ABSTRACT:

Background: Several commercial and academic autologous chimeric antigen receptor T-cell products targeting CD19 have been approved in Europe for relapsed/refractory B-cell acute lymphoblastic leukemia, high-grade B-cell lymphoma, and mantle cell lymphoma. Products for other diseases such as multiple myeloma and follicular lymphoma are likely to be approved by the European Medicines Agency in the near future.

Design: EBMT-JACIE and the European Haematology association (EHA) proposed to draft best  practice  recommendations  based  on  the  current  literature,  to   support  healthcare professionals in delivering consistent, high-quality care in this rapidly moving field

Results: Thirty-six CAR-T experts (medical; nursing; pharmacy/laboratory) assembled to draft recommendations to cover all aspects of CAR-T patient care and supply chain management, from  patient  selection  to  long-term  follow-up,  post-authorisation  safety  surveillance  and regulatory issues.

Conclusions: We provide practical, clinically relevant recommendations on the use of these high-cost,  logistically  complex  therapies  for  hematologists/oncologists,  nurses  and  other stakeholders including pharmacists and health sector administrators involved in the delivery of CAR-T in the clinic.

KEYWORDS:

CART-cells (CAR-T), B-cell acutelymphoblastic leukemia (B-ALL),adult relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL),Multiple myeloma (MM), Mantle cell lymphoma (MCL), follicular lymphoma (FL), cytokine release syndrome (CRS), Macrophage activation syndrome (MAS), immune effector cell-associated neurotoxicity syndrome (ICANS), JACIE, long-term follow-up (LTFU).

INTRODUCTION:

CD19   chimeric   antigen   receptor   T-cell    (CAR-T)   therapeutics   are   widely   used   for relapsed/refractory (r/r) B-cell malignancies including acutelymphoblastic leukemia (B-ALL), large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL)1-3. CAR-T are also under evaluation in multiple myeloma, acute myeloid leukemia and solid tumors4,5 .

Three CAR-T products are licensed: Tisagenlecleucel (Kymriah®, Novartis) for r/rpaediatric B-ALL and adult LBCL; Axicabtagene ciloleucel (Yescarta®, Gilead), for r/r adult LBCL and Tecartus (brexucabtagene autoleucel), for r/r adult MCL. In the US, lisocabtagene maraleucel (Liso-Cel, BMS) is approved for r/r LBCL andidecabtagenevicleucel [ide-cel, Abecma]6  and JNJ-45287 have been approved for r/rmultiple myeloma4. In Spain, regulators have approved academic CD19 CAR-T (ARI-0001) for r/r B-ALL8.

CAR-T   confers   a   risk    of   potentially   life-threatening    immunological   toxicities    and comprehensive training of personnel involved in CAR-T delivery, including intensive care unit (ICU) and neurology specialists, is key9.

Post-marketing pharmacovigilance over 15 years post-infusion is mandated by EMA to ensure ongoing evaluation of efficacy and safety of licensed CAR-T in the real-world setting via the EBMT registry. CIBMTR (Center for International Blood and Marrow Transplant Research) fulfils a similar role. Further, the PASS (post-authorization safety studies) initiative makes an assessment of the value of CAR-T in relation to standard-of-care treatments.

METHODOLOGY

In 2021, EBMT and EHA proposed an expanded revision of the 2019 EBMT-JACIE CAR-T guidelines10,11 . Thirty-six CAR-T experts (medical; nursing; pharmacy/laboratory) assembled to draft recommendations based on the current literature, to reflect current best practice in this rapidly moving field and to support healthcare professionals in delivering consistent, high- quality care. Given the absence of randomized trial evidence, a decision was made not to grade these recommendations. They therefore represent the consensus view of the authors.

The recommendations principally apply to licensed CAR-T therapies. For CAR-T clinical trials, healthcare teams should follow relevant trial protocols.

PATIENT ELIGIBILITY

Patient eligibility should be assessed by a CAR-T centre multi-disciplinary team including cellular therapy and haematology/oncology disease specialists. Medical history, performance status and CAR-T product should be considered with respect to tolerability (Table 1).

Table 1. Patient eligibility criteria for CAR-T

SCREENING LABORATORY TESTS AND IMAGING

To ensure patient eligibility and fitness, the screening tests in Table 2 should be considered. This list is not exhaustive, and, in the trial setting, trial protocols should be followed.

Table 2. Screening tests prior to CAR-T therapy. Key. NHL: Non-Hodgkin Lymphoma; ALL: acutelymphoblastic leukemia;  ULN: upper limit of normal;  TTE: transthoracic echocardiogram; LVEF: left ventricular ejection fraction; ECG: electrocardiogram;  CMR: cardiac magnetic resonance; PMBCL: primary mediastinal B cell lymphoma; MRI: Magnetic resonance  imaging;  CNS: central nervous system;  VoD:  veno-occlusive disease; GvHD: graft versus host disease.

WORK-UP FOR LEUKAPHERESIS

Leukapheresis procurement in the European Union must comply with the Tissue and Cell Directives (2004/23/EC; 2006/17/EC; 2006/86/EC). Shipment across borders requires current viral serology and compliance with regulations in both the countries of origin and destination 13,14

.

A pre-leukapheresis checklist and suggested washout periods for pre-leukapheresis therapeutics are listed in Tables 3 and 4.

Table 3. Checklist prior to leukapheresis

Table 4. Washout period before leukapheresis [adapted from Kansangra et al, BBMT 201915] Key. Allo-SCT: allogeneic stem cell transplantation; GvHD: graft versus host disease; DLI: donor lymphocyte infusion; ALC Absolute Lymphocyte Count.

PERFORMING LEUKAPHERESIS

To be a CAR-T delivery site, accreditation with FACT-JACIE is recommended. Pharmaceutical providers and health service commissioners may have additional requirements.

CAR-T product prescription/order and non-mobilised leukapheresis 16,17  scheduling/shipping, is  co-ordinated  with  the  CAR-T  manufacturing  facility  (often  via  proprietary  web-based platforms),

Most manufacturers stipulate storage of fresh leukapheresis at 2-8 °C prior to shipping. Novartis additionally accept locally cryopreserved starting material (within 30 months).

Accredited, validated leukapheresis testing methods should be compatible with manufacturer’s requirements   and   authorizations.   An   absolute   lymphocyte   count   (ALC)   threshold   of 0.2x109/L17,18    is   generally  recommended19,20,  but   emerging  evidence   supports   CAR-T leukapheresis in paediatric and adult patients with low ALC21,22 .

Infectious  disease  markers  must  be  tested  on  peripheral  blood  (PB)  within  30  days  of leukapheresis (with results available on the day of shipment). Microbial contamination is rare and the presence of leukemic blasts is acceptable to manufacturers.

Based on the observation that T-cells suffer qualitative damage from chemotherapy, feasibility of pre-emptive  leukapheresis  in  high-risk  patients  is  currently  being  explored,  but  with significant regulatory, infrastructural, and cost implications.

BRIDGING THERAPY

‘Bridging’  therapy,   administered  in  the   4-6  weeks  between   leukapheresis  and  CAR-T admission, aims to reduce disease burden and in doing so, improve intention-to-treat and reduce CAR-T immunotoxicity23-25.

Patient-specific  bridging  recommendations  should  be  made  by  a  multi-disciplinary  team following review of response to prior therapy, overall tumour burden, and anatomical sites of disease. Bridging can be omitted if the CAR-T ‘vein-to-vein’ time is short and the disease

burden is low. Otherwise, bridging is broadly split into: (a) high-dose chemotherapy; (b) low- dose chemotherapy; (c) radiotherapy; and (d) novel agents/approaches. Radiation therapy can be an effective bridge, but the impact on circulating lymphocytes should be considered if radiation  is  commenced prior to  leukapheresis.  Further  studies  are  warranted  to  optimise bridging approaches26.

Bridging can be delivered at the CAR-T centre or at the referring center, provided there is clear communication regarding the selected bridging strategy, management of complications, and scheduling of bridging in relation to CAR-T admission, fastidiously observing recommended washout periods (Table 5).

Table 5. Washout period between the bridging therapy and the onset of LD conditioning [Expert opinion]. Key:

TKI: tyrosine-kinase inhibitor.

HOSPITALIZATION

Outpatient CAR-T administration can be done safely, provided clear policies, appropriate infrastructure,  well-trained  staff,  and  capacity  for  24/7  hospitalization  in  the   event  of complications is in place14. As such facilities are not available in most European centers, we recommend that patients remain hospitalized for at least 14 days following infusion (Table 6).

Table 6. Recommendations on hospitalisation in the first 28 days after CAR-T infusion.

LYMPHODEPLETING CONDITIONING (LD)

LD acts to enhance CAR-T proliferation by modulating cytokine and immune pathways. Fludarabine and cyclophosphamide (FC) is widely used27. Fludarabine dosing is consistent between  products  and  indications  (25-30  mg/m2/day  x3  days)  whilst  cyclophosphamide schedules differ28. Bendamustine (+/- fludarabine) has been tested in CD30CAR-T for Hodgkin Lymphoma as an alternative to FC29.

LD is administered following CAR-T product release, the week prior to CAR-T infusion with a minimum of two rest days. Where CAR-T infusion is delayed by ≥4 weeks, repeat LD is recommended, with consideration given to patient fitness, blood counts and prior fludarabine exposure10.

Potential  complications  from  LD  include  pancytopenia,  immunosuppression,  infection, neurotoxicity, haemorrhagic cystitis, pericarditis and secondary malignancy. Renal or hepatic impairment should prompt appropriate dose modification. Considerations prior to LD are outlined in Table 7.

Table 7. Checklist before starting LD conditioning. Key. LD: lymphodepletion;  WBC: white blood cell count; ALC: absolute lymphocyte count; ULN: upper limit of normal; VoD: veno-occlusive disease; GvHD: graft versus host disease; TTE: trans-thoracic echography; ECG: electrocardiogram

PRODUCT RECEIPT

Oversight and responsibility for this process varies nationally. Country-specific guidance is beyond the scope of this document. Centers should have regulatory approval for storage of genetically modified organisms (GMOs).

Transport tracking on the manufacturer’s website enables the date and time of product shipment to be known in advance. At receipt, checks should include: (1) inspection of the dry shipper seal for breaches; (2) review of the temperature log throughout transportation; (3) inspection of product integrity; (4) CAR-T identity label checks, prior to completion of receipt forms. In the  event  of  non-conformance,   cells   are  quarantined,  and  the  hospital   delegate  and manufacturer should be immediately informed. Back-up bags are sometimes available as a replacement for defective products.

Out-of-specification (OOS) CAR-T may still be used (exceptions include microbial/noxious contamination), provided the release certificate lists the OOS details, written agreement is provided from the manufacturer (with acceptance of responsibility by both manufacturer and physician), and the patient has given written consent30-32.

THAWING AND INFUSION

Prior to thawing and infusion, patients are medically assessed to ensure they are fit to proceed (Table 8); identity and consent is confirmed; the prescription reviewed, and vital signs and IV access (central venous catheter or a newly inserted and pre-tested peripheral cannula) checked at the bedside. Pre-medication with paracetamol and antihistamine (NOT corticosteroids) is recommended. No concurrent medication should be administered during CAR-T infusion.

Product thawing is performed in a pharmacy clean room, cell therapy unit or patient bedside, double wrapped in a watertight plastic bag, using thawing devices according to manufacturer’s instructions and local regulations (automated thawing device, 37±2°C water bath, or dry-thaw method). CAR-T is stable at room temperature for 30-90 minutes after thawing33.

CAR-T should be administered rapidly after thawing during working hours by competent medical or nursing personnel13,14 . Vital signs should be assessed and recorded before, during and following infusion. Using aseptic non-touch technique (ANTT), cells in vials are drawn up into a syringe to be administered as a slow bolus. CAR-T infusion bags are infused through a non-filtered giving set.   Fluid infusion sets with sub-micrometre bacterial filters and blood transfusion sets with leukocyte depletion filters should NOT be used for CAR-T infusion.

Infusion reactions are rare but should be treated symptomatically. Corticosteroids should be avoided unless the patient becomes critically unwell.

Following infusion, the vial/bag and giving set should be disposed of as a GMO biohazard in compliance with institutional policies and country-specific regulations.

Table  8. Potential complications to be ruled out before product thawing and CAR-T infusion. Key. LD: lymphodepletion

SHORT-TERM COMPLICATIONS: ADMISSION TO DAY +28

Tumour lysis syndrome: TLS can occur following LD/CAR-T, and should be prevented and managed with standard local protocols.

Infection: Active infection should be controlled prior to initiation of LD.  Following LD, all patients will be neutropenic. A fever should prompt empiric antimicrobial therapy (based on institutional protocols) and investigation with blood and urine cultures; chest x-ray and/or high- resolution CT chest, when indicated; respiratory viral screening including COVID-19 and other tests such as influenza A PCR testing; cytomegalovirus (CMV) and Epstein-Barr virus (EBV) nucleic acid testing (NAT); and lumbar puncture and brain MRI in selected cases. The specific risk profile of the patient (duration of neutropenia, prior allo-HCT, previous infections, and local  antibiotic  resistance  profiles)   should  guide  diagnostic  work-up  and  selection  of antimicrobial agents.

Cytokine release syndrome (CRS): CRS affects 30-100% of all patients, with ≥grade 3 CRS reported in 10-30%34. Incidence depends on the CAR-T product, disease characteristics and CRS grading system used. Typical onset is between  1-14  days post-CAR-T  infusion and duration is commonly between 1-10 days. Rare delayed cases are reported9.

CRS is characterised by fever ≥ 38°C, hemodynamic instability and hypoxemia. Severity is graded according to the ASTCT consensus criteria (Figure 1)35  and the differential diagnosis includes neutropenic sepsis. Empiric, broad-spectrum IV antibiotics should be commenced.

CAR-T activation leads to effector cytokine release (IFN-γ, TNF-α, IL-2) which can trigger pro-inflammatory cytokine release (IL-1, IL-6, IFN- γ, IL-10 and MCP1), with increased CRP and hyperferritinemia24. Risk factors for high-grade CRS include tumor burden, concurrent infection, CAR-T dose and product, and LD conditioning intensity36.

CRS management combines symptomatic measures (antipyretics, fluids) with tocilizumab (IL6 receptor antagonist) +/-corticosteroids10. When two doses of tocilizumab (8mg/kg) fail to control CRS, dexamethasone should be administered. Tocilizumab should be used earlier in older, co-morbid patients. Early/prophylactic tocilizumab has been studied in CRS but there is insufficient data to date to support a formal recommendation of this approach.

Clinicians should be vigilant for occult sepsis emerging post-tocilizumab. Gastrointestinal perforation has also been reported37. Corticosteroids should be subject to rapid taper once CRS is controlled.

If CRS does not respond to tocilizumab/corticosteroids, alternative therapeutic options include siltuximaband anakinra, but limited clinical data is available38. There should additionally be a high  index  of  suspicion   for  underlying/concurrent  infection  or  macrophage  activation syndrome (MAS). A suggested CRS management algorithm is shown in Figure 1.

Figure 1: Algorithm outlining the grading and management of CRS

Macrophage  Activation  Syndrome  (MAS):  Persistent  fever  despite  tocilizumab  with organomegaly,  cytopenias  (+/-  hemophagocytosis  in  the bone marrow), hyperferritinemia (>10,000ng/mL),     liver     dysfunction,     coagulopathy      (hypofibrinogenemia     requiring cryoprecipitate/fibrinogen concentrate) and hypertriglyceridemia, favors a CRS/MAS overlap syndrome rather than CRS39.

Patients should be monitored with twice daily blood tests (WBC, liver function, ferritin, CRP) and treated with anakinra, a recombinant humanised IL-1 receptor antagonist, in combination with corticosteroids40  (Figure 2). In refractory CRS/MAS, chemotherapy can be used, albeit there is  a  lack  of data  and  a  high risk  of ablating CAR-T. Where there is neurological involvement, intrathecal chemotherapy can be considered41,42 .

Figure 2: Management of CRS/MAS (expert opinion based on literature review) 38-42

Immune effector cell-associated neurotoxicity syndrome (ICANS): ICANS affects 20-60% of CD19CAR-T patients (grade ≥3, 12-30%). Onset is typically three to five days after CAR- T but can occur concurrently with/shortly after CRS, and 10% of patients develop ‘delayed ICANS’ more than three weeks after infusion43,44 . Classical ICANS is also reported, to a lesser extent, with CD22- and BCMA-targeting CAR-T4. On the CARTITUDE study of LCAR- B38M,   a   series   of  movement/neurocognitive   disorders/nerve   palsies/peripheral   motor neuropathies have been observed (12% of cases), not temporally associated with CRS, that are of later onset (median day 27), and take longer to resolve (median 75 days)45. These will require ongoing evaluation in clinical trials. ICANS is rarely reported in solid tumor CAR-T studies.

The pathophysiology of ICANS is likely to be due to a combination of inflammatory cytokines increasing  vascular  permeability;  endothelial  activation  leading  to  blood-brain  barrier breakdown;  increased  CSF  cytokines  and  in  some  cases  leading  to   cerebral  edema40. Pharmacokinetics indicate that greater, earlier CAR-T expansion in vivo correlates with higher ICANS risk46. Risk factors for ≥ grade 3 ICANS include CD28-CAR-Tproducts, higher CAR- T doses, high disease burden, pre-existing neurological conditions, low platelet count, and early, severe CRS47. High fever (≥38.9°C) and hemodynamic instability within 36 hours of CAR-T infusion predicts for severe ICANS with high sensitivity48.

Symptoms include tremor, confusion, agitation, and seizures. Dysphasia, hesitant speech and deterioration  in  handwriting  is  prominent  and  can  progress  to  expressive  and  receptive aphasia48. Routine anti-convulsant prophylaxis is not recommended except in high-risk cases. Fatal cerebral  edema has been described48. Late psychiatric presentations have also been reported49.

ICANS is a clinical diagnosis, but MRI brain and CSF examination can exclude alternative diagnoses10. Electroencephalogram (EEG) can be normal but can also demonstrate slowing and non-convulsive  status  epilepticus.  Diagnostic  work-up  should  include  CT  head,  clotting screen/fibrinogen and EEG, MRI, and lumbar puncture (LP) in severe ICANS, or steroid- refractory cases.

Duration and frequency of ICANS monitoring should be conducted as per product label/trial protocol. The ten-point Immune Effector Cell Encephalopathy (ICE) score in adults (Table 9) and the CAPD assessment in children (Table 10) is usually performed twice daily. ICANS grading integrates the ICE/CAPD scores into an overall assessment of neurological function (Figure 3).

Management is supportive for grade 1 ICANS. Corticosteroid therapy with a rapid taper is indicated for grade ≥2 ICANS and ICU transfer should be considered (Figure 3). Evidence suggests that steroids do not impact CAR-T efficacy, although longer courses can be associated with  shorter  PFS50.  Seizures  are  treated  with  levetiracetam  and  status  epilepticus  with benzodiazepines. There is no clear therapeutic role for tocilizumab in ICANS and it has been suggested that it may contribute to ICANS through increase circulating IL-646. In the specific setting of grade 1 CRS with concurrent ≥ grade 2 ICANS, it is appropriate that steroids (and not tocilizumab) be administered, but this does not apply in higher grade CRS. Alternative agents include siltuximab and anakinra, but clinical data on their utility in ICANS is limited. A management algorithm for ICANS is shown in Figure 3.

Table 9. ICE score for neurological toxicity assessment. Adapted from Lee et al48

Table 10. CAPD for encephalopathy assessment in children < 12 years, Adapted from Traubeet al51,52.

Figure 3: Grading and management of ICANs

Cardiovascular    Toxicity:10-20%     of    CAR-T     patients     experience     cardiovascular complications53. Hypotension requiring vasopressor support was the main cardiac complication observed in pediatric CAR-T studies, but arrhythmias, myocardial impairment, left ventricular systolic dysfunction (LVSD), decompensated cardiac failure, and cardiovascular death are reported54.

Risk factors for CAR-T-cardiotoxicity include ≥ grade 2 CRS55, more often in high disease burden  and  patients  with  pre-existing  cardiac   dysfunction  following  prior  exposure  to cardiotoxins including anthracyclines, radiation, and tyrosine kinase inhibitors.

Thorough  pre-CAR-T  cardiovascular  assessment  with  appropriate   surveillance  and  risk reduction  strategies  may  reduce  CAR-T-cardiovascular  complications.  Elevated  baseline serum cardiac biomarkers (troponin, N-terminal-pro-brain-natriuretic-peptide (NT-proBNP), may signal a greater risk of CAR-T-cardiotoxicity. Electrocardiograph (ECG) will exclude underlying   arrhythmias   and   QT   interval    (as   a   surrogate   for   cardiac   repolarisation abnormalities)  and transthoracic  echocardiography  (TTE)  defines baseline  left ventricular ejection  fraction  (LVEF)  and diastolic  function to identify pre-existing LVSD  (using 4D volumetric, 2D Simpson’s Biplane, and global longitudinal strain assessment tools). Cardiac magnetic resonance (CMR) can be considered in the setting of poor image quality, including patients with pericardial/myocardial involvement on PET to assess lymphomatous infiltration.

On admission, baseline ‘dry weight’ and daily weights should be recorded, where weight increase as a surrogate for fluid overload should prompt repeat cardiac assessment with serum biomarkers, ECG, TTE and cardiologist review.

Tocilizumab is also associated with rapid improvement in cardiovascular complications56. One retrospective analysis showed a 1.7-fold increased risk of CAR-T-cardiotoxicity with each 12- hour delay intocilizumab administration from CRS onset55. Current experience suggests that CAR-T-cardiotoxicity is an early, largely reversible phenomenon, with rare LVSD beyond 6 months and no late cardiovascular effects at one year49.

Laboratory testing:  CRP,  fibrinogen,  liver  function tests  and  ferritin  are  checked  daily. Cytokine  testing  is  not  routinely  performed  in  most  centers.  Atypical  lymphocytes  that resemble   leukemic   blasts    are   not    uncommon   at    peak   CAR-T    expansion.   Repeat microbiological testing and imaging to exclude infection is recommended in febrile patients.

MEDIUM TERM COMPLICATIONS: DAY +28 TO DAY +100

Delayed TLS/CRS/ICANS: Although rare, delayed events can occur and should be managed according to standard protocols (Figures  1, 2 and 3). Table 11 outlines recommended testing during this period to monitor for complications.

Table 11. Patient monitoring during medium-term follow-up. Abbreviations. FBC: full blood count; CMV: cytomegalovirus; EBV; Epstein-Barr virus; IV: intravenous; SC: subcutaneous.  * Additional tests and imaging can be performed as clinically indicated.

Infections  and  antimicrobial  prophylaxis:  Opportunistic  infections  are  common  and prophylaxis  is  warranted  until  immune  reconstitution  (Table   12)43.    Risks  include  prior autologous/allo-HCT, bridging therapy, and steroids/tocilizumab for CRS/ICANS. Prolonged neutropenia, CD4 T-cell lymphopenia and B-cell aplasia/hypogammaglobulinemia (affecting up to 46% of patients at Day +90) also contribute. Neutropenia beyond day +30 and beyond

day +90 affects 30% and 10-20% of patients, respectively. Prolonged CD4 T-cell lymphopenia resolves to >200/µL by 1 and 2 years in 65% and 86% of patients, respectively57.

Most early infections (first 30 days) are bacterial, or respiratory viral infections. Invasive fungal infections are rare. Risk factors include B-ALL with prior allo-HCT; prior fungal infection and prior long-term/high-dose steroid exposure58.

Beyond Day +30, viral infections predominate. HSV and VZV reactivation is uncommon in patients on valaciclovir prophylaxis. CMV, EBV, adenovirus, HHV6, BK-virus and JC-virus reactivation are rare and routine monitoring is not advised, except in high-risk patients (allo- HCT; high-dose/long-term corticosteroids)59. COVID-19 is a formidable challenge in CAR-T patients and consortium guidance on how to maintain CAR-T delivery through the pandemic is available.

Evidence suggests that CAR-T manufacturing is feasible in HBV, HCV and HIV infection and treatment is safe provided the virus is undetectable before apheresis and prior to starting LD. In    hepatitis    B    infection     (especially    if    HBsAg+     and    HBV-DNA+),    long-term entecavir/tenofovir/equivalent prophylaxis is recommended.

Table 12. Infection prophylaxis post-CAR-T. Abbreviations.  G-CSF: granulocyte colony stimulating factor; CRS:  cytokine  release syndrome: LD:  lymphodepleting  conditioning; NCCN:  The National  Comprehensive Cancer Network, allo-HCT: allogeneic hematopoietic cell transplantation.

* A negative impact of G-CSF applied earlier after CAR-Thas not been clearly demonstrated; a recent report starting G- CSF on day 5 after CAR-T infusion showed no increase in CRS orICANS, indicating that earlier application may be safe and may reduce duration of neutropenia. Further data to support this observation is required.60

**  In  patients  with  neutropenic  fever,   empiric   treatment  with  broad-spectrum  antibiotics  is  strongly recommended.

B-cell aplasia & hypogammaglobulinemia: Following CAR-T, B-cell aplasia is associated with sino-pulmonary infections and should be measured regularly10. 83% of paediatric B-ALL patients on the ELIANA study had ongoing B-cell aplasia at 6 months. In ZUMA-1 responders, 25% had ongoing B-cell aplasia at 12months.

Due to immunological immaturity, immunoglobulin replacement is routine in pediatric CAR- T. In adults, long-lived plasma cells following CD19 CAR-T may confer an immune-protective effect, but a common approach is immunoglobulin replacement for hypogammaglobulinemia (<4g/L) with serious or recurrent/chronic infections. Data on efficacy of immunoglobulin replacement in CAR-T is limited and current recommendations are mainly extrapolated from primary immunodeficiencies (e.g., Bruton’s agammaglobulinemia). One study showed that increased serum IgG led to significantly less sinopulmonary infection post-CAR-T61.

Immunoglobulin replacement aims to maintain serum levels above 4 g/L in adults and within age-adapted normal ranges for children, titrated to the incidence of breakthrough infection.

Intravenous immunoglobulins (IVIGs) (0.4g/kg) and subcutaneous (SCIGs) (0.1–0.15g/kg) are administered 3-6 weekly, and weekly, respectively. After reaching steady state, levels should be measured three monthly.

Cessation of immunoglobulin replacement should be guided by recovery offunctional B-cells. This also provides a surrogate for functional CAR-T persistence and maybe useful in clinical decision making, particularly in B-ALL.

Vaccinations: General guidance is outlined in Table  13, applicable to adults and children. Incomplete immune reconstitution or ongoing immunosuppression confers a high likelihood of lower vaccine responses (including COVID-19), but consensus view is that vaccination may reduce infection rates and improve clinical outcome. Recommendations and adherence to national schedules require individualized assessment based on infection history and laboratory assessments of cellular/humoral immunity, where available. Specific antibody responses to vaccination should be assessed where possible. A recent analysis in adults following CD19- and BCMA-CAR-T indicated that despite hypogammaglobulinemia, CD19-CAR-T patients developed seroprotection comparable to the general population (with the exception of specific pathogens e.g., pneumococcus), but that in BCMA-CAR-T patients, fewer pathogen-specific antibodies  were   detected62.  This  highlights   the  need   for  vaccine   and  immunoglobulin replacement studies in this at-risk population. Further analysis of T-cell vaccine responses is also warranted in this group.

Table 13. Eligibility Criteria for Vaccination inpatients receiving CD19-targeted CAR T-cell therapy [adapted from Hill and Seo. 63.  * Absolute  CD4 T cells  > 0.2x109/L, CD19 or CD20 positive B-cells > 0.2x109/L, no concomitant immunosuppressive or cytotoxic therapy

Graft-versus-Host Disease  (GvHD):  CAR-T post-allo-HCT  is  generally  considered  safe without  increased  risk  of  high-grade  GvHD.  However,  a  recent  publication   describes histologically confirmed GvHD in 4 pediatric patients post-KymriahTM.64  GvHD post-CAR-T should be diagnosed and managed using standard protocols, balancing the benefits of systemic immunosuppression against the adverse impact on CAR-T viability.

Delayed cytopenias: Haematological toxicity has a cumulative one-year-incidence of 58%, post-CD19-CAR-T, is often prolonged and can follow a biphasic temporal course, with initial neutrophil recovery followed by a ‘second dip’65.

Duration and severity vary between products and indications, but there is a high incidence of persistent grade ≥3 neutropenia (30-38%), thrombocytopenia (21-29%), and anemia (5-17%) after Day +2866-69. Risk factors include baseline cytopenias, pre-treatment bone marrow disease burden, an inflammatory state, prior allo-HCT within one year, and severe CRS/ICANS65,70 . Protracted cytopenias are less pronounced in BCMA- and solid tumor targeting CAR-T.6,68

The pathophysiology remains poorly understood and there may be product-intrinsic and/or disease-specific     factors.      Investigations      should     consider      hematinic      deficiency, myelosuppressive medications (co-trimoxazole), and viral infections (HHV6,Parvovirus B19). Bone  marrow  biopsy  may  be  useful  beyond   day  28  to   exclude   recurrent   disease, hemophagocytosis and rarely, myelodysplasia.

G-CSF can be used for severe neutropenia (<0.5 x 109/L) from day +14 onwards, following resolution of CRS/ICANS. Recent data on earlier prophylactic G-CSF found no effect on immunotoxicity, CAR-T expansion, or prognosis.60

G-CSF-refractory neutropenia (ANC <100/µl) lasting ≥30 days affects 5-10% of patients, with a risk of fungal infection71. Autologous stem cell rescue is an option, where cells are available72 and donor-derived, unconditioned CD34+-selected ‘top-up’ can be considered in post-allo- HCT patients73.  Anti-inflammatory therapies (dexamethasone) and EPO/TPO-agonists may have a role. Allo-HCT is the last resort inpatients with refractory cytopenias.

LONG-TERM FOLLOW-UP (LTFU): FROM DAY +100

LTFU should be conducted by a multi-disciplinary team (CAR-T physicians; disease-specific specialists; LTFU nursing staff; data managers; clinical trial staff) to capture disease status and late effects14.

Prolonged cytopenias, hypogammaglobulinemia and infections  are common. Neurological complications  and  pulmonary  toxicity  confer  an  increased  mortality  risk.   Secondary malignancy is rare: a single report of relapse following transduction of a leukemicB-cell during the manufacturing process is described74 and a case of myelodysplastic syndrome was reported

in the ZUMA-1 trial.  A recent publication described CAR-T-derived malignancies following genome-edited CD19-CAR-T due to insertional mutagenesis75. This area requires ongoing surveillance.

CAR-T centres should liaise with referral centres, providing protocols and policies for LTFU, to sustain shared care arrangements.

A recommended LTFU schedule of attendance and testing schedule are outlined in Tables 13 and 14.

Table 13. Recommended minimum frequency of attendance at CAR-T centre for patients in remission for Late Effect monitoring.

Table 14. Recommended tests to be performed at LTFU clinic. Key: PB:peripheral blood; NPA: nasopharyngeal aspiration. *Equivalent test methods for other immune effector cells as they become available.