Mobilization of Autologous Peripheral Blood Stem Cells (PBSC) in CD20+ Lymphoma Patients Using RICE, Granulocyte-Colony Stimulating Factor (G-CSF) and Plerixafor

Leona A. Holmberg ^1,2,*, Michael Linenberger ^1,2, Laura Connelly-Smith ^1,2

1. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

2. Department of Medicine, University of Washington, Seattle, WA, USA

* Correspondence: Leona A. Holmberg

Academic Editor: Kamyar Afshar

Received: May 06, 2022 | Accepted: February 17, 2023 | Published: March 30, 2023

OBM Transplantation 2023, Volume 7, Issue 1, doi:10.21926/obm.transplant.2301178

Recommended citation: Holmberg LA, Linenberger M, Connelly-Smith L. Mobilization of Autologous Peripheral Blood Stem Cells (PBSC) in CD20+ Lymphoma Patients Using RICE, Granulocyte-Colony Stimulating Factor (G-CSF) and Plerixafor. OBM Transplantation 2023; 7(1): 178; doi:10.21926/obm.transplant.2301178.

© 2023 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

1. Introduction

Patients with advanced non-Hodgkin’s lymphoma (NHL) can be successfully treated with high-dose therapy followed by infusion of autologous peripheral blood stem cells (ASCT). The reported disease-free survival (DFS) rates for patients with diffuse NHL in second complete remission or not in remission at time of ASCT, for patients with mantle cell NHL, and for patients with transformed follicular NHL are 38% at three years, 28% at three years, 39% at five years, and 49% at four years, respectively [1,2,3].

Autologous peripheral blood stem cells (PBSCs) are routinely measured by fluorescence-activated cell sorting (FACS) analysis for CD34+ cells [4]. PBSCs are known to be increased in the peripheral blood by treatment with granulocyte-colony stimulating factor (G-CSF) before collection of PBSCs by apheresis [5,6]. PBSCs in NHL patients are routinely collected off either growth factor support alone or after chemotherapy and growth factor.

The successful engraftment of PBSCs post ASCT is well correlated with the number of CD34 cells infused in the stem cell product [7]. The optimum number of CD34 cells utilized for transplantation varies between institutions, but is suggested to be 5 × 10⁶ CD34 cells/kg, with 2 × 10⁶ CD34 cells/kg being the minimum number required. However, the infusion of 5 × 10⁶ CD34 cells/kg results in faster engraftment and improved survival [8,9]. The success rate of engraftment after autologous PBSC transplantation is ≥90% [8].

Patients are considered “poor mobilizers” if they do not collect 2 × 10⁶ CD34 cells/kg in a certain number of days of apheresis (e.g., 5 days or less) [8,10,11]. In the literature, up to 20% of NHL patients are reported to have a hard time initially mobilizing PBSC [8,10,11,12]. Consequently, this may result in multiple attempts to collect stem cells with increased costs.

We had looked previously at our center at 370 lymphoma patients in which we attempted to mobilize autologous PBSC without using Plerixafor. The median age was 52 years old. Median number of previous regimens was two. Overall, 69% had persistent disease in their pre-transplant evaluation. Only 10% of the patients were mobilized with growth factor (G-CSF) alone. The factors associated with increased number of days of apheresis were previous fludarabine therapy (estimate effect (EE) 0.5, 95% confidence interval (CI) -0.03-1), and mobilization with G-CSF alone, EE 0.7, (95% CI. 0.24-1.12). What correlated with a decreasing number of CD34 cells/kg collected on the first day of apheresis was age, (EE 0.1, 95% CI 0.03-0.96), number of cycles of Hyper-CVAD previously received (EE 0.5, 95% CI 0.02-0.96), previous fludarabine therapy, (EE 3.4, 95% CI 0.11-6.63) and mobilization with G-CSF alone, (EE 3.9, 95% CI 1.1-6.74). What correlated with overall decreased total number of CD34 cells/kg collected was age (EE 0.1, 95% CI 0.06-0.17), number of cycles of Hyper-CVAD previously received (EE 0.5, 95% CI 0.12-0.93), previous fludarabine therapy (EE 2.8, 95% CI 0.04-5.6) and mobilization with G-CSF alone (EE 4.1, 95% CI 1.7-6.45). In these patients, we collected a median of 3.7 × 10⁶ CD34 cells/kg and 5% of all of our lymphoma patients failed to mobilize on the initial attempt but subsequently were re-challenged and were able to get overall an adequate total number of PBSC on further recollection attempts. Factors associated with neutrophil engraftment post ASCT was only a history of previous radiation therapy (EE -1.2, 95% CI -2--0.04), and for platelet engraftment was previous therapy with fludarabine (EE 3.4, 95% CI 0.7-6.1), number of cycles of Hyper-CVAD previously received (EE 0.6, 95% CI 0.2-1.0) and total number of CD34 cells/kg infused (EE -0.25, 95% CI -0.4--0.05). Mobilization off of RICE and G-CSF did not impact statistically in our univariate analysis models for any of these factors above that we assessed.

Hematopoietic stem cells express on their surface the cognate receptor chemokine (C-X-C motif) receptor 4 (CXCR4) that attracts them to and helps them to anchor to BM stromal cell surface stromal cell derived factor -1α (SDF-1α) [13]. Plerixafor is a selective, reversible inhibitor of the binding of SDF-1α to CXCR4. Plerixafor-induced elevations in circulating hematopoietic progenitor cell levels result from the disruption of the chemo attractant and cell adhesion effects. Plerixafor, given with G-CSF, is known to increase the number of circulating blood CD34 cells and results in a higher number of CD34 cells being collected during apheresis [14]. When Plerixafor (160 or 240 micrograms/kg) was given after 4 days of G-CSF (10 micrograms/kg/day) to NHL patients, apheresis yields were significantly better compared to when the patients were given G-CSF alone [15]. PBSC collection with plerixafor and G-CSF thus has a major benefit by increasing the circulating number of PBSC and consequently decreasing the number of apheresis sessions required to collect a sufficient number of PBSCs for ASCT. Plerixafor and G-CSF also provide a level of predictability for the successful timing of apheresis collection, resulting in better optimizing of clinical resources.

Many patients with NHL, though, need to receive salvage chemotherapy for tumor debulking to make them better candidates for ASCT. RICE (rituximab, ifosfamide, carboplatin, and etoposide) is a standard debulking therapy that has been routinely used to control disease. RICE and G-CSF can also be used as in vivo purging to collect a stem cell product without tumor contamination. As a result, we felt that it would be more efficient to collect autologous PBSC off RICE, G-CSF and Plerixafor to increase the CD34 cell product yield and to minimize the number of apheresis sessions. In this paper, we report on the results of our study in using RICE/G-CSF and Plerixafor to mobilize autologous PBSC. Also, since relapse remains a problem after ASCT for NHL and to date the characterization of lymphocyte subpopulations mobilized with G-CSF and Plerixafor is poorly studied, we do also descriptively report on the immune content of the product collected in our study.

2. Materials and Methods

2.1 Patients

After giving informed consent, twenty patients were treated on the Fred Hutchinson Cancer Center IRB-approved protocol.

Inclusions criteria for getting study therapy included: diagnosis of CD20+ NHL, ≥18 years old, having adequate organ function as measured by: left ventricular ejection fraction at rest ≥50%, bilirubin ≤2.0 mg/dL (except for isolated hyperbilirubinemia attributed to Gilbert syndrome), ALT and AST ≤3× the upper limit of normal (ULN) and creatinine clearance >50 mL/min. Patient needed to be planned to undergo ASCT within 3 months after collection of PBSCs.

Exclusion criteria included: Karnofsky performance score <70%, uncontrolled bacterial, viral, or fungal infection (defined as currently taking medication with progression or no clinical improvement), pregnant or breastfeeding, unwilling to use contraceptive techniques, had prior autologous or allogeneic transplant, was HIV positive or was hepatitis B carrier. Patients could not have been treated on another investigational therapy within 4 weeks of enrolling on our study. Patients were also deemed ineligible for therapy if they had history of a prior other malignancies, with exception of resected basal cell carcinoma or treated cervical carcinoma or breast cancer in situ. Patients with previous history of cancer that treated with curative intent >5 years were allowed on the study. There was nothing stipulated in the exclusion criteria for the percent of BM lymphoma involvement that would prevent enrollment on the study.

See Table 1 for characteristics of patients.

Table 1 Patients Characteristics.

2.2 Treatment Plan

See Table 2 for treatment schema.

Table 2 Treatment Plan.

Patients received hydration, anti-emetics, standard infectious surveillance and prophylactic antibiotics per institutional guidelines. Of note, patients were also prescribed Imodium prn for diarrhea to use after starting Plerixafor. There was no dose reduction of therapy allowed.

Twenty-four hours after recovery of counts from their nadir, CD34 blood cell counts were started to be tested daily in the morning. When the CD34 blood count was ≥5 cells/microliter, Plerixafor 240 microgram/kg SC, rounded to nearest 2 mg, was added that evening, at most 10 to 14 hours prior to the planned initiation of apheresis. Plerixafor was daily continued until completion of collection, with plan for a maximum of 4 doses. G-CSF was administered in the morning approximately 1 hour prior to each daily apheresis. Plerixafor and G-CSF were administered as SC injections at separate sites. All apheresis was done as large volume per standard operating procedure at our institution. If clinically indicated, the dose of G-CSF could be increased at attending physician’s discretion. All G-CSF and Plerixafor were stopped if WBC reached ≥90,000. Recommended goal of collection was ≥5 × 10⁶ CD34 cells/kg, with a minimum collection goal of 2.5 × 10⁶ CD34 cells/kg over 4 days acceptable.

The Plerixafor dose calculation was based on the patient’s weight at screening pre therapy. The dose of Plerixafor was adjusted for patients who weigh >30% over their IBW and then we used adjusted body weight (ABW = IBW + 0.4 (actual weight - IBW). Otherwise, the dose was based on actual body weight. G-CSF dosing was based on actual weight unless >125% of ideal weight then dosing was based on adjusted weight per institutional guidelines.

2.3 Lab Testing

Six color flow cytometry staining was done for assessing for tumor contamination in the product and flow cytometry was done for assessing immune cell subpopulations [16]. NK and LAK activity were measured by a standard 4 hour ⁵¹Cr-release assay using K562 and Daudi as target cells. Briefly, 1-2 × 10⁶ target cells (K562 or Daudi) in 500 µl of CRA medium were labeled with 50-150 uCi Na₂ ⁵¹CrO₄ (New England Nuclear, Dupont, Boston, MA) at 37°C in 5% CO₂ in humidified air for one hour. After labeling, the cells were washed four times with warm medium before being re-suspended at a concentration of 5 × 10⁴ cells/ml. Five × 10³ target cells were added to the U-bottom wells of a 96 well tissue culture plate (Falcon). The NK-sensitive tumor cell line, K562, was used as a target for NK activity and lysis of the NK-insensitive tumor cell line, Daudi, was used as a measurement of LAK activity. Effector cells in 100 µl of CRA medium were then added to the wells at various effector-to-target ratios (E/T). After addition of the effector cells, every plate was centrifuged at room temperature for ten seconds at 1000 rpm. After incubation at 37°C 5% CO₂ in humidified air for four hours, the plates were centrifuged for 2 minutes at 1000 rpm. Then, 100 µls of supernatant was removed from each well and the amount of radioactivity counted in a gamma counter. Triplicate wells were assayed for each E/T. Spontaneous release was determined by evaluating the supernatant from tumor targets not incubated with effector cells. Spontaneous release was <10% per hour. Total release was determined by lysing the tumor targets with 1% NP40 detergent. The percentage cytotoxicity at a given E/T was calculated according to the following formula:

\[ \% \ \text{Specific} \ \text{Lysis}=100\times\dfrac{\left(\text{Test samples CPM}-\text{Medium control CPM}\right)}{\left(\text{Detergent control CPM}-\text{Medium control CPM}\right)} \]

Lytic units were determined as the number of effector cells required to cause 20% lysis of 5000 target cells.

2.4 Statistical Analysis

As we expected with chemotherapy to have hematological toxicity, they were not graded. Other toxicity though was graded by the NCI’s Common Terminology Criteria for Adverse Events (CTCAE), version 3. The probabilities of OS and PFS were estimated from the date of study therapy to the date of death or the date of disease progression. Data were censored at date of last contact according to Kaplan and Meier [17].

3. Results

3.1 Patients’ Characteristics

As outlined in Table 1, the patients treated on our study were older individuals and heavily treated before receiving mobilization therapy. Overall, 40% had received > four different previous chemotherapy regimens (range 4-8). They also were older as median age was 66 years, with 60% ≥65 years and 20% ≥70 years old. The majority also had diffuse large cell NHL but 3 patients had diagnosis of Burkitt’s or Richter transformation from CLL. Forty-five percent of the patients had primary refractory NHL and 40% had chemo-resistant disease when they received RICE/G and Plerixafor for mobilization.

3.2 Toxicity

No patients died of complications of the study therapy. No one met the stopping rules of the study for significant toxicity. Hematological toxicities are not reported for the study as all patients were expected to get low counts due to chemotherapy. See Table 3 for number of episodes of non-hematological, non-infectious grade 3-4 toxicity with RICE and G-CSF and Plerixafor. Table 3 also shows the number of episodes of any toxicity seen after Plerixafor was added. As expected, diarrhea grade 1-2 was most common side effect associated with the addition of Plerixafor. Of note, since patients were given Imodium prior to use after starting Plerixafor for diarrhea symptoms, this use of Imodium may have impacted on severity of grade and episodes of diarrhea reported by the patients. See Table 3 for number of infection episodes seen in all study patients. Of note, no patient’s PBSC product was contaminated with bacteria on sterility testing.

Table 3 Toxicities.

3.3 Response to Therapy

Three patients were taken off study therapy by their attending physicians as CD34 blood count was <5 cells/µL and did not get Plerixafor as per study. All of these patients collected after additional therapy, including Plerixafor and G-CSF. The number of days of collections of these three patients was 2, 3 and 5 days, respectively. The median number of cells collected was 4.44 × 10⁶ CD34 cells/kg (range 2.76-4.58).

In the remaining 17 patients collected per study therapy, the apheresis product was flowed for contaminating NHL cells and 15 out of 17 were flow negative. Two products were flow positive: one with history of Richter transformed from CLL and second with history of transformed follicular. The overall increase in CD34 blood counts from blood checked day before Plerixafor was added to day after was 2.6-9 fold increase. In the 17 patients, 15 (88%) out of 17 required only one day of collection. Two patients required two days of collections to reach the collection goal. The number of CD34 cells collected was 12.92 × 10⁶ CD34 cells/kg (range 5.44-83.76).

One patient got treated with RICE/G and Plerixafor and did not go to transplant due to disease progression. Thus, sixteen patients underwent transplant. The median number of CD34 cells/kg infused were 7.29 × 10⁶ (range 4.63-28.10). The median day to reaching ANC ≥500 was 12 days (range 10-19) in absence of G-CSF support and platelets ≥20K was 11 days (range 0-19) and platelets count ≥50K was 16.5 days range 11-42 days. There was no primary or secondary graft failures post ASCT.

Seven (41%) out of 17 patients have now relapsed NHL disease. Ten out of 17 (58%) are still in remission, including one patient though with history of Waldenström’s who has now had reoccurrence of Waldenström’s but has no relapse of the diffuse large cell NHL for which they underwent the ASCT. Eleven patients out of the 17 who completed study therapy are alive (65%). There have been 6 deaths, from NHL (n = 5) and from ARDS/heart attack and infection (n = 1). Median follow-up is 4.17 years (range 3.7-6.69 years).

3.4 Descriptive Lab Correlative Studies

With respect to the apheresis PBSC product, see Table 4. Only two patients had NK activity in their PBSC product and six patients had LAK activity. NK activity was the most recovered lytic activity seen in day +28 blood post TX (n = 14). There was no correlation between lytic activity in apheresis product and day +28 blood expression post TX. Table 5 shows the number of lymphocytes subsets per 100,000 cells in collected apheresis product and day + 28 post ASCT. Only three patients had absolute lymphocyte count >500 on day 14 post TX, with a median absolute lymphocyte count (ALC) of 295 (range 180-1,160). All patients, though, did have an ALC ≥500 by day 28, with a median of 1170 (range 680-5510). There was no significant correlation between apheresis product with the number of immune cells seen in blood on day +28 post-ASCT or with NHL relapse or remission status.

Table 4 LAK (Daudi) and NK (K562) Lytic Activity*.

Table 5 Lymphocyte Populations in Apheresis Stem Cell Product and Day +28 Blood Post ASCT.

4. Discussion

The best way to mobilize autologous PBSC remains controversial. Many prefer the convenience of growth factor alone. But historically, chemotherapy and growth factor have been more successful at collecting higher number of stem cells at a price of increased toxicity and higher resource utilization [18]. RICE therapy remains the major chemo-mobilization regimen used for lymphoma control and mobilization of autologous PBSC in CD20+ lymphoma patients.

Little has been published, though, on the efficacy and safety of comparing RICE with G-CSF vs Plerixafor growth factor alone regimens. Dhakal et al showed that chemo-mobilization with ICE was superior to routine Plerixafor use with G-CSF or just in time approach for use of Plerixafor [19]. The median collected apheresis product was 5.35 × 10⁶ CD34 cells/kg for chemotherapy vs routine Plerixafor 3.15 × 10⁶ CD34 cells/kg and just in time Plerixafor 3.6 × 10⁶ CD34 cells/kg, p > 0.001. There was no significant difference in the number of apheresis sessions, with a median of two sessions in each group. Median time to ANC and platelet engraftment post ASCT was faster with chemoembolization product 10.3 vs. 12.1 vs. 11.6 days for ANC and for platelets 13.7 vs. 20.3 vs. 17.1, respectively for chemo-mobilization, Plerixafor routine and just in time Plerixafor. The differences in days to engraftment most likely were due to differences in the median number of CD34 cells/kg infused in the groups.

Recently, Jagirdar et al reported on the addition of Plerixafor to chemotherapy, including twenty lymphoma patients who got RICE therapy and seventeen patients who got cyclophosphamide [20]. Plerixafor was added after the ANC recovered to 1.5 × 10⁹ cells/L. In their study, 33% of MM or lymphoma patients required more than one day of apheresis and Plerixafor treated patients collected a total of 7.8 × 10⁶ CD34 cells/kg.

In lymphoma patients in order to offer the curative therapy of an ASCT, an adequate number of autologous PBSC need to be collected. Historically, 10-30% of patients are not able to collect a minimum number of >2 × 10⁶ CD34 cells/kg and previous therapy with bendamustine, fludarabine and Hyper-CVAD as well as age have all contributed to this failure [21,22,23].

In our paper, we presented data on using RICE and G-CSF and Plerixafor for treating CD 20+ NHL patients. The median number of cells collected was 12.92 × 10⁶ CD34 cells/kg and higher than that reported in Jagirdar and Dhakal et al studies [19,20]. Toxicity in our study included 5 episodes of diarrhea (grades 1-2) and one episode of site injection irritation (grade 1) after Plerixafor was added. Collection was completed in one apheresis session in 15 (88%) out of 17 patients treated per study. It is hard to comment on the three patients taken off our study by their attending physician as they did not meet the criteria to be removed from the study and in fact might have been able to collect if CD34 blood cells had been checked again in a couple of days, especially if G-CSF dose had been increased as allowed on the study. With a median of 7.29 × 10⁶ CD34 cells/kg infused in our patients, the median time to ANC engraftment was 12 days (in the absence of post TX G-CSF supportive care) and the median day to Platelet ≥20K was 11 days and ≥50K was 16.5 days, respectively. Engraftment was what would have expected for the number of CD34 cells/kg infused. There were no primary or secondary graft failures on our study. It should also be pointed out that we used a CD34 blood cell count of equal or >5 cells/µL to add Plerixafor and that a CD 34 blood count of 5 is lower than most use in their real time formula for adding Plerixafor. The patients on our study also were an older population, with a median age of 66 years old and 12 (60%) of the patient were ≥65 years old and 4 (20%) were ≥70 years old. We and others have shown that age is an important factor in determining ability to get a good stem cell product. Our patients were also heavily treated, with 90% of them having a history of two or more regimens before getting study therapy and 40% of them with a history of >4 regimens. Given the small number of patients it was hard for us to comment on the effect of getting previously fludarabine, bendamustine or four or more cycles of Hyper-CVAD and also take into account the age of the patient and number of different regimens. Indeed, for the NHL patient, it is easier if they are getting chemotherapy for debulking therapy to take advantage of the therapy and use our study therapy approach as the majority collected in only one day.

Limitations of our study though, include the small number of patients treated on the study, and variability in previously received treatments and different NHL pathology included in the study. In addition, given the number of cells collected, it is not obvious that all patients really needed Plerixafor and many would have not received it under our real-time risk adapted rescue strategy [24]. Another limitation is the lack of control for intra-physician variability in decision making about how to treat patients with low blood CD34 cell counts after salvage chemotherapy and G-CSF as show with 3 patients coming off study at the discretion of attending physician caring for them before G dose was increased as recommended in the study. Finally, our approach to how to manage these patients today would be different given our recent publication [25].

5. Conclusions

In summary, the combination of Plerixafor to RICE/G-CSF overall was well tolerated, was highly effective in mobilizing autologous PBSC and allowed collection in one day of apheresis in 88% of our elderly and heavily treated NHL patient population.

Acknowledgments

Funding for the study was provided by Sanofi/Genzyme. The authors wish to thank Julia Montes for her help and support as study staff.

Author Contributions

LAH was the principal investigator, designed the study, analyzed the data, recruited patients and wrote the manuscript. LCS and ML oversaw the apheresis and cryopreservation of the autologous PBSC. All authors reviewed the manuscript for intellectual content and approved the final version. Sanofi/Genzyme provided funding for the study.

Funding

Dr. Holmberg received funding for studies from Millennium-Takeda, Sanofi/Genzyme, Seattle Genetics and Merck Sharp & Dohme, Inc. and royalty from UpToDate. She is also a consultant for NCCN and Jazz Pharmaceuticals.

Competing Interests

The other authors have declared that no competing interests exist.