Antibody responses to tetanus toxoid and Haemophilus ... - Nature

Bone Marrow Transplantation, (1997) 20, 33–38  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

Antibody responses to tetanus toxoid and Haemophilus influenzae type b conjugate vaccines following autologous peripheral blood stem cell transplantation (PBSCT) CY Chan1, DC Molrine1, JH Antin2, C Wheeler3, EC Guinan4 , HJ Weinstein4, NR Phillips1, C McGarigle2, S Harvey3 , C Schnipper1 and DM Ambrosino1 1

Laboratory of Infectious Diseases and 4 Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA; 2Division of Hematology-Oncology, Brigham and Women’s Hospital, and 3Division of Hematology-Oncology, Beth Israel Hospital, Boston, MA, USA

Summary: Accelerated granulocyte and platelet recovery following peripheral blood stem cell transplantation (PBSCT) are well documented. We hypothesize that functional immunity may also be enhanced in PBSCT and performed a phase II trial of immunizations in patients with lymphoma undergoing autologous transplantation with peripheral blood stem cells or bone marrow. Seventeen BMT and 10 PBSCT recipients were immunized at 3, 6, 12, and 24-months post-transplantation with Haemophilus influenzae type b (HIB)-conjugate and tetanus toxoid (TT) vaccines. IgG anti-HIB and anti-TT antibody concentrations were measured and compared between the two groups. Geometric mean IgG anti-HIB antibody concentrations were significantly higher for PBSCT recipients compared to BMT recipients at 24 months post-transplantation (11.3 mg/ml vs 0.93 mg/ml, P = 0.051) and following the 24 month immunization (66.2 mg/ml vs 1.30 mg/ml, P = 0.006). Similar results were noted for IgG anti-TT antibody with significantly higher geometric mean antibody concentrations in the PBSCT group at 24 months post-transplantation (182 mg/ml vs 21.6 mg/ml, P = 0.039). Protective levels of total anti-HIB antibody were achieved earlier in PBSCT recipients compared with those of BMT recipients. PBSCT recipients had higher antigen-specific antibody concentrations following HIB and TT immunizations. These results suggest enhanced recovery of humoral immunity in PBSCT recipients and earlier protection against HIB with immunization. Keywords: peripheral blood stem cell transplantation; immunization, active; vaccines, conjugate; vaccines, protein

Bone marrow transplantation (BMT) is a well established treatment modality for hematologic malignancies, aplastic anemia, and immunodeficiency disorders.1–4 In recent Correspondence: Dr DM Ambrosino, Dana-Farber Cancer Institute, Laboratory of Infectious Diseases, 44 Binney Street, JFB Room 424, Boston, MA 02115, USA Received 8 November 1996; accepted 28 March 1997

years, many centers have also employed peripheral blood stem cell transplantation (PBSCT). Most peripheral blood stem cell transplants have been autologous,5–9 although allogeneic peripheral blood stem cell transplants have also been studied.10–13 Peripheral blood stem transplantation has offered marrow rescue therapy for patients whose marrows were unsuitable for transplantation and it provides more rapid reconstitution of granulocytes and platelets. 14–16 More rapid hematopoietic recovery has resulted in reduced transplant-related morbidity with less febrile days, fewer documented infections, less antibiotic usage, and fewer red cell and platelet transfusions. While granulocytes recover more rapidly, the kinetics of immune recovery after autologous PBSCT are less well defined. Limited data suggest that immunological reconstitution following autologous BMT and PBSCT is similar.14,17–19 To evaluate functional humoral immune reconstitution following autologous PBSCT as compared with autologous BMT, we immunized 17 autologous BMT and 10 autologous PBSCT recipients at 3 months, 6 months, 12 months, and 24 months post-transplantation with Haemophilus influenzae type b (HIB)-conjugate vaccine and tetanus toxoid vaccine. Total immunoglobulin concentrations and antigen-specific antibody responses of the two transplant groups were compared.

Patients and methods Study population Adult patients undergoing autologous bone marrow transplantation (ABMT) or peripheral blood stem cell transplantation (PBSCT) for non-Hodgkin’s lymphoma or Hodgkin’s disease between July 1990 and April 1993 at the Brigham and Women’s and Beth Israel Hospitals in Boston, MA, USA were recruited. Seventeen ABMT and 10 PBSCT patients were enrolled in the study. Of the 10 PBSCT patients, six received autologous bone marrow in addition to PBSC. Clinical characteristics of PBSCT and ABMT patients comprising the study population are shown in Table 1. Of the 17 autologous ABMT patients, 10 patients had a diagnosis of Hodgkin’s disease and seven patients had a diag-

Active immunization in PBSC transplantation CY Chan et al

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nosis of non-Hodgkin’s lymphoma. Of the 10 autologous PBSCT patients, four patients had a diagnosis of Hodgkin’s disease and six patients had a diagnosis of non-Hodgkin’s lymphoma. PBSCT patients had more advanced stage of disease than the ABMT patients, nine PBSCT patients (90%) and 10 ABMT patients (59%) had either stage III or IV at the time of diagnosis. More patients in the PBSCT group (50%) as compared to the ABMT group (18%) had tumor involvement of the bone marrow. Limited regional radiotherapy was given to one PBSCT patient (10%) and six AMBT patients (35%) within 3 months of transplantation. None of the differences in clinical characteristics of the two groups was statistically significant as noted in Table 1. Patients who survived without relapse until at least 3 months post-transplant were enrolled in the study. Patients who relapsed, died, missed immunizations, or were lost to follow-up after 3 months post-transplant were included in the analysis until the time that these events occurred. Two PBSCT patients had not reached the 24 month time-point at the time of analysis. The study was approved by the Institutional Review Boards of both participating hospitals and informed consent was obtained from each enrolled patient. Collection of PBSC and bone marrow Autologous PBSC were collected by three to six aphereses performed during the rapid rise in blood counts following myelosuppressive chemotherapy. Four PBSCT patients also received GM-CSF mobilization therapy. A Fenwall CS3000 Plus cell separator (Fenwall Laboratories, Dearfield, IL, USA) with a granulocyte chamber was used for automated isolation of mononuclear cells after bone marrow harvest or PBSC collection. Mononuclear cells were then cryopreserved in dimethyl-sulfoxide (DMSO) using a controlled rate liquid nitrogen freezer. The number of mononuclear cells pre-freezing per kilogram of patient’s lean body weight ranged from 1.12 3 108 to 12.0 3 108 in the PBSCT group (median = 6.83 3 108) and from 0.29 3 108 to 2.98 3 108 in the ABMT group (median = 0.92 3 108). Immunization schema All study patients received 0.5 ml Haemophilus influenzae type b (HIB)-conjugate vaccine (HibTITER; Lederle-Praxis Biologicals Division, Pearl River, NY, USA) and 0.5 ml tetanus toxoid aluminum phosphate-adsorbed vaccine (Wyeth Laboratories, Philadelphia, PA, USA) intramuscularly at 3 months, 6 months, 12 months, and 24 months following transplantation. In addition, patients were immunized with 23-valent pneumococcal polysaccharide vaccine (Pnu-Immune 23; Lederle-Praxis Biologics) or an investigational pneumococcal-conjugate vaccine (Merck Research, West Point, PA, USA) at 12 and 24 months after transplant. Antibody responses to the pneumococcal vaccines were minimal in both transplant groups and these data are not reported. Serum was obtained from each patient prior to the administration of vaccines and 3–6 weeks following the 24 month immunization. A subset of patients who had a scheduled visit between 12 and 24 months had

an additional serum sample collected. All sera were stored at 270°C until assayed. Antibody assays Total binding anti-HIB antibody was measured by radioimmunoassay (RIA) using a standard Food and Drug Administration protocol with tritiated polyribosylribitol phosphate (provided by Dr Porter Anderson, University of Rochester, NY, USA). The RIA was standardized using the Center for Biologic Research and Review standard serum pool with an assigned value of 70 mg/ml. IgG and IgM anti-HIB antibody concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using HIB oligosaccharide coupled to human albumin (provided by Dr Porter Anderson) and standardized using a serum reference pool with assigned values of 27.2 mg/ml for HIB IgG and 1.62 mg/ml for HIB IgM. IgG and IgM anti-tetanus toxoid antibody concentrations were measured by ELISA as previously described.20 Total serum IgG and IgM concentrations at 12 and 24 months post-transplant were determined by ELISA using goat anti-human IgG and IgM (Tago, Burlingame, CA, USA) and goat anti-human IgG and IgM alkaline phosphatase conjugates (Tago) as described previously.20 All serum samples for a given time-point from both transplant groups were measured in the same assays to reduce effects of interassay variation. Statistics Data organization and analysis were performed by the PROPHET system, a national computer system sponsored by the Chemical/Biological Information Handling Program of the National Institute of Health (Bethesda, MD, USA). Logarithms of the antibody concentrations were used for statistical calculations. Antibody concentrations that were below the limit of assay sensitivity were assigned values of one half the lower limit. Comparisons of geometric means of antibody concentrations were performed by the two-tailed t-test for parametric analysis and by the Mann– Whitney rank sum test for non-parametric analysis. The Fisher exact test was used to compare the frequency of individuals with protective concentrations of total anti-HIB antibody following HIB-conjugate immunization. Multiple linear stepwise regression analyses were performed to examine the effect of selected variables on total immunoglobulin concentrations and antigen-specific antibody concentrations post-transplantation. Partial correlation coefficients presented are calculated with all the significant variables included in the regression model.

Results Patient population Patients were conditioned for transplantation with cyclophosphamide (1500 mg/m2/day 3 4 days), etoposide (400 mg/m2/day 3 4 days), and BCNU (112.5 mg/m2/day 3 4 days), except for one PBSCT patient who received high dose ifosfamide, carboplatin, and etoposide. All patients


received granulocyte–macrophage colony-stimulating factor (GM-CSF) post-transplantation, starting either on the day of transplant or 2 days after transplantation until the absolute neutrophil count (ANC) was >0.5 3 109/l for 2 consecutive days. None of the patients received intravenous immunoglobulin (IVIG) following transplantation. After transplantation, patients of the PBSCT group had a more rapid neutrophil recovery defined as the number of days to reach ANC > 0.5 3 109/l compared to patients in the ABMT group (16 vs 21 days, respectively) (Table 1). The number of evaluable patients in the PBSCT group was 10 at 3 months, nine at 6 months, eight at 12 months, and five at 24 months. The number of evaluable patients in the ABMT group was 17 at 3 months, 17 at 6 months, 12 at 12 months, and nine at 24 months.

Table 1

Clinical characteristics of PBSCT and BMT study patients

Characteristic Number Median age years (range) Male/Female Diagnosis (%) Hodgkin’s disease non-Hodgkin’s lymphoma Stage at initial diagnosis I/II/III/IV Prior relapse (%) None/1/2 or more Pre-harvest radiotherapy Prior marrow involvement Post-transplant radiotherapy b Median days to achieve ANC >0.5 3 109/l

PBSCT

BMT

10a 35 (21–47) 6/4

17 31 (21–57) 10/7

4 (40) 6 (60)

10 (59) 7 (41)

0/1/1/8

1/6/5/5

2/7/1 5 (50) 5 (50) 1 (10) 16 (10–27)

2/10/5 8 (47) 3 (18) 6 (35) 21 (11–42)

Total IgG and IgM immunoglobulin reconstitution a

To determine immune reconstitution following transplantation, we measured total serum IgG and IgM concentrations in both transplant groups. Total serum IgG concentrations were similar for PBSCT and ABMT patients at 12 months and 24 months following transplantation. Total serum IgG geometric mean antibody concentrations (95% confidence interval) in the PBSCT group were 686 mg/dl (226–2080) at 12 months and 520 mg/dl (192–1400) at 24 months compared with 685 mg/dl (276–1700) and 723 mg/dl (284–1840) respectively in the ABMT group. In contrast, total serum IgM concentrations were significantly lower in the PBSCT group compared with patients in the ABMT group at 12 and 24 months. Geometric mean total serum IgM concentrations (95% confidence interval) at 12 months after transplant were 28.2 mg/dl (4.70–169) in the PBSCT group compared with 100 mg/dl (15.8–635) in the ABMT group (P = 0.007). At 24 months, geometric mean total serum IgM were 42.4 mg/dl (11.2–161) in the PBSCT group compared with 152 mg/dl (62.1–370) in the ABMT group (P = 0.001). Antibody responses to HIB-conjugate vaccine PBSCT recipients had higher anti-HIB antibody concentrations at all measured time-points following transplantation compared to ABMT recipients. Geometric mean IgG anti-HIB antibody concentrations as measured by ELISA were significantly higher in the PBSCT group at 24 months (11.3 mg/ml vs 0.93 mg/ml, P = 0.051) and following the 24 month immunization (66.2 mg/ml vs 1.30 mg/ml, P = 0.006). The difference in IgG anti-HIB antibody concentrations trended towards significance at 12 months and following the 12 month dose (Table 2). Similar results were noted for total anti-HIB antibody as measured by RIA with significantly higher geometric mean antibody concentrations at 24 months post-transplantation (8.30 mg/ml vs 0.22 mg/ml, P = 0.01) and following the 24 month immunization (74.0 mg/ml vs 1.14 mg/ml, P = 0.02) (Figure 1). Measurements of HIB antibody by RIA and ELISA assays were highly correlated at each measured time-point (r = 0.51 to r = 0.96). IgM concentrations to HIB were also compared between the two transplant groups before and after HIB-conjugate immunization. Geometric mean IgM

Six patients received PBSC as well as bone marrow; four patients received PBSC only. b Receipt of regional radiation treatment within 3 months of transplant. None of the clinical characteristics was significantly different between the two groups. Comparisons of age and ANC >0.5 3 109/l were performed by two-tailed t-test. The other characteristics were compared by Fisher exact test. For stage at initial diagnosis, patients with stages I and II were combined vs stages III and IV for each group and comparison of the number of prior relapses was none vs one or more relapses for each group.

anti-HIB antibody concentrations were similar for patients in the PBSCT group as compared with patients in the ABMT group at each measured time-point (data not shown). The frequency of patients with presumed protective concentrations of total anti-HIB antibody was also compared for the two transplant groups. A concentration of total antiHIB antibody of >1.00 mg/ml is considered to be predictive of long-term protection.21 More patients in the PBSCT group had protective levels of total anti-HIB antibody following immunization compared with patients in the ABMT group. At 6 months, 12 months, and following the 12 month dose, 44, 25, and 75% of patients in the PBSCT group had protective antibody concentrations compared to 24, 17 and 43% respectively of patients in the BMT group. The frequency of patients with protective antibody concentrations at 24 months declined to 60% in the PBSCT group and 11% in the ABMT group. Following the 24 month dose, the frequency of patients protected increased to 80% in the PBSCT group and 44% in the ABMT group. The differences in the frequency of patients protected in each group were not statistically significant at any of the measured time-points. Antibody responses to tetanus toxoid vaccine Patients in the PBSCT group also achieved higher IgG antitetanus toxoid antibody concentrations following immunization as compared to those in the ABMT group (Figure 2). Geometric mean IgG anti-tetanus toxoid antibody concentrations were significantly higher in the PBSCT group at 24 months post-transplantation (182 mg/ml vs 21.6 mg/ml, P = 0.039) and trended toward significance at 12 months and following the 24 month immunization (Table 2). Although IgG anti-tetanus toxoid antibody concentrations

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Table 2

Antibody responses to Haemophilus influenzae type b (HIB)-conjugate and tetanus toxoid vaccines post-transplantation Geometric mean antibody mg/ml (95% confidence intervals)

Antibody

Months

n

HIB IgG

3 6 12 post 12 24 post 24

10 9 8 4 5 5

1.35 1.30 4.76 13.2 11.3 66.2

(0.077–24.0) (0.044–38.5) (0.170–132) (0.068–2580) (0.031–4140) (0.875–5010)

17 17 12 7 9 9

Tetanus Toxoid IgG

3 6 12 post 12 24 post 24

10 9 8 4 5 5

49.6 20.4 105 337 182 418

(8.13–302) (2.52–165) (3.06–3570) (35.2–3240) (57.2–581) (171–1020)

17 17 12 7 9 9

a

PBSCT

BMT

P-value a

0.84 0.67 1.69 0.91 0.93 1.30

(0.104–6.82) (0.034–13.2) (0.178–16.2) (0.010–80.5) (0.066–13.2) (0.024–71.8)

NSb NS 0.121 0.113 0.051 0.006

33.5 20.8 35.2 141 21.6 51.1

(8.05–140) (2.93–148) (2.54–489) (7.02–2480) (0.447–1040) (0.311–8410)

NS NS 0.139 NS 0.039 0.104

n

t-Test for normally distributed data and Mann–Whitney rank sum test for non-normally distributed data. Values .0.150 are identified as not significant (NS).

b

1000

100

Tetanus toxoid Geometric mean IgG antibody µ g/ml

Total HIB Geometric mean antibody µ g/ml

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PBSCT group 10

BMT group 1

PBSCT group

100

BMT group

10

1

0.1 0

3

6

12

24

Months post-transplantation Figure 1 Total anti-HIB antibody concentrations following HIB-conjugate immunization of patients who received PBSCT as compared with patients who received BMT. Open squares represent geometric mean antibody concentrations of patients in the PBSCT group. Closed squares represent geometric mean antibody concentrations of patients in the BMT group. The solid line represents presumed protective level of total antiHIB antibody as measured by radioimmunoassay. Arrows indicate the times post-transplantation when HIB-conjugate vaccine was administered to all patients.

were higher in the PBSCT group, all patients in each group had IgG anti-tetanus toxoid antibody concentrations above the presumed protective level of > 0.01 IU/ml (equivalent to 0.43 mg/ml in our assay) at all measured time-points post-transplantation.22 Similar to HIB IgM antibody results, IgM anti-tetanus toxoid antibody concentrations were not significantly different between the two transplant groups both before and following immunizations. Variables affecting total serum immunoglobulins and antigen-specific antibody concentrations after transplantation Because of the significant differences in antigen-specific antibody concentrations of patients in the PBSCT group

3

6

12

24

Months post-transplantation Figure 2 Antibody responses to tetanus toxoid immunization following PBSCT or BMT. Open squares represent geometric mean antibody concentrations of patients in the PBSCT group. Closed squares represent geometric mean antibody concentrations of patients in the BMT group. Arrows indicate the times post-transplantation when tetanus toxoid vaccine was administered to all patients.

compared with patients in the ABMT group at 24 months and post 24 months transplantation, multiple linear regression analyses were performed to examine the effect of selected clinical variables on IgG anti-HIB and antitetanus toxoid antibody concentrations at these time-points. The influence of these variables on total serum IgG and IgM concentrations at 12 and 24 months after transplantation were also examined. The variables included in the analyses were: type of transplant, age, gender, underlying diagnosis, number of relapses, stage of disease, tumor involvement of the marrow, GM-CSF mobilization therapy, number of prior chemotherapy regimens, pre-transplant radiation therapy, post-transplant radiation therapy, and total number of mononuclear cells infused. PBSCT was independently significantly associated with higher total anti-HIB antibody concentrations at 24 months (partial correlation coefficient 0.662, P = 0.009) and post


24 months (partial correlation coefficient 0.611, P = 0.019), and with higher IgG anti-HIB antibody concentrations at 24 months (partial correlation coefficient 0.531, P = 0.034) and following the dose at 24 months (partial correlation coefficient 0.695, P = 0.005). PBSCT was also independently associated with higher IgG anti-tetanus toxoid antibody concentrations at 24 months (partial correlation coefficient 0.556, P = 0.072). In addition, younger age of recipients was independently associated with higher IgG anti-tetanus toxoid antibody concentrations at 24 months and post 24 months transplantation. No other variables were consistently associated with antigen-specific antibody concentrations. Finally, PBSCT was independently significantly associated with lower total serum IgM concentrations at 12 months (partial correlation coefficient 0.569, P = 0.007) and 24 months (partial correlation coefficient 0.771, P = 0.001) after transplantation. None of the variables was consistently independently associated with total serum IgG concentrations. Discussion PBSCT results in more rapid reconstitution of hematopoiesis as compared to bone marrow transplantation. However immune reconstitution has been less well studied.14–16 Descriptions of reconstitution of lymphocyte subsets over time and in vitro functional assays, as well as immunoglobulin concentrations, have been published.14,18,19,23 These initial studies evaluate reconstitution following PBSCT and make comparisons to those previously reported for marrow recipients. In general these studies have indicated similar immune reconstitution for PBSCT recipients and recipients of autologous marrow cells.14,18,19 However, these studies are difficult to interpret as the patient populations are heterogeneous, the numbers of patients studied are small and the comparisons are made using in vitro assays or lymphocyte phenotype analysis. In addition, these comparisons are not direct correlates of protection from infectious pathogens. We therefore examined functional humoral immunity following transplantation using antigen-specific responses to immunizations. The protective correlates of responses to these vaccines are well established and commonly employed to determine immune function for immunodeficient patients. Ten patients who received PBSCT and 17 patients who received ABMT were immunized with HIB-conjugate and tetanus toxoid vaccines following transplantation at 3, 6, 12 and 24 months. The PBSCT patients had consistently higher antibody concentrations compared to the ABMT group after the first three doses. The differences in IgG anti-HIB antibody reached significance following the final immunization at 24 months (P = 0.006). For IgG antitetanus toxoid antibody, the PBSCT patients had higher concentrations before the final dose at 24 months and following that dose (P = 0.039, P = 0.104 respectively). Our study demonstrates that antigen-specific responses are enhanced in PBSCT patients as compared to autologous marrow recipients. We speculate that the mechanism for this difference relates to the number of antigen-specific

memory cells infused. Both the carrier protein for the HIBconjugate vaccine, diphtheria toxoid, and the tetanus toxoid vaccine are administered as routine childhood vaccines. Thus the harvested peripheral blood and marrow would contain memory B and T cells for both these proteins. The representation of memory B cells and T cells in peripheral blood as compared to marrow is not clearly defined. However, the total number of mononuclear cells/kg infused was at least seven-fold greater in the PBSCT group than the number infused in the ABMT group. Thus we suggest that more memory B and/or T lymphocytes were likely to be infused in the PBSCT group as compared to the marrow recipients. We also noted that the geometric mean total serum IgM concentrations were significantly lower at 12 and 24 months post-transplant in the PBSCT patients. One previous study has reported a similar percentage of patients following PBSCT or ABMT had low total serum IgM concentrations; however, the time after transplantation when the measurements were determined was not equivalent between the transplant groups.18 Our finding will need to be confirmed and the significance of lower total serum IgM is unknown. It is possible that PBSCT results in fewer IgM naive B cells being transferred compared to marrow transplantation. Primary antibody responses would then be impaired despite enhanced secondary antibody responses. We doubt any clinical significance of this finding. The small number of patients in this report is a limitation. One reassuring fact was that antibody responses were enhanced for both vaccines studied. In addition, our groups were not randomized due to obvious practical considerations. The predominant indication for PBSCT during the study period was the inability to obtain adequate marrow. We would not expect PBSCT patients to be ‘better responders’ as these patients were at a more advanced stage of disease. Finally, we included patients who received both PBSC and marrow in the PBSCT group. This decision is supported by other investigations which have demonstrated that hematopoietic reconstitution of patients who receive both PBSC and BM is similar to patients who receive PBSC only.24,25 In summary, increased antibody responses to HIB-conjugate and tetanus toxoid vaccines administered after transplant were documented in PBSCT patients as compared to ABMT patients. We speculate that PBSCT patients have enhanced immune reconstitution and are at less risk for infections. Of note, our study examined T cell-dependent antibody responses and not cellular immune function. The effect of PBSCT on reconstitution of antigen-specific cytotoxic lymphocytes (CTL) will need to be defined although it is likely that increased numbers of CTLS were also transferred. Different cytokine and chemotherapy mobilization techniques, positive selection of CD341 cells for infusion, ex vivo expansion of CD341 cells, and the use of umbilical cord blood cells are all being actively investigated for transplantation.24–29 Our data suggest that immune reconstitution will likely be affected by these manipulations. Our results may also have importance for tumor vaccine studies in the transplant setting. We speculate that enhanced tumor vaccine responses would be seen in PBSCT compared to auto-

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logous marrow transplants. Initial studies may wish to focus on this transplant group.

Acknowledgements This work was supported by grants A129623, CA01730 and CA39542 from the National Institutes of Health, Friends of DanaFarber Cancer Institute, and the Claudia Barr Program at DFCI. CYC is a Pfizer Fellow in the Beth Israel Hospital/HarvardMassachusetts Institute of Technology Division of Health Sciences and Technology: The Clinical Trial Investigator Training Program.

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