), and both methods are complementary. Clonal evolution may occur, and blinatumomab immunotherapy may impact MFC B-ALL MRD evaluation.

Unusual B-Lymphoid Blastic Crisis as Initial Presentation of Chronic Myeloid Leukemia Imposes Diagnostic Challenges.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder, characterized by reciprocal translocation t(9,22) (q34; q11), leading to increased myeloid proliferation. Most cases are diagnosed in the chronic phase (CP). However, a minority of cases can be present in the blastic phase (BP). In most patients with CML-BP, the blasts have a myeloid phenotype, however, in 20-30% of cases, the blasts have a lymphoid phenotype, mostly a B-cell phenotype. It is challenging to differentiate CML B-lymphoblastic phase (CML-BLP) from Ph + primary B-acute lymphoblastic leukemia (B-ALL) especially when the CML-BLP is the initial presentation of the disease, which is uncommon. We report here an unusual case of CML-BLP as an initial presentation of the disease without typical CML morphological findings. This case demonstrates diagnostic challenges and emphasizes the importance of an integrated approach using morphology, multiparametric flow cytometry, cytogenetic studies, and molecular studies to render an accurate diagnosis.

Ofatumumab plus HyperCVAD/HD-MA induction leads to high rates of minimal residual disease negativity in patients with newly diagnosed mantle cell lymphoma: Results of a phase 2 study.

BACKGROUND: Ofatumumab is a humanized type 1 anti-CD20 monoclonal antibody. Preclinical studies show improved complement-mediated cytotoxicity (CMC) compared to rituximab in mantle cell lymphoma (MCL). This study evaluates the safety and efficacy of combining ofatumumab with HyperCVAD/MA (O-HyperCVAD) in newly diagnosed MCL. METHODS: In this single-arm phase 2 study, 37 patients were treated with the combination of O-HyperCVAD for 4 or 6 cycles, followed by high dose chemotherapy and autologous stem cell transplant. Primary objectives were overall response rate (ORR) and complete response (CR) rate at the end of therapy. Secondary objectives included minimal residual disease (MRD) negativity, progression-free survival (PFS), and overall survival (OS). RESULTS: Median age was 60 years; ORR was 86% and 73% achieved a CR by modified Cheson criteria. The MRD negativity rate was 78% after 2 cycles of therapy, increasing to 96% at the end of induction; median PFS and OS were 45.5 months and 56 months, respectively. Achieving a post-induction CR by both imaging and flow cytometry was associated with improved PFS and OS. Early MRD negativity (post-2 cycles) was also associated with an improved PFS but not OS. There were 3 deaths while on therapy, and grades 3 and 4 adverse events (AEs) were observed in 22% and 68% of the patients. CONCLUSION: The addition of ofatumumab to HyperCVAD/HD-MA led to high rates of MRD negativity by flow cytometry in patients with newly diagnosed MCL. Achieving a CR post-induction by both imaging and flow cytometry is associated with improved overall survival.

Deciphering spatial genomic heterogeneity at a single cell resolution in multiple myeloma.

Osteolytic lesions (OL) characterize symptomatic multiple myeloma. The mechanisms of how malignant plasma cells (PC) cause OL in one region while others show no signs of bone destruction despite subtotal infiltration remain unknown. We report on a single-cell RNA sequencing (scRNA-seq) study of PC obtained prospectively from random bone marrow aspirates (BM) and paired imaging-guided biopsies of OL. We analyze 148,630 PC from 24 different locations in 10 patients and observe vast inter- and intra-patient heterogeneity based on scRNA-seq analyses. Beyond the limited evidence for spatial heterogeneity from whole-exome sequencing, we find an additional layer of complexity by integrated analysis of anchored scRNA-seq datasets from the BM and OL. PC from OL are characterized by differentially expressed genes compared to PC from BM, including upregulation of genes associated with myeloma bone disease like DKK1, HGF and TIMP-1 as well as recurrent downregulation of JUN/FOS, DUSP1 and HBB. Assessment of PC from longitudinally collected samples reveals transcriptional changes after induction therapy. Our study contributes to the understanding of destructive myeloma bone disease.

CD319 (SLAMF7) an alternative marker for detecting plasma cells in the presence of daratumumab or elotuzumab.

BACKGROUND: Daratumumab is an anti-CD38 immunotherapeutic drug that has increasingly been used to treat patients with heavily pre-treated and relapsed/refractory multiple myeloma. In so doing, the detection of CD38 antigen on plasma cells by flow cytometry is impeded. We hypothesized that alternative markers can be used in place or in addition to CD38 when detecting plasma cells post-treated with daratumumab. METHODS: A total of 16 alternative markers were tested using 22 bone marrow aspirates from patients with plasma cell neoplasm. The ability of selected markers to discern plasma cells from other hematopoietic cells were evaluated. The stability of tested markers when stored at 4 or 25°C after T = 0, 24, 48, and 72 h was also established. Finally, selected markers were incorporated into a panel used for monitoring multiple myeloma measurable residual disease to test their utility to identify plasma cells in the presence of daratumumab and/or elotuzumab (anti-CD319) drugs. RESULTS: Out of the 16 tested markers, CD319, CD54, CD229, CD317, and p63 were expressed by >90% of the plasma cells. Only CD319, CD54, and CD229 achieved 100% detection sensitivity. Further analysis showed that CD319 was better than CD229 and CD54 at resolving plasma cells from background hematopoietic cells, with CD54 being the worst (resolution metric, mean ± SD: CD319 [2.04 ± 0.86]; CD229 [1.47 ± 0.45]; and CD54 [1.22 ± 0.60]). CD229 was expressed by >90% of T lymphocytes, whereas CD319 was expressed preferentially by the CD8+ T cells and less frequently in CD4+ T cells. Additionally, CD229 was found on >60% of B and NK cells, as well as minor subsets of monocytes and granulocytes. CD319 was expressed on most NK cells and a minor subset of B cells, granulocytes, and monocytes. Even though CD229 and CD319 were expressed by different leukocyte subsets, their expression levels were highest on plasma cells. The expression of CD138 on plasma cells was significantly lower after storage at 4°C, while the expression levels of CD38, CD229, and CD319 remained stable at 4 or 25°C. Using limiting dilution experiments, the treatment of cells with daratumumab severely impeded the detection of CD38 antigen on plasma cells, whereas elotuzumab treatment did not block detection of CD319 on plasma cells. CONCLUSIONS: CD319 is a suitable alternative to CD38 for identifying plasma cells. Our results showed that a panel used for monitoring multiple myeloma measurable residual disease could be modified by using CD319 alone or in combination with CD38 to detect PCs in daratumumab or elotuzumab treated patients.

Circulating CD14+ HLA-DRlo/- monocytic cells as a biomarker for epithelial ovarian cancer progression.

PROBLEM: Previous studies identified circulating CD14+ HLA-DRlo/- monocytic cells as an immune suppressive subset in solid malignancies, such as prostate, renal cell carcinoma, and pancreatic cancer. Such monocytic cells have been implicated not only in tumour progression but also as a potential barrier for immunotherapy. This study examined the relationship between the frequency of circulating monocytic cells and epithelial ovarian cancer (EOC) progression pre- and post-frontline chemotherapy, defined by disease stage, which is a leading prognostic factor for this malignancy. METHOD OF STUDY: Incident cases of 236 women with EOC were recruited and comprehensive flow cytometry was utilized to assess the frequency of peripheral blood CD33+ CD11b+ HLA-DR-/low CD14+ CD15- monocytic cells, henceforth termed CD14+ HLA-DRlo/- monocytic cells, prior to and after completion of frontline chemotherapy. Multivariable odds ratios (OR) were used to estimate the association between CD14+ HLA-DRlo/- monocytic cell percentages and disease stage. Wilcoxon signed-rank tests evaluated changes in these monocytic cell levels pre- and post-chemotherapy in a patient subset (n = 70). RESULTS: Patients with elevated frequencies of circulating CD14+ HLA-DRlo/- monocytic cells at diagnosis were at 3.33-fold greater odds of having advanced stage (III/IV) EOC (CI: 1.04-10.64), with a significant trend in increasing CD14+ HLA-DRlo/- monocytic cell levels (P = .04). There was a 2.02% median decrease of these monocytic cells post-chemotherapy among a subset of patients with advanced stage disease (P < .0001). CONCLUSION: These findings support the potential clinical relevance of CD14+ HLA-DRlo/- monocytic cells in EOC for prognosis and may indicate a non-invasive biomarker to measure disease progression.

Phase I Clinical Trial of Combination Propranolol and Pembrolizumab in Locally Advanced and Metastatic Melanoma: Safety, Tolerability, and Preliminary Evidence of Antitumor Activity.

PURPOSE: Increased ß-adrenergic receptor (ß-AR) signaling has been shown to promote the creation of an immunosuppressive tumor microenvironment (TME). Preclinical studies have shown that abrogation of this signaling pathway, particularly ß2-AR, provides a more favorable TME that enhances the activity of anti-PD-1 checkpoint inhibitors. We hypothesize that blocking stress-related immunosuppressive pathways would improve tumor response to immune checkpoint inhibitors in patients. Here, we report the results of dose escalation of a nonselective ß-blocker (propranolol) with pembrolizumab in patients with metastatic melanoma. PATIENTS AND METHODS: A 3 + 3 dose escalation study for propranolol twice a day with pembrolizumab (200 mg every 3 weeks) was completed. The primary objective was to determine the recommended phase II dose (RP2D). Additional objectives included safety, antitumor activity, and biomarker analyses. Responders were defined as patients with complete or partial response per immune-modified RECIST at 6 months. RESULTS: Nine patients with metastatic melanoma received increasing doses of propranolol in cohorts of 10, 20, and 30 mg twice a day. No dose-limiting toxicities were observed. Most common treatment-related adverse events (TRAEs) were rash, fatigue, and vitiligo, observed in 44% patients. One patient developed two grade ≥3 TRAEs. Objective response rate was 78%. While no significant changes in treatment-associated biomarkers were observed, an increase in IFNγ and a decrease in IL6 was noted in responders. CONCLUSIONS: Combination of propranolol with pembrolizumab in treatment-naïve metastatic melanoma is safe and shows very promising activity. Propranolol 30 mg twice a day was selected as RP2D in addition to pembrolizumab based on safety, tolerability, and preliminary antitumor activity.

Low-Level Cytomegalovirus Antigenemia Promotes Protective Cytomegalovirus Antigen-Specific T Cells after Allogeneic Hematopoietic Cell Transplantation.

Previous studies have reported a beneficial effect from cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation (alloHCT) on immune reconstitution. We determined the CMV antigenemia level associated with increased CMV antigen-specific T cells (CASTs) at day +100 and decreased CMV reactivation after day +100. CMV reactivation and CASTs were measured with CMV antigenemia and CMV-specific major histocompatibility complex multimers. The analysis consisted of 775 CAST measurements obtained before and 30, 100, and 365 days post-alloHCT from 327 consecutive patients treated between 2008 and 2016. Detectable CASTs correlated with recipient (P < .0001) and donor (P < .0001) CMV seropositivity pre-alloHCT. CMV reactivation before day +100 was associated with a higher proportion of patients who achieved ≥3 CASTs/µL by day +100 (61% with versus 39% without reactivation, P < .001). In alloHCT recipients at high risk for CMV reactivation (R+D±) with a maximum of grade II acute graft-versus-host-disease, reactivating CMV before day +100 and achieving ≥3 versus <3 CASTs/µL at day +100 was associated with reduced CMV reactivation from day +100 to +365 (27% versus 62%, P = .04). This protective effect was observed with low-level but not high-level CMV reactivation (<5 versus ≥5/50,000 polymorphonuclear leukocytes + pp65, respectively). These findings suggest low-level CMV reactivation may be beneficial and that treatment may be delayed until progression. These findings will need validation in prospective clinical trials using CMV PCR and antigenemia assays.

Quantification of Early-Stage Myeloid-Derived Suppressor Cells in Cancer Requires Excluding Basophils.

Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature cells that accumulate in the peripheral blood and tumor microenvironment and are barriers to cancer therapy. MDSCs serve as prognostic biomarkers and are targets for therapy. On the basis of surface markers, three subsets of MDSCs have been defined in humans: granulocytic, monocytic, and early stage (e-MDSC). The markers attributed to e-MDSCs overlap with those of basophils, which are rare circulating myeloid cells with unrecognized roles in cancer. Thus, we asked whether e-MDSCs in circulation and the tumor microenvironment include basophils. On average, 58% of cells with e-MDSC surface markers in blood and 36% in ascites from patients with ovarian cancer were basophils based on CD123high expression and cytology, whereas cells with immature features were rare. Circulating and ascites basophils did not suppress proliferation of stimulated T cells, a key feature of MDSCs. Increased accumulation of basophils and basogranulin, a marker of basophil degranulation, were observed in ascites compared to serum in patients with newly diagnosed ovarian cancer. Basophils recruited to the tumor microenvironment may exacerbate fluid accumulation by their release of proinflammatory granular constituents that promote vascular leakage. No significant correlation was observed between peripheral basophil counts and survival in patients with ovarian cancer. Our results suggest that studies in which e-MDSCs were defined solely by surface markers should be reevaluated to exclude basophils. Both immaturity and suppression are criteria to define e-MDSCs in future studies.

Correction: Immune profiling in diffuse large B-cell lymphoma and mantle cell lymphoma patients treated with autologous hematopoietic cell transplant.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.