We report the case of a 19-year-old female diagnosed with Ph-negative B-cell ALL in February 2019, who presented with hyperleukocytosis (WBC count 317 x 109/L). Flow cytometry on a bone marrow (BM) aspirate showed that 93% of cells were positive for CD45, CD10, CD19 and CD22, and had an aberrant expression of CD33. FISH performed using the Cytocel probe detected a deletion at 6q21/SEC63 in 43.5% of the analysed nuclei. The BCR::ABL1-like predictor was positive and showed a CRLF2 upregulation and an IKZF1 deletion. Molecular-cytogenetic analysis, performed using the ZytoLight  SPEC CRLF2 Dual Color Break Apart probe, and the LSI IGH Dual Color, Break Apart Rearrangement Probe (Vysis-Abbott) showed hybridization patterns consistent with the presence of an IGH : CRLF2 rearrangement. Targeted RNA sequencing detected no mutations or rearrangements.
The patient was enrolled in the chemo-immunotherapy GIMEMA LAL2317 protocol, which exploits a risk-oriented strategy based on disease characteristics and MRD evaluation at fixed time-points, intercalating a maximum of two cycles of blinatumomab into a pediatric-like chemotherapy backbone (clinicaltrial.gov NCT03367299).
Our patient, classified as very high risk, underwent three cycles of chemotherapy, obtaining a complete morphologic remission (CR) after induction. Central nervous system (CNS) prophylaxis was carried out as per protocol. MRD assessment by RQ-PCR Ig gene rearrangement remained strongly positive (>10-2) after all three chemotherapy cycles. After a single cycle of blinatumomab which induced the molecular remission (<10-5), the patient underwent an allogeneic hematopoietic stem cell transplant (HSCT) from an HLA-identical sister. Conditioning consisted of treosulfan, fludarabine and TBI 4 Gy, and the graft versus host disease (GvHD) prophylaxis included post-transplantation cyclophosphamide and sirolimus. A post-transplant aspirate documented a CR with full donor chimerism, FISH and a molecular MRD negativity. Sirolimus was discontinued six months later. The patient never developed GvHD.
In January 2021, 18 months after the HSCT, a BM evaluation detected a relapse (5% blasts). The patient had also a palpable mass in her right breast, whose histology was compatible with an ALL localization. No CNS disease was detected.
The patient was deemed fit for anti-CD19 CAR-T cell therapy with tisagenlecleucel. After lymphapheresis in early February, we started a bridging treatment with ponatinib 45 mg daily for 30 days on compassionate use and 1 mg/kg prednisone for 14 days. No cardiac, hepatic or hematologic toxicity was reported. In mid-February, we repeated a BM aspirate that confirmed a morphologic relapse (12% blasts). A third aspirate after a month of ponatinib showed a stable disease (8% blasts) and on physical examination a reduction of the palpable breast nodule was documented. We withdrew bridging drugs and started lymphodepletion with fludarabine-cyclophosphamide, followed by a CAR-T cell infusion on March 2021. The patient developed a grade 4 neutropenia and received three doses of tocilizumab for grade 1 cytokine release syndrome (CRS). No neurotoxicity occurred. Three months later the patient was in CR with a full donor chimerism and a MRD negativity. A breast ultrasound revealed a regression of her nodule. In June 2021, maintenance with ponatinib at a lower dose (15 mg/day) was initiated. In November 2022, 20 months after the CAR-T cell infusion the patient is in good clinical conditions and in persistent molecular CR. She still receives ponatinib maintenance with excellent tolerance, except for a 10-day discontinuation due to a transient G4 neutropenia.  Figure 1  summarizes the case timeline. We longitudinally monitored the patient's CAR-T cell expansion and their subsequent persistence by flow cytometry and plotted the data over time in  Figure 2A . After a marked expansion peak at day 7 after infusion (1819.6/mcl), CAR-T cell counts decreased, though persisting over time. At late time-points (day 180 and 270), the patient still had circulating anti-CD19 CAR-T cells (4.0 and 4.3/mcl, respectively). At the last available follow-up (365 days, April 2022), circulating CAR-T cells were no longer detectable. However, a concomitant B-cell aplasia is ongoing and we are monitoring it as a decisional tool for future treatment. Peak levels of inflammatory cytokines and chemokines occurred within the first week after the CAR T-cell infusion, with particularly high levels of monocyte chemoattractant protein-1 (MCP-1), interferon gamma-induced protein 10 kDa (IP-10), interleukin-6 (IL-6) and interferon-gamma (IFN- gamma,  Figure 2B ).