Unlocking Sanctuary Sites: The Efficacy of Brexucabtagene Autoleucel in Ocular B‐<scp>ALL</scp> Relapse
Authors: Ludovica Calabretta, Patrizia Zappasodi, Carlo Di Biase, Cristina Picone, Elisa Roncoroni, Marianna Rossi, Antonio Bianchessi, Irene Defrancesco, Caterina Zerbi, Beatrice Ferrari, Erica Consensi, Gianluca Martini, Claudia Patricia Tobar Cabrera, Claudia Battista, Alessandro Mazzacane, Francesco Romano, Giulia Losi, Alessia Taurino, Luca Arcaini, Nicola Polverelli
Journal: American Journal of Hematology (2024)

A 52-year-old male with Philadelphia-negative (Ph−), CD20-positive B-cell acute lymphoblastic leukemia (B-ALL) achieved measurable residual disease (MRD)-negative complete remission (CR) after immunochemotherapy according to the Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA) LAL 1913 regimen (consisting of vincristine, cyclophosphamide, idarubicin, pegylated-asparaginase, cytarabine, methotrexate, 6-mercaptopurine and rituximab), and CNS-directed prophylaxis with periodical medicated lumbar punctures with intrathecal cytarabine and methotrexate [1]. During standard 2-year maintenance therapy with 4-week cycles of cyclophosphamide, 6-mercaptopurine (6-MP) and methotrexate (MTX) alternating with vincristine, 6-MP and MTX, the patient complained of recurrent episodes of conjunctivitis. Conjunctivitis in a patient with B-ALL undergoing active treatment is worrisome and can be caused by either non-infectious or infectious agents. The former include leukemic infiltration and chemotherapy side effects; however, these options seem less probable. First, our patient's disease was in MRD-negative CR; second, antiblastic drugs (e.g., 6-MP and MTX) which he took during maintenance therapy, rarely cause conjunctivitis. Due to his immunocompromised state, an infectious etiology is more probable. I would ask for an ophthalmologic examination with detailed history, thorough clinical examination, and appropriate diagnostic testing. The comprehensive ophthalmologic evaluation was normal; after a course of empirical topical antibiotic treatment, the symptoms resolved, and he resumed maintenance therapy. Unfortunately, the patient experienced a first relapse with MRD-positivity in the bone marrow (BM) during his seventh cycle of maintenance therapy. No CNS involvement was detected by lumbar puncture. In adults with standard-risk B-ALL who achieved MRD-negative CR, the relapse rate is roughly 30% and it occurs mainly within 2 years from diagnosis [2]. Our patient was diagnosed with B-ALL in March 2022, and experienced the first disease relapse after approximately 17 months. Patients with B-ALL relapsed after initial therapy should undergo allogeneic hematopoietic stem cell transplant (Allo-HSCT) after achieving a second remission [3]. Fortunately, novel therapies such as bispecific T-cell engager (BiTE) blinatumomab, antibody-drug conjugate Inotuzumab-ozogamicin and anti-CD19 chimeric antigen receptor-T (CAR-T) cells induce high rates of disease remission and can be a potential bridge to allo-HSCT. A month after his first disease relapse, taking into account CD19-positivity, he received two cycles of blinatumomab, obtaining a second MRD-negative remission. However, treatment was complicated by COVID-19 pneumonia that delayed the planned allo-HSCT. In December 2023, the patient developed rapidly worsening ocular symptoms, including bilateral pseudohypopyon, hyphema, and conjunctival hyperemia. The clinical finding of pseudohypopyon suggests leukemic blast infiltration and requires prompt evaluation and management. Aqueous humor (AqH) aspiration and flow cytometry confirmed the presence of B-ALL blasts in the anterior chamber of the eye (Figure 1, panel A2). Further systemic workup, including BM aspirate, lumbar puncture, contrast-enhanced brain MRI, and whole-body PET scan, revealed the presence of CD19-positive ALL blasts in the BM (> 5% of total cells) and extramedullary relapse in the anterior chamber of the eye, left palpebral soft tissues, and left latero-cervical lymph nodes. Given the evidence of ocular and systemic relapse, the patient was deemed eligible for anti-CD19 CAR-T cell therapy with brexucabtagene autoleucel (brexu-cel, Tecartus, Kite Pharma, CA, USA). As a bridging therapy, he received high dose cytarabine (Ara-C) and idarubicin in combination with local ophthalmologic therapy (dexamethasone and tobramycin). He achieved MRI/PET-negative status, ocular CR as shown by the AqH assessment (Figure 1, panel B2) but persistent BM MRD-positivity. Five months after his second disease relapse, he underwent lympho-depletion with fludarabine and cyclophosphamide, followed by brexu-cel infusion. The procedure was complicated by Grade 1 cytokine release syndrome and transient ocular symptoms, including palpebral ptosis, conjunctival hyperemia, and blurry vision. The development of ocular symptoms after CAR-T cell infusion in a patient with a documented ocular relapse that was in remission before treatment initiation might be due to: (i) a local inflammatory response; (ii) CAR-T cell trafficking secondary to low-level residual disease that was not detected during the AqH assessment; or (iii) ocular disease progression. To understand the reason for the patient's ocular symptoms, I would ask for an ophthalmic evaluation and a cytofluorimetric analysis of the aqueous humor. Cytofluorimetric analysis of AqH revealed no detectable B-ALL blasts but presence of 36% CAR-T cells in the AqH (Figure 1, panel C2-C3), with a predominance of CD8+ T cells and 13.5% CD4+ T cells. Two months later, cytofluorimetric disease reassessment confirmed MRD-negative CR, with no detectable blasts and CAR-T cells in the AqH (Figure 1, panel D2-D3). The evidence of CAR-T cells in the AqH demonstrates that they can reach sanctuary sites such as the anterior chamber of the eye after their expansion and release of cytokines like IL-1 and IL-6 in the peripheral blood (Figure 2) and may contribute to the eradication of the residual disease. Moreover, the complete symptom resolution coupled with absence of CAR-T cells at a follow-up AqH assessment (Figure 1, panel D3) suggests that CAR-T cells may act locally without permanently compromising the organ function. Ten months after his second disease relapse, the patient received allo-HSCT and is now in CR, with BM MRD-negativity, full donor chimerism and lack of circulating CAR-T cells. Clinically, ocular symptoms have not occurred again. Ophthalmic localization is an atypical but well-described manifestation of B-ALL. There are three patterns of direct ocular leukemic infiltration: anterior segment, uveal and orbital. In ALL patients the prevalence of symptomatic and asymptomatic ophthalmic manifestations at diagnosis is roughly 30% [4], slightly higher than in pediatric patients [5, 6]. Data regarding ophthalmic relapse are scarce, mainly from pediatric B-ALL case series [7], and treatment remains heterogeneous and challenging due to the difficulty in delivering therapeutic drug doses. In physiologic conditions, due to its exposure to environmental and bloodborne insults the eye is protected by selective blood barriers. Since such insults may lead to excessive innate and adaptive immune reactions that can compromise ocular integrity, the eye has an "immune privileged" environment [8]. Specifically, the blood-aqueous barrier (BAqB), localized at the level of the ciliary body, which is formed by tight junctions and fenestrated endothelium, is not absolute. As a result, immune cells and molecules such as immunomodulatory neuropeptides, cytokines, growth factors, soluble cell-surface receptors, can enter the AqH. These elements can inhibit innate immunity by reducing the production of nitric oxide by activated macrophages, limiting NK cells lysing ability and the activation and effector function of neutrophils and suppressing complement activation. Regarding adaptive immunity, effector CD4+ T cells are locally converted into regulatory cells that suppress bystander T cells. In addition, to prevent ocular damage, the epithelial and endothelial cells of the BAqB express PDL1 and CD86, immunoregulatory factors that suppress T cell proliferation. Some pathologic conditions, such as inflammation, lead to transient loss of barrier control and subsequent leukocyte infiltration [9]. In this case report, during maintenance therapy, the patient had multiple episodes of conjunctivitis, treated with local medication, which may have been caused by low-level leukemic involvement of the eye. On the other hand, these recurrent inflammatory processes may have caused a transient breakdown of the BAqB, allowing the infiltration by leukemic blasts. Unfortunately, the blood-barriers with tight junctions, such as the BAqB, limit the availability of drugs into ocular compartments. Moreover, the capillary endothelium of the eye expresses efflux transporter proteins such as P-glycoproteins, multidrug resistance proteins, and breast cancer resistance proteins [10], providing an additional barrier to drug absorption. Therapeutic strategies include systemic chemotherapy, intravitreal MTX, Ara-C and corticosteroids, and ocular radiotherapy [11-13]. All the approaches have limitations: systemic cytotoxic drugs have scarce penetration in ocular tissues, intravitreal MTX increases the risk of ocular infection, and radiotherapy might induce cataracts or ptosis, which may require surgical intervention. Regarding novel treatment approaches such as immunotherapy, blinatumomab is approved for use in ALL Ph− patients with measurable residual disease, with rates of MRD-negative CR after one cycle of up to 78% [14]; however, BiTEs cannot access sanctuary sites such as the CNS. By contrast, CAR-T cells are not approved for use in patients with MRD-positive CR but can penetrate the blood brain barrier and thus may be effective for treatment of extramedullary disease in sanctuary sites. CAR-T cell therapy has expanded treatment options for relapsed B-ALL, but its efficacy in cases with ocular involvement is not yet well-established. CAR-T cell therapy was used in pediatric and young adult ALL patients with ocular relapse, as documented by some case series [15-17]. In the case report by Gomel et al. [18], the authors demonstrated that a protocol consisting of radiotherapy and high-dose Ara-C followed by the administration of Tisagenlecleucel (Kymriah, Novartis, Switzerland) was effective in clearing blasts from the anterior chamber in a young adult with relapsed B-ALL, leading to long-lasting remission. However, the authors did not demonstrate that CAR-T cells penetrated the ocular barrier and exerted their function locally. On the other hand, there are no data on the use of brexu-cel as therapy for ocular localization of B-ALL. As mentioned above, there are no consensus guidelines on how to treat ocular localizations of B-ALL. Our therapeutic strategy consisted of high-dose Ara-C and idarubicin coupled with local steroid treatment. We chose to administer systemic chemotherapy because our patient had bone marrow involvement; in addition, the left palpebral soft tissue localization and its proximity to the CNS warranted the use of high-dose Ara-C, which penetrates the blood–brain barrier and achieves therapeutic drug concentrations in the CNS. Since local steroid therapy alone proved effective in rapidly reducing the pseudohypopyon, we continued with the same therapeutic approach. The patient achieved CR with BM MRD-positivity and was treated with brexu-cel. The use of sequential targeted immune therapies may drive evolving phenotypes of extramedullary disease. Fortunately, although the patient had previously received blinatumomab, he experienced a CD19+ extramedullary relapse and remained eligible for anti-CD19 CAR-T cell therapy. As previously shown, CAR-T cells can travel to sites of disease: in patients with non-Hodgkin Lymphoma CAR-T cells move from the bloodstream to nodal and extranodal sites and, upon activation, expand and release inflammatory cytokines, sometimes inducing tumor flares or pseudoprogression [19]. Moreover, CAR-T cells can gain access to sanctuary sites such as the CNS: multiple studies have demonstrated that CAR-T cell therapy is effective, with response rates and safety profiles comparable to those reported in the ELIANA pivotal trial [20]. Furthermore, in the ZUMA-3 study five patients had CNS-2 disease (defined as detectable blasts in the CSF with less than five white blood cells per mm3), and 80% achieved CR or CR with incomplete hematologic recovery. For the first time, we showed here that CAR-T cells can gain access to the AqH; in fact, we observed the presence of 36% of CAR-T cells, with a predominance of CD8+ T cells and 13.5% of CD4+ T cells (Figure 1, panel C3). Given that blasts were absent in the aqueous humor before CAR-T cell infusion and that the patient experienced ocular inflammation with ptosis, blurry vision and conjunctival hyperemia, it is possible that CAR-T cell's entry in the AqH was favored by the local inflammatory reaction. Another possibility is that CAR-T cells indirectly altered the permeability of BAqB's tight junctions and increased their paracellular permeation by stimulating cytokine production, such as IL-6 and IL-1 [21]. Moreover, CAR-T cells' production of IL-4, IL-5, and IL-13 may have favored lymphocyte migration in the AqH [22]. Lastly, it is possible that residual leukemic cells were too few to be detected by flow cytometry and CAR-T cells targeting these cells entered the AqH and were activated locally, causing ocular tumor flare. In summary, this is the first case of an adult patient with ocular relapse of B-ALL who was successfully treated with a combination of chemotherapy and brexu-cel, with documentation of CAR-T cells in the aqueous humor. It exemplifies how CAR-T cells reach even sanctuary sites, including the anterior chamber of the eye and how they can be effective in eradicating residual disease without permanently compromising the organ function. Written informed consent was obtained from the patient for publication of this case report and any accompanying images. All identifying information has been anonymized to protect the patient's privacy. As this is a single case report, formal ethical approval was not required in accordance with the guidelines of Fondazione I.R.C.C.S. Policlinico San Matteo. The authors declare no conflicts of interest. The data supporting this case report are not publicly available to protect patient privacy. De-identified data may be available from the corresponding author upon reasonable request, subject to institutional and ethical review and compliance with privacy regulations.