Introduction

Graft failure is a major concern for patients undergoing reduced-intensity allogeneic hematopoietic SCT. Initial studies using 200 cGy (centigray) of TBI followed by post-graft immune suppression resulted in a 20% graft failure rate.¹ The addition of fludarabine has reduced the incidence of graft failure to generally less than 3%.² Neither primary graft failure nor secondary graft rejection is lethal in the non-myeloablative setting as endogenous hematopoiesis returns;^{3, 4} however, the patient does not derive the benefit of a new immune system to treat the underlying illness, thus subsequent therapies are still needed. A second attempt at allogeneic immunotherapy is often contemplated, though data on success are limited. There are also questions of whether the same donor can/should be used and whether a second attempt at a reduced-intensity regimen is sufficient in a patient who has already experienced one episode of rejection. We therefore report on 11 patients with hematologic malignancies, renal cell cancer or marrow failure who underwent a second reduced-intensity regimen for primary or secondary graft failure.

Methods

Patients

Patients were consented and treated for the first non-myeloablative transplant attempt on one of our institutionally approved in vivo T-cell depleted protocols. Patients received their second non-myeloablative allogeneic transplant between 2000 and 2006 with all reported outcomes current to March 2007. Table 1 details the patient characteristics. The ages ranged from 20 to 68 years. Two patients underwent an HLA-matched related donor allograft, while nine received donor cells from partially HLA-matched family members. All patients met minimum criteria for protocol including both a cardiac ejection fraction and diffusing capacity of the lung for carbon monoxide greater than 40% as we have published previously in detail.⁵ Patients were not eligible for a myeloablative transplant secondary to organ dysfunction, known uncontrolled fungal infection with aspergillus or infection with hepatitis C.

Table 1 - Patient characteristics and outcomes.

Full table

Treatment regimens

First transplant attempt

Nine patients were treated with a 5-day preparative regimen that included fludarabine 30 mg/m² on days -5 to -1 (150 mg/m² total), cyclophosphamide 500 mg/m² on days -5 to -2 (2000 mg/m² total) and alemtuzumab 20 mg on days -4 to 0. Patients who received an HLA-matched related donor received no additional GVHD prophylaxis, whereas patients who underwent an HLA-mismatched transplant received mycophenolate mofetil 1000 mg twice daily for 6 weeks. This preparative regimen is associated with a 1% graft failure rate in the HLA-matched patients and 6% graft failure rate in the mismatched HLA population.^{5, 6} Two patients were treated with a reduced-intensity preparatory regimen of fludarabine 30 mg/m², cyclophosphamide 2000 mg/m², alemtuzumab 20 mg and TBI 200 cGy all delivered on day -1. These two patients also received GVHD prophylaxis with mycophenolate mofetil 15 mg/kg and cyclosporine 6.25 mg/kg.

Second transplant attempt

For the second transplantation attempt, all patients were treated with the 5-day fludarabine, cyclophosphamide and alemtuzumab regimen described above.

PBSC collections

All cells for both transplants were peripherally collected from the donor via apheresis. The average CD34 cell dose for the first transplant was 14.3 10⁶ with a range of 4.29–21.3 10⁶. The average CD34 cell dose for the second transplant was 15.4 10⁶ with a range of 7.68–31.3 10⁶.

Outcome assessments

Regimen-related toxicity

Toxicities were formally graded twice weekly using the NCI Common Toxicity Criteria (version 3.0) through at least day 45 and once patients were discharged home, then assessment was performed at a minimum of every other week with doctor visits or phone assessments through day 100 and thereafter as clinically indicated.⁷ GVHD was assessed using standardized criteria as well. Any new toxicity not present before initiation of therapy or a pre-existent toxicity that worsened by at least once grade was scored as being at least possibly related to the transplant. Follow-up is current to time of submission of this manuscript for any long-term complications that may have been encountered in the surviving patients.

Engraftment

For consistency reasons, we followed our program's previously published outline for assessing donor engraftment.^{5, 8} We perform RFLP analysis using eight microsatellite markers plus X and Y chromosome-specific markers and assessed for donor-derived hematopoiesis beginning at the time the ANC was at least 0.5 10⁹, the platelets >20 10⁹ and the hemoglobin >8 g/dl un-transfused. For purposes of our assessment of meaningful allogeneic hematopoiesis, we consider at least partial donor engraftment to have occurred if there are >2.5% donor cells detected with this method. Patients who never have donor cells detected following this definition of engraftment are considered to have 'primary graft failure'. Those who met this definition at least once for having had at least partial donor engraftment but later decreased donor percentage to <2.5% were scored as having secondary graft failure regardless of cause. Engraftment is checked at the first sign of early hematopoietic recovery (around day 14), 6 weeks later than at 3-month intervals.

Disease response was determined using recently published standardized criterion for those with hematologic malignancies and documentation of donor engraftment with normal hemoglobin electrophoresis in the thalassemia patient.^{9, 10}

Results

Six patients had primary graft failure from the first attempt at transplantation with one having persistent pancytopenia due to persistence of myelodysplastic syndrome (MDS). Four of the five with secondary graft failure developed this within 2 months of transplant. Their initial levels of donor hematopoiesis ranged from 5 to 63%. One had respiratory syncytial virus treated with ribavirin and a second patient was treated with ganciclovir for CMV infection, which might have contributed to the graft failure. The reason for graft failure in the third and fourth patients is unknown. One patient had late secondary graft failure with relapsed myelofibrosis at 9 months. He had 10% donor cells at the time of relapse and was documented.

Engraftment with the second transplant attempt: nine of the 11 patients initially engrafted with this second attempt, including two of the four patients who had the same donor. Six patients initially experienced primary graft failure prior to this second transplant attempt. Five of these patients engrafted on this second attempt. The one patient with the same donor failed to engraft with the second transplant. Five patients experienced secondary graft failure prior to this second transplant attempt. Four of these patients engrafted on the second attempt including two of the three patients who used the same donor. Two of the three patients who used the 5-day preparatory regimen described above with both transplants engrafted. Two patients who used the 1-day preparatory regimen with the first transplant regimen engrafted on the second attempt after the 5-day preparatory regimen was used. Figure 1 shows the initial donor percentage at the time of engraftment and at 4 months in those with available data.

Figure 1.

Percent donor cells at initial engraftment and at 4 months.

Full figure and legend (34K)

The two patients with persistent graft failure, one with refractory MDS/AML (myelodysplastic syndrome) and one with renal cell cancer, had cell doses of 7.68 10⁶ and 10.52 10⁶. The patient with refractory MDS/AML initially had 5% donor engraftment with the first transplant prior to secondary graft rejection with persistent disease. The patient with renal cell cancer had primary graft failure with the first transplant. Both used the same donor and the same 5-day preparatory regimen for both transplants. The patient with MDS/AML eventually achieved allogeneic engraftment following a third attempt using a different partially matched family member and the 1-day preparatory regimen above.

A patient with nodal marginal zone lymphoma had only 3% donor hematopoiesis at initial engraftment analysis from this second attempt. He died 34 days after transplant of diffuse alveolar hemorrhage having rejected the donor cells. A 59-year-old patient with CML had 9% donor hematopoiesis with the day 14 RFLP, but developed CMV infection and subsequent graft rejection by day 30 despite a donor lymphocyte infusion. The same 5-day preparatory regimen was used both times with a different HLA-mismatched donor in both cases described above. A patient with mantle cell lymphoma and MDS initially had 97% donor cells on day 22, but experienced progression of his MDS/AML by day 47 and subsequently died from disease.

Toxicities

GVHD: this partially T-cell depleted approach resulted in at least grade 1 acute GVHD occurring in five of the nine engrafting patients. Two patients, including one after a donor lymphocyte infusion, developed grade II GVHD involving the skin and gastro intestinal tract. There was no grade 3–4 aGVHD in these nine patients. One patient who did not engraft with this second attempt engrafted following a third non-myeloablative attempt (counted as one of the two with graft failure) developed grade 3 severe skin and gut GVHD after the third attempt.

Other toxicities: patients tolerated the preparative regimen well with two patients experiencing severe nausea or vomiting, two with transient renal insufficiency with creatinines ranging from 2.4 to 3.0. Six patients developed reactivation of CMV and one developed CMV disease. One each had respiratory syncytial virus pneumonitis and zoster as well. Four of the 11 patients experienced other severe infections including Streptococcus viridians, mucor, aspergillus, Stenotrophomonas pneumonia and a mold infection. Two of these patients with active pneumonia developed secondary diffuse alveolar hemorrhage as well, one of which contributed to early death. No other significant toxicities were encountered.

Survival as of June 2007: with a median follow-up of survivors of 29 months, 5 of the 11 patients are still alive and in remission, 7 months to 5 years from second T-cell depleted non-myeloablative transplantation. This includes patients with marrow failure, leukemia and myelofibrosis. There were two treatment-related deaths. One patient died at day 33 from infections and diffuse alveolar hemorrhage. One with MDS/AML died 4.5 months after her third transplant from GVHD and infections. Four patients died from progressive disease 1–9 months following therapy.

Discussion

The goal of a stem cell transplant is to develop a mutual tolerance between host and recipient cells to prevent both host versus graft disease (rejection) and GVHD.¹¹ In the absence of myelosuppressive drug therapy, poor donor cell function, or infections, primary graft failure is rejection of a graft by residual host immunocompetent cells.¹² It has been found that donor-specific CD8+ cytotoxic T cells appear in the blood at the time of rejection in many patients.¹³ In addition, patients who have host lymphocytes detected after transplant are more prone to graft failure than those who do not. The immune status of the host at the time of transplant is even more important in the non-myeloablative setting where a mixed chimerism outcome is common.¹⁴ The patient's ability to reject donor stem cells can be affected by age, disease and amount of prior therapy.^{1, 14} Early ablative allogeneic transplant data indicate that 30–60% of patients with aplastic anemia had graft failure from alloantibodies from previous blood transfusions.¹⁵ The amount of host T cells at the time of engraftment may be a critical feature with a mixed chimerism associated with higher numbers of host circulating CD8+ and CD4+ T cells.^{14, 16} One of the reasons patients might have engrafted with the second transplant is that the second preparatory regimen following so closely to the first (a median of 6 months for those with primary graft failure) might have further suppressed an already-weakened host immune system, assuring acceptance of the donor cells. The choice of preparatory regimen is also an important factor in determining engraftment. Less-intensive regimens and possibly T-cell depletion lead to increased rates of graft failure.¹⁷ For instance, two patients initially treated with the 1-day preparation noted above, engrafted after the 5-day regimen. They are both engrafted and long-term survivors 2.5 and 3 years later. Engraftment during the first transplant may have been affected by other factors such as infections with certain viruses and the need for myelotoxic drugs (ganciclovir) to treat these infections.¹⁸ Fortunately, for some patients in this case series, they were not affected by the same viruses during the subsequent transplant. It is helpful to note that two of the four patients with the same donor in this case series engrafted following this second non-myeloablative transplant. The patients would already have been exposed to mHAs on the donor cells. However, the second regimen given in close proximity to the first must have immunosuppressed the host sufficiently and, in combination with the generally high donor cell dose, allowed engraftment despite the continued use of T-cell depletion.

Other small case series have suggested that a second non-myeloablative transplant is beneficial in a subset of patients who experience graft failure. Ruiz-Arguilies et al.,¹⁹ reported the largest series of such patients to date (12 patients) who underwent a second reduced-intensity transplant. The series included a mixture of pediatric and adult patients. Eleven patients had the same donor for the second transplant, including two patients initially treated with umbilical cord blood cells from a sibling and subsequently transplanted with peripheral blood from the same sibling. They received the same preparatory regimen for both transplants, which consisted of oral busulphan 4 mg/kg on days -6 and -5, cyclophosphamide 350 mg/m² on days -4, -3 and -2, fludarabine 30 mg/m² on days -4, -3 and -2. Oral cyclophosphamide (5 mg/kg) and IV methotrexate (5 mg/m² on days 1, 3, 5 and 11) was used for GVHD prophylaxis. Five patients who experienced primary graft failure failed to engraft with the second transplant. There were 3 of the 12 patients, a 3-year-old with ALL, a 24-year-old with aplastic anemia and a 49-year-old with AML, who remained engrafted without evidence of disease at 10, 52 and 41 months respectively. Narimatsu et al.³ reported engraftment in three of the four patients who underwent a second reduced-intensity regimen with a cord blood transplant for graft failure. The fourth patient died at day 8 from pneumonia. Two of the three who engrafted died early of treatment-related toxicity (thrombotic microangiopathy on day 57, sepsis on day 160). The remaining patient is without disease progression at over 9.0 months. The importance of this case series is to demonstrate that it is possible for patients who experience graft failure to successfully engraft following a second T-cell depleted non-myeloablative conditioning regimen and remain in remission long term. It also shows the same donor can be used for the subsequent transplant with a reasonable possibility of obtaining engraftment. Thus, though sometimes requiring a second attempt, patients have the ability to obtain a new hematopoietic and immune system to fight their underlying disease. In our cohort of patients, 9 of the 11 patients successfully engrafted including two of the four patients who used the same donor. There are long-term survivors, including patients with both malignant and non-malignant diseases. One patient with -thalassemia is alive and engrafted with a stable mixed chimeric state and phenotypically free of disease 5 years from the second transplant. A patient with myelofibrosis has been in remission for over 2.5 years and another patient with AML is alive and in remission for over 3 years without recurrence. Two other patients are without evidence of disease at 7 and 10 months from transplant. With the use of alemtuzumab, the rate of severe GVHD is very low and there were only two treatment-related deaths, one within 30 days and the other 7 months later. As a retrospective review, our case series is subject to a number of biases. It is a small series of patients who underwent transplantation for a diverse array of hematologic malignancies at different disease statuses, making it impossible to identify which group benefits most from a second transplant. The patient population is further selected for a group felt to be able and willing to undergo a second transplant. In conclusion, this case series suggests that patients are capable of long-term engraftment following a second non-myeloablative regimen. The second transplant can be well-tolerated and there is a meaningful chance of prolonged survival. Further work needs to be done to identify better patients who are the best candidates for this salvage technique.

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