The second case we present is a 16-year-old girl with a late, isolated BM ALL relapse after allogeneic HSCT (Figure 2 and Table 1). The patient was initially treated according to the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP)-BFM ALL 2009 protocol (Clinicaltrials.gov registration number: NCT01117441) and was transplanted from an unrelated donor matched at 9 out of 10 loci in CR1 after administration of blinatumomab. The latter was used to lower her pre-transplant MRD and led to an MRD load of 2 x 10-4 prior to allogeneic HSCT. Conditioning consisted of 12 Gy TBI in combination with etoposide (60 mg/kg). BM was used for the graft. Pre- and post-transplant organ toxicities included severe necrotic pancreatitis with concomitant stomach perforation and polyomavirus BK-associated haemorrhagic cystitis requiring prolonged antiviral treatment. After allogeneic HSCT, the patient developed acute GvHD of the skin (grade IV), which responded well to multimodal immunosuppressive therapy. Nephrotoxic side effects of both immunosuppressive and antiviral treatment resulted in stage IV renal insufficiency (glomerular filtration rate 20 mL/min/1.73 m2; serum creatinine 2.0-2.5 mg/dL).
After successful haematopoietic engraftment with full donor chimerism at Day + 28, the BM at 21 months after allogeneic HSCT revealed a late, isolated BM relapse (partially CD19 +, CD22 +). Despite the CD19- subclone (49% of the cells), the multidisciplinary leukaemia and cellular therapy board opted for CAR T-cell therapy with tisagenlecleucel as an attempt for remission induction therapy followed by allogeneic HSCT.
While awaiting CAR T-cell production, the patient received bridging chemotherapy derived from the ALL-BFM protocol III variant (Clinicaltrials.gov registration number: NCT00430118)   under continuous veno-venous haemodiafiltration (CVVHDF). MRD as measured by PCR remained at a level of 1 x 10-1. During chemotherapy, the patient developed multiple pulmonary lesions with pleural effusion and both hepatic and intracerebral lesions, which were considered of fungal origin and finally treated with isavuconazole. Due to rapid deterioration of the patient's general condition, tisagenlecleucel was given without prior lymphodepleting chemotherapy. It was well-tolerated with only grade I CRS and no neurotoxicity. Follow-up imaging showed clearance of the hepatic and pulmonary lesions; the intracerebral lesions remained unchanged. BM puncture on Day + 28 showed molecular remission of the CD19+ clone; however, as expected, the CD19- clone persisted with an MRD level of 2 x 10- by PCR.
The presence of the CD19- clone led us perform the second allogeneic HSCT very soon after tisagenlecleucel infusion (Day + 59), using the patient's haploidentical mother. Due to stage IV renal insufficiency, the patient received a reduced-intensity dose-adjusted chemoconditioning with fludarabine, treosulfan and thiotepa under concomitant CVVHDF. Chemoconditioning was a reduced-toxicity regimen based on the For Omitting Radiation Under Majority age (FORUM) study in paediatric ALL.   Post-transplant cyclophosphamide, mycophenolate mofetil and tacrolimus were used as GvHD prophylaxis. Overall, the second allogeneic HSCT was well-tolerated. BM on Day + 28 showed full donor chimerism; however, the CD19- leukaemic clone persisted with an MRD level of 1 x 10- by PCR. Thus, immunosuppression was reduced to the lowest possible level. Three months after allogeneic HSCT, MRD by PCR in the BM was negative for the first time.
Secondary graft failure prompted us to administer a stem cell boost (CD3/19 and alpha/beta depleted PB stem cells) on Day + 107, leading to stable engraftment. Unfortunately, a second isolated BM relapse of the patient's CD19+ BCP-ALL occurred on Day + 180. After a profound discussion with the patient and her family regarding her dismal prognosis, a mutual decision for another CAR T-cell infusion, using allogeneic T-cells transplanted at the second HSCT, was reached and induced a third CR at the time of writing.