Apheresis for chimeric antigen receptor T-cell production in adult lymphoma patients

2.1 Patients

In this retrospective study, 23 patients suffering from relapsed/refractory Non-Hodgkin's lymphoma treated at the University Hospital Regensburg were included. All patients underwent autologous lymphapheresis as preparation for subsequent treatment with CD19-specific CAR T cells. We retrieved epidemiologic, demographic, clinical, laboratory, management, and outcome data from patients' medical records. Aphereses were conducted between April 2019 and December 2021. This retrospective study was approved by the Ethics Committee of the University Regensburg (ethics statement number: 21-2716-104), and written informed consent was obtained from all patients prior to apheresis.

2.2 Apheresis

This calculations will be done on the day of the apheresis itself, and these preliminary values can then be used to more easily adjust the blood volume to be processed based on the latest lymphocyte counts. Parameters defining a successful apheresis run comprised a TNC value of at least 2 × 10⁹ (viability ≥70%) with a minimum number of 1 × 10⁹ CD3-positive lymphocytes. After apheresis, TNC and CD3-positive lymphocytes were determined using a multi-parameter automated hematology analyzer (XN-550, Sysmex, Kobe, Japan) and flow cytometry (Navios, EX, Beckman Coulter, Indianapolis, IN, USA) respectively.

2.3 Laboratory analysis prior to lymphapheresis

Prior to apheresis, a multi-parameter automated hematology analyzer was utilized to generate complete blood counts with automated differentials. Lymphocyte subpopulations were distinguished via flow cytometry (Navios EX, Beckman Coulter) using the following markers: CD3 (T cells), CD19 (B cells), and CD16/56 (NK cells).

2.4 Statistical analysis

The collection efficiency for CD3-positive lymphocytes was calculated according to the following formula: CD3-positive lymphocytes collected (×10⁶)/(Pre-apheresis CD3-positive lymphocytes (×10⁶) × liters (L) processed) × 100. As indicated, variables were documented as mean, median, and range (minimum/maximum) values. Significant differences between study variables were determined using two-tailed Student's t-test. A p-value less than .05 was deemed statistically significant.

3.1 Patient characteristics

In aggregate, 23 adult patients (12 females and 11 males) with histologically confirmed Non-Hodgkin's lymphoma treated at our medical center underwent lymphapheresis as preparation for CAR T-cell therapy with Tisagenlecleucel. All patient characteristics are depicted in Table 1. The majority of patients suffered from diffuse large B cell lymphoma not otherwise specified (DLBCL-NOS). Moreover, three patients with transformed follicular lymphoma, one patient with transformed chronic lymphocytic leukemia (CLL) and one patient with follicular lymphoma were included. Median age of patients was 72 years (range 37–77 years, only five patients were younger than 60 years) at the time of apheresis. The median weight was 78 kg (range 54–110 kg). All patients completed at least one line of rituximab-based standard immunochemotherapy. Furthermore, 12 patients presented with relapsing or refractory disease after two lines of standard chemotherapy, and two patients underwent apheresis after pretreatment with more than two different standard chemotherapy regimens. Overall physical condition at the time of apheresis according to ECOG performance status indicated a slightly reduced performance (ECOG 1) in 18 patients, full function (ECOG 0) in two patients, and moderately reduced performance (ECOG 2) in three patients. Initial lymphapheresis was successful in 22 of 23 patients. One patient had to undergo a second apheresis owing to an insufficient lymphocyte count after the first run. Hence, 24 apheresis runs were available for analysis.

3.2 Apheresis process

Upon informed consent, the venous status of apheresis patients was assessed to determine which venous access was appropriate for lymphocyte collection. A hemodialysis catheter was required for half of the apheresis runs, while the other half was conducted with peripheral venous access (Table 2). Neither central venous access nor peripheral venous access was associated with any significant complications, such as vasovagal reactions. The processed blood volume varied from 5.0 to 13.9 L with a median of 9.9 L (Table 2). The median total blood volume (TBV) processed amounted to 1.9 TBV (range 1.3–3.0 TBV) (Table 2), taking into account the German regulations (“Richtlinie zur Gewinnung von Blut und Blutbestandteilen und zur Anwendung von Blutprodukten”) in their most recent version, which generally limits leukapheresis to 3 TBV. Anticoagulation was performed with ACD-A using a median volume of 1012 ml per apheresis (range 518–1538 ml) (Table 2). The evaluation of the cell separators' internal time-tracking protocols yielded a median collection time of 167 min (range 103–259 min). Possible serious side-effects reported in the context of lymphapheresis including paresthesia, nausea, headache, pain, hypotension, or cardiovascular events were not observed in any of the evaluated patients.

3.3 Preapheresis laboratory values

Automated differential blood counts as well as flow-cytometry based analyses of lymphocyte subpopulations and circulating malignant cells were obtained from all patients within 2 weeks prior to apheresis (Table 3). Flow-cytometric analysis of CD3-positive lymphocytes, NK cells, and B cells are available from 22 of 24 runs. Before apheresis, the patients showed a median WBC of 4610 cells/μL (range: 1670–19,200 cells/μl) with a median neutrophil count of 2800 cells/μl (range: 950–15,400 cells/μl) and a median monocyte count of 520 cells/μl (range: 250–2740 cells/μl) (Table 3). Moreover, the median lymphocyte count of 740 cells/μl (range: 120–1490 cells/μl) at the time of apheresis was found below the reference range, which was largely attributed to B-cell aplasia (median B-cell count 0 cells/μl) following CD20-directed chemoimmunotherapy (Table 3). Median CD3-positive lymphocyte counts and NK cell counts were within reference range at 565 cells/μl (range: 70–1345 cells/μl) and 140 cells/μl (range: 31–504 cells/μl) respectively (Table 3). Circulating lymphoma cells as assessed by flow cytometry were only detected in one patient with DLBCL. Preapheresis laboratory requirements were set by institutional guidelines encompassing a measurable CD3-positive cell count in the peripheral blood, and immediately prior to apheresis hemoglobin levels greater than 8 g/dL as well as a platelet count of greater than 30 G/L. Apheresis collection yields.

Apheresis success, defined as meeting the requirements of the manufacturer (Novartis) comprising a target TNC of ≥2 × 10⁹, a CD3-positive lymphocyte count of ≥1 × 10⁹ with an overall viability of ≥70%, was achieved in 21 of 23 patients (Table 4). Apheresis collection yields are summarized in Table 4. The median TNC count in the apheresate was 9.2 × 10⁹ (range: 2.9 × 10⁹–47.4 × 10⁹). The median CD3-positive lymphocyte count in the apheresate amounted to 2.55 × 10⁹ (range: 0.370 × 10⁹–6.915 × 10⁹) with an overall viability of 94% (range: 82.7%–97.2%). In general, the peripheral blood CD3-positive cell count correlated with the product CD3-positive cell yield (Figure 1). The median collection efficiency for CD3-positive lymphocytes was 63.7% (range: 9.56–93.6%). In total, 17 apheresis products obtained from 16 patients (including the two patients formally not achieving apheresis success) were dispatched to Novartis for Tisagenlecleucel manufacturing. Unfortunately, three patients died from rapidly progressing disease during CAR T-cell production. Due to death, CAR T-cell manufacturing was prematurely aborted in all three patients, and no CAR T-cell products were generated. Consequently, CAR T-cell products were successfully administered to a total number of 13 patients, while seven patients continued with standard chemotherapy treatment. Their remaining apheresis products were stored below −140°C in our transfusion medicine facility for CAR T-cell production in case of relapse or chemo-refractory disease. At the time of this writing, those seven patients do not require CAR T-cell therapy due to ongoing responses, thus their apheresis products are still frozen. An early lymphapheresis with subsequent on-site storage has become standard of care at our medical center, as this strategy permits CAR T-cell production even for patients that become lymphopenic upon sequential immunochemotherapies. Post-apheresis analysis of the apheresis products' composition revealed a median lymphocyte percentage of 38.3% (range: 8.4%–60.3%), a median CD3-positive cell percentage of 35.9 (range: 4.4%–55.6%), a median granulocyte percentage of 7% (range: 1.7%–62.5%), and a median monocyte percentage of 50.1% (range: 11.0%–62.4%) (Table 4). Hemoglobin levels well below 1 g/dl in the apheresate demonstrated the successful targeting of the WBC layer during the apheresis process (Table 4). In aggregate, T-cell apheresis and subsequent CAR T-cell production were feasible in the majority of patients included in this study.

3.4 Apheresis related changes in hematocrit, hemoglobin, and platelet count

Blood counts were closely monitored before and after apheresis. The median starting hematocrit prior to apheresis was 33.1% (range: 25.2%–42.2%) and the mean post-apheresis hematocrit amounted to 28.6% (range: 21.3%–38.6%) (Table 5). Moreover, a hemoglobin level of greater than 8 g/dl and a platelet count of greater than 30 G/L were prerequisites to begin apheresis, which were fulfilled in all patients without the need to administer blood products. The lowest hemoglobin level and the lowest platelet count at the start of apheresis were 8.4 g/dl and 63 G/L respectively (Table 5). The median hemoglobin level prior to apheresis was 11.3 g/dl (range: 8.4–14.4 g/dl), and the median pre-apheresis platelet count was 234 G/L (range: 63–318 G/L) (Table 5). Post-apheresis median hemoglobin levels and platelet counts amounted to 9.8 g/dl (range: 7.1–13.2 g/dl) and 150 G/L (range: 51–244 G/L) (Table 5). While no patient required thrombocyte transfusion after apheresis, four patients received RBCs post apheresis due to hemoglobin levels dropping below 8 g/dl. In all those four patients, pre-apheresis hemoglobin levels ranged between 8.4 and 8.7 g/dl. Among others, one reason for the drop in hemoglobin levels may rely on dilution effects owing to ACD-A addition during apheresis. Irrespective of symptoms of anemia, which were not present in any of those four patients, blood transfusions will be administered as soon as hemoglobin levels drop below 8 g/dl as stipulated by institutional guidelines. In sum, lymphapheresis expectedly caused drops in hematocrit, hemoglobin, and platelet counts, and close monitoring ensured appropriate RBC substitution before patients became symptomatic.

3.5 Apheresis related changes in selected electrolytes

Calcium and potassium levels were also regularly checked during the apheresis process to avoid hypocalcemia and hypokalemia, which may both result in potentially relevant arrhythmias or even cardiogenic shock. The median total calcium level measured before apheresis was 2.32 mM (range: 2.08–2.45 mM). All patients, including those with normal calcium levels, received prophylactic calcium substitution during apheresis by calcium gluconate infusions (usually 2–6 g calcium). Hence, total serum calcium levels post-apheresis were significantly higher than pre-apheresis with a median calcium level at the end of apheresis of 2.47 mM (range: 2.27–2.73 mM). Potassium was also prophylactically substituted during apheresis (usually 20–40 mM potassium chloride were administered) ensuring similar median potassium levels pre- (3.75 mM, range: 3.2–4.5 mM) and post-apheresis (3.56 mM, range: 3.0–4.56 mM). In sum, close monitoring and prophylactic substitution prevented severe electrolyte dysbalances during apheresis. Moreover, no side-effects associated with electrolyte dysbalances were observed.