Immunological recovery Immunologic recovery after ... - Nature

Bone Marrow Transplantation (2001) 28, 1105–1109  2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00 www.nature.com/bmt

Immunological recovery Immunologic recovery after autologous blood stem cell transplantation in patients with AL-amyloidosis G Akpek1–3, G Lenz1, SM Lee4, V Sanchorawala1,2, DG Wright1,2, T Colarusso1, K Waraska1, A Lerner1, E Vosburgh1,2, M Skinner2 and RL Comenzo1,2,5 1

Section of Hematology and Oncology, Department of Medicine, Boston University Medical Center, Boston, MA, USA; 2Amyloid Program, Department of Medicine, Boston University Medical Center, Boston, MA, USA; 3Division of Hematologic Malignancies, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Division of Biostatistics, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; and 5Hematology Service, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Summary: We prospectively studied absolute lymphocyte (ALC) and monocyte counts (AMC), lymphocyte subsets and proliferative in vitro responses to mitogen and antigen in 12 patients with AL-amyloidosis (AL) undergoing autologous blood stem cell transplantation (SCT) with high-dose i.v. melphalan. Myeloid and lymphoid recovery (⬎500 per ␮l) occurred in a median of 10 days post SCT. While there was a continuous decline in the number of CD4ⴙ T cells at 3 months, ALC, AMC, B cells (CD19ⴙ), CD8ⴙ T cells, and NK cells (CD16ⴙ/56ⴙ) returned to baseline. While T cell proliferative responses to phytohemagglutinin (PHA) remained depressed, B cell function measured by the proliferative response to staphylococcal antigen returned to baseline by 3 months. To supplement our findings, we retrospectively evaluated ALC, AMC and serum immunoglobulin levels in a separate group of patients treated with the same protocol at our institution. ALC and AMC recovery was similar to the pattern observed in the initial study group. Immunoglobulin levels remained within normal ranges at 3 and 12 months after SCT. Of 50 patients who were followed for a minimum of 1 year following SCT, seven (14%) developed shingles and one (2%) had PCP pneumonia. In conclusion, cellular immune function, reflected by absolute numbers of CD4ⴙ T cells and PHA responsive T cell proliferation, is significantly suppressed at 3 months after SCT in patients with AL, and this post-transplant immunosuppression is associated with a low but clinically meaningful occurrence of opportunistic infections typical of T cell immunosuppression. Bone Marrow Transplantation (2001) 28, 1105–1109. Keywords: amyloidosis; transplantation; stem cell; immunologic; reconstitution

Correspondence: Dr G Akpek, Johns Hopkins Oncology Center, Division of Hematologic Malignancies, The Bunting-Blaustein Cancer Research Building, Floor 2M, 1650 Orleans Street, Baltimore, MD 21231, USA Received 17 October 2000; accepted 24 September 2001

High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (SCT) has emerged as an effective treatment modality in patients with AL-amyloidosis (AL). We have shown that high-dose melphalan (HDM) chemotherapy with autologous SCT prolongs survival in this disease.1,2 As in other diseases, immunologic recovery after SCT is an essential factor affecting the outcome of patients with AL. It is well known that post-transplant immune recovery is influenced by the type of transplant (allo vs auto),3 the severity of graft-versus-host disease,4,5 the type of graft6 (bone marrow vs peripheral blood stem cell) and graft manupulation7,8 (T cell-depleted vs unmanipulated graft). A recent report also indicates that immune reconstitution after allogeneic SCT may vary depending on the underlying hematologic malignancies.9 Although auto-SCT has been utilized in patients with AL, immune recovery following SCT has not been well studied in this patient population. Our clinical observations of infections during the 6 months following transplant in patients with AL10 led us to investigate the character and timing of post-SCT immune reconstitution in these patients. Patients and methods Between January 1998 and June 1998, we conducted a prospective study on 12 patients to investigate the pattern of quantitative and functional immune recovery after doseintensive chemotherapy and autologous SCT. The study was approved by Institutional Review Board and required written informed consent. All patients had biopsy proven amyloid deposits and a plasma cell dyscrasia by bone marrow, serum, and/or urinary studies. The patients received either 200 mg/m2 of melphalan over 2 consecutive days if they were less than 60 years of age or 140 mg/m2 if they were more than 60, followed by auto-SCT as previously described.2,11 Peripheral blood stem cell collection was performed as previously described.1,2,12 The stem cell products with a minimum of 2 ⫻ 106 CD34⫹ cells/kg were infused on the day after the completion of melphalan administration. All patients were given G-CSF at a dose of 5 ␮g/kg/day starting on the day after stem cell infusion until

Post-transplant immune recovery in amyloidosis G Akpek et al

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the absolute neutrophil count (ANC) was more than 500 per ␮l for 2 consecutive days, and erythropoietin at a dose of 100 U/kg three times week. Oflaxocine 200 mg p.o. twice a day and acyclovir 400 mg p.o. three times a day were begun on the day following stem cell infusion and continued until the myeloid recovery was complete. To supplement findings from this initial study group, we retrospectively collected additional data on ALC and AMC in 42 patients who received 200 mg/m2 (n ⫽ 28) and 140 mg/m2 (n ⫽ 14) of i.v. melphalan followed by SCT at our institution between December 1997 and February 2000. Serum quantitative immunoglobulin G, A and M levels were also retrieved from the clinical records of 19 patients whose Ig levels prior to treatment had been normal. These retrospective data were evaluated separately. The incidence and the type of opportunistic infections seen after auto-SCT were also evaluated in 50 patients who had been followed for a minimum of 1 year after auto-SCT. Quantitation of peripheral blood cell subsets Total white blood cell (WBC) counts and WBC differential counts were determined by an automatic counter. For the evaluation of mononuclear cell subsets flow cytometric immunophenotyping was performed. Specimens were analyzed on an EPlCs XL3 (Coulter, Miami, FL, USA) cell sorter. Monoclonal antibodies (MoAbs) directly conjugated to fluorochromes including CD3, CD4, CD8, CD16, CD56 and CD19 (Coulter and Becton Dickinson, San Jose, CA, USA) were used in immunophenotypic analysis. Cell culture and lymphocyte proliferation assays Twenty ml of heparinized blood was collected in a tube containing sodium heparin. Light-density mononuclear cells were separated on Ficoll–Paque density gradients, washed, counted, and suspended in RPMI-1640 (GIBCO, Carlsbad, CA, USA) and 10% fetal bovine serum at 106 cells/ml. The cells were plated in equal volumes in 96-well trays at 5 ⫻ 104 cells per well with medium alone and with optimal concentration of mitogen (PHA) and antigen (SAC). 0.01% Staphylococcus aureus Cowen strain A (SAC) (Pansorbin) and 1 mg/ml phytohemagglutinin (PHA; Difco, Detroit, MI, USA) were used for B cell and T cell stimulation, respectively. Cultures were grown for 3 days in quadruplicates for each situation. For the last 16 h, cultures were labeled with 1 mCi 3H-thymidine per well, then harvested on to fiber filtermats, and evaluated using a liquid scintillation ␤-counter. For evaluation, the mean activity of the mitogen and antigen cultures were calculated. For correction of outliers, the cultures exhibiting lowest and highest activity were disregarded. ‘Background’ activity was determined as the mean activity of autologous controls. Results were expressed as mean counts per minute (cpm) ‘increment’ ie the mean of the measured activities (cpm) minus ‘background’ (cpm) [Net CPM ⫽ Measured CPM ⫺ Background CPM].6 Study intervals In vitro studies including immunophenotypic analysis and proliferation assays were performed in 12 patients at three

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time points: at baseline prior to mobilization, at discharge after the engraftment is completed, and at 3-month followup post transplant. Data on ALCs and AMCs at these intervals were also collected prospectively. ALCs and AMCs at 12 months were retrospectively retrieved and added to the results. Data collection in the retrospective group was extended to 12 months post SCT. Statistics The immune recovery data are summarized as median and the first and third quartiles. Box plots of selected outcomes are also provided. Due to small sample size, multiple tests were done as a limitation. Pair-wise testing of baseline immune recovery data and the values at discharge, 3 months and 12 months post transplant were performed using the sign rank test. The measures of summary and the sign rank test were chosen due to the lack of normality of the data in some circumstances. The number of patients may vary by visit due to missing data. No comparative analysis by melphalan dose was performed due to the limited number of participants. Results Patient characteristics Between January and June 1998, immune reconstitution in 12 patients with AL-amyloidosis who received HDM preparative regimen followed by autologous SCT at Boston University Medical Center was studied prospectively. Five patients received high-dose (200 mg/m2) and seven patients, who were more than 60 years of age, received intermediate dose (140 mg/m2) intravenous melphalan over 2 consecutive days. Patient characteristics are summarized in Table 1. Hematologic recovery Recoveries of neutrophil, monocyte and lymphocyte counts (⬎500 cells/␮l) occurred by 2 weeks in all patients. Median Table 1

Patient characteristics Total

No. of patients Median age (range), years Male/Female Primary involvement Renal Nervous system Liver Lymph node/soft tissue Median number of stem cells transplanted (CD34⫹ cell/kg) Dose of melphalan prior to SCT 140 mg/m2 200 mg/m2

12 53 (38–71) 6/6 9a 2 1 1 5.1 ⫻ 106 (2.4–8.8 ⫻ 106) 7 5

a One patient had both renal and peripheral nervous system involvement by AL.


absolute lymphocyte counts (ALC) returned to baseline levels at 3 months. Absolute monocyte counts (AMC) and NK cell counts (CD16⫹ and CD56⫹), following an increase at the time of discharge following myeloid recovery, had also returned to baseline levels at 3 months. Median ALC and AMC in the retrospective series were also at baseline levels by 3 months. Following a sharp decline at the time of discharge, median B cell (CD19⫹) counts returned to baseline by 3 months (Table 2). However, median T cell (CD3⫹) counts were not different from baseline at discharge following SCT, nor at 3 months. There was a significant decrease both in the subset of CD4⫹ T cells and in the CD4/CD8 ratio at 3 months following SCT (Figure 1a, b and c).

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Clinical correlation Fifty patients who underwent SCT in 1998 and 1999 were followed for a minimum of 1 year. Opportunistic infections that occurred in these patients included Herpes zoster reactivation (shingles) in seven patients (14%) and pneumonias requiring hospitalization in six patients (12%). No microbial diagnosis was made in five of the patients who developed pneumonia, but in one Pneumocystis carinii was identified (Table 3). Fifteen patients treated with HDM/SCT during the same time period survived less than 1 year. Pneumonia (at 1 and 7 months), and sepsis (at 1.5 and 7 months) were the causes of death in four of these patients.

Cellular response to mitogen and antigen in vitro Figure 2a and b show functional studies related to T cell and B cell function after transplant. While T cell proliferative responses to PHA were depressed at 3 months (Figure 2a), B cell function measured by the proliferative response to staphylococcal antigen had returned to baseline by this time (Figure 2b). Serum immunoglobulin levels following SCT Data on immunoglobulin levels were collected retrospectively from 19 patients who received 200 mg/m2 (n ⫽ 14) or 140 mg/m2 (n ⫽ 5) of melphalan. Each of these patients had normal Ig levels prior to treatment. Although, serum immunoglobulin levels were significantly lower at 12 months as compared to baseline levels, absolute values of all three immunoglobulin fractions remained within the normal range at all times of evaluation (Table 2).

Table 2

Discussion The rate, type and severity of infectious complications are closely related to the recovery of a functional immune system following hematopoietic stem cell transplantation. While quantitative aspects of the immune system generally return to normal within the first 3–6 months following transplant, prolonged delays in the recovery of qualitative immune functions have been documented, particularly in patients who have received allo-SCT. T cell proliferation is impaired immediately after transplantation and may not recover until more than a year. There is also a documented deficit in IL-2 producing cells post BMT.13 Our results demonstrate that recipients of autologous peripheral blood stem cell grafts after high-dose melphalan had full recovery of B cells by 3 months post transplant. Functional studies of B lymphocyte proliferative responses to antigen were also normal by 3 months following SCT.

Immunologic parameters studied before and after SCT

Parameter

Median (1st quartile, 3rd quartile) Baseline

At discharge

3 months

12 months

Prospective data (n ⫽ 12) ALC (⫻1000/mm3) AMC (⫻1000/mm3) CD19⫹ cells (%) CD3⫹ cells (%) CD4⫹ cells (%) CD8⫹ cells (%) CD16⫹/56⫹ (%)

1.9 (1.7, 2.45) 0.5 (0.3, 0.6) 0.12 (0.08, 0.15) 0.73 (0.61, 0.76) 0.44 (0.38, 0.49) 0.23 (0.19, 0.29) 0.13 (0.07, 0.21)

1.8 (0.95, 2.55) 0.7 (0.45, 1.25) 0.02 (0.01, 0.035)* 0.62 (0.56, 0.66) 0.36 (0.30, 0.49) 0.19 (0.14, 0.33) 0.31 (0.26, 0.37)*

2.45 (1.25, 3.35) 0.5 (0.3, 0.6) 0.1 (0.4, 0.12) 0.69 (0.63, 0.75) 0.28 (0.23, 0.35)* 0.35 (0.33, 0.45) 0.13 (0.08, 0.17)

1.7 (1.6, 1.8) 0.5 (0.3, 0.6) NA NA NA NA NA

T cell thymidine uptake after PHA stimulation (cpm)

79 801 (64 725, 97 135)

21 221* (3370, 35 287)

62 791 (25 095, 88 358)

NA

B cell thymidine uptake after Staph-A stimulation (cpm)

4271 (2755, 5395)

459 (272, 1017)

4614 (1380, 19 389)

NA

Retrospective data ALC ⫻1000/mm3 (n ⫽ 42) AMC ⫻1000/mm3 (n ⫽ 42) Serum Ig G mg/dl (n ⫽ 19) Serum Ig A mg/dl (n ⫽ 19) Serum Ig M mg/dl (n ⫽ 19)

1.6 (1.1, 2.2) 0.6 (0.4, 0.8) 932 (864, 1410) 131 (97, 150) 82 (71, 122)

1.4 (1.0, 2.3) 0.6 (0.5, 1.0) NA NA NA

1.65 (1.4, 2.5) 0.5 (0.4, 0.7) 884 (633, 1280) 96 (76, 137) 87 (57, 119)

1.7 (1.2, 2.2) 0.5 (0.4, 0.7) 881* (720, 1058) 91 (55, 121) 66 (42, 119)

*P ⬍ 0.01; NA ⫽ not assessed. Bone Marrow Transplantation


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a

a

150 000

CD4+ cells (%)

60

100 000 cpm

40

50 000

20

0

0 Before transplant

b

At discharge

3 Months

b

At discharge

Before transplant

At discharge

3 months

30 000

60

20 000

40 cpm

CD8+ cells (%)

Before transplant

10 000

20

0

0 Before transplant

At discharge

3 Months

c

CD4+ /CD8+ ratio

8

3 months

Figure 2 Cellular in vitro responses to PHA mitogen (a) and Staphylococcus Cowen strain-A antigen (b) after SCT. cpm: 3H-Thymidine uptake increment. Vertical axis shows the cpm reading after radioactive labeling with thymidine. Horizontal axis is a time axis.

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4

Table 3 Opportunistic infections in AL patients treated with HDM/SCT (n ⫽ 50) 2

Type of infection

0 Before transplant

At discharge

3 months

Herpes zostera (shingles) ⭐6 months post SCT 6–12 months post SCT

Melphalan 200 Melphalan 140 Overall 7 (14%) 4 —

2 1

Figure 1 Phenotypic reconstitution of mononuclear cells subsets after SCT. Peripheral blood cell counts of CD4⫹, CD8⫹ lymphocytes, and CD4⫹/CD8⫹ ratio are shown in box plots (a to c). Vertical axis shows the percentage of particular cell subsets in total lymphocyte population. Horizontal axis is a time axis.

Pneumonia of undetermined etiologyb ⭐6 months post SCT 6–12 months post SCT PCP pneumoniac ⭐6 months post SCT

Although there was an overall decline in serum immunoglobulin levels at 12 months following SCT, the absolute levels remained within normal ranges at all times following SCT. NK cells appeared early after myeloid engraftment and recovery was completed by 3 months as was the case

a Total documented cases of Herpes zoster infections following SCT among 223 patients who received HDM/SCT between September 1994 and March 2001 was 17 patients (7.6%). b Requiring hospitalization. c Total documented cases of Pneumocyctis carinii (PCP) infections following SCT among 223 patients who received HDM/SCT between September 1994 and March 2001 was four patients (1.8%).


5 (10%) 3 1

— 1

1

—

1 (2%)


with B cell and monocyte recoveries. Given the normalization of B cell number and functional activities by 3 months, it would appear that no post-SCT prophylaxis for infections associated with humoral immune defects (eg Pneumococcus or Hemophilus influenza) is warranted based on transplant-related effects on immune reconstitution per se. On the other hand, such prophylaxis could be justified if hypogammaglobulinemia associated with underlying disease was present prior to SCT and persisted following treatment. In contrast, T cell recovery was found to be delayed following SCT, as significant depressions in CD4⫹ T cell counts were observed at 3 months. CD8⫹ T cells, on the other hand, recovered to baseline levels or to levels higher than baseline at 3 months following SCT in all patients, as has been reported by others.14–16 The duration of this cellular immune defect beyond 3 months after SCT in patients with AL is uncertain; however, the first 6 months post transplant in these patients appears to be the most vulnerable period of time for infectious complications. Given the quantitative and qualitative T cell deficits following HDM/SCT, together with the documented occurrence of opportunistic infections, including PCP pneumonia, in patients with AL following SCT, PCP prophylaxis should be considered during the 6 months post transplant. Patients should also be followed expectantly for the occurrence of Herpes zoster infections. There has been recent interest in the possibility that immune recovery may serve as a prognostic indicator in patients undergoing autologous hematopoietic stem cell transplantation. Mayo Clinic investigators recently showed enhanced survival for auto-transplant patients whose absolute lymphocyte counts were greater than 500/␮l at day 15 post transplant (Luis Porrata; personal communication). Bensinger et al17 from Seattle also reported that early lymphoid recovery has an impact on reducing infectious complications following allogeneic hematopoietic SCT. In summary, we have found that peripheral blood counts of T helper cells (CD4⫹) are significantly decreased and T cell functions depressed at 3 months following SCT in patients with AL amyloidosis, unlike T suppressor (CD8⫹) cell, monocyte, B cell, and NK cell counts as well as levels of B cell functional activity, which have returned to baseline by this time following SCT.

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Acknowledgements We thank Dr Thomas Rothstein, who provided his immunology laboratory for us to perform in vitro assays and Dina Pelley from the Clinical Flow Cytometry Laboratory at Boston University Medical Center for the immunophenotypic analyses. This work was supported by JoAnn McCaleb Amyloid Research Award, Boston University School of Medicine, Boston MA and published in the Proceeding of American Society of Hematology Meeting in Miami FL, 1998.

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