IKAROS gain of function disease: allogeneic hematopoietic cell transplantation experience and expanded clinical phenotypes

Abstract

IKAROS, encoded by IKZF1, is a tumor suppressor and a key hematopoietic transcription factor responsible for lymphoid and myeloid differentiation. IKZF1 mutations result in inborn errors of immunity presenting with increased susceptibility to infections, immune dysregulation, and malignancies. In particular, patients carrying IKZF1 gain-of-function (GOF) mutations mostly exhibit symptoms of immune dysregulation and polyclonal plasma cell proliferation. Herein, we describe seven new IKAROS GOF cases from two unrelated families, presenting with novel infectious, immune dysregulation and hematologic diseases. Two of the patients underwent allogeneic hematopoietic cell transplantation (HCT) due to poorly responsive complications. HCT was well-tolerated achieving full engraftment in both patients receiving reduced intensity, matched unrelated donor grafts, with no severe acute or chronic graft-vs-host-disease, and in remission from their diseases 2.5 and 4 years post-HCT, respectively. These results suggest that HCT is a valid and curative option in patients with IKAROS GOF disease and severe clinical manifestations.

1. Introduction

IKZF1 encodes IKAROS, an essential transcription factor for lymphopoiesis and hematopoiesis. IKAROS mostly negatively regulates the gene transcription machinery during lymphocyte as well as myeloid, megakaryocyte, and erythroid differentiation. Specifically, IKAROS interacts with a variety of transcriptional complexes and factors, including the nucleosome remodeling and deacetylase (NuRD) complex, the polycomb repressive complex 2 (PRC2), signal transducer and activator of transcription (STAT) family members, and interferon regulatory factor 4 (IRF4), to modulate gene expression during hematopoietic cell development.[1–4] Germline IKZF1 mutations have been implicated in human disease involving immunodeficiency, immune dysregulation, and hematologic malignancy; somatic variants were also related to the latter.[5–7] Germline heterozygous IKZF1 variants have been classified through their different mechanisms of action as IKZF1 haploinsufficiency (HI), IKZF1 dimerization defective (DD), IKZF1 dominant negative (DN), and IKZF1 gain-of-function (GOF) mutations. Each of these allelic variants are associated with particular clinical phenotypes. Patients with IKZF1 HI usually present with a progressive decline of B-cell numbers and serum immunoglobulin, are more likely to develop bacterial infections, autoimmune diseases/immune dysregulation, and B-cell acute lymphoblastic leukemia (B-ALL) in a common variable immunodeficiency (CVID)-mimicking phenotype.[6, 8] Patients with IKZF1 DD variants share several clinical features with IKZF1 HI patients, are commonly associated with hematopoietic cytopenia and lymphoproliferative disorders such as T-cell lymphoblastic leukemia, and Burkitt lymphoma, but are less likely to get recurrent or severe infections.[7, 9, 10] Patients presenting with IKZF1 DN mutations experience more severe and invasive complications of bacterial, viral, and opportunistic infection; Pneumocystis jirovecii pneumonia is highly prevalent among them, and can also develop hematologic malignancies, all in the context of a combined immunodeficiency (CID) phenotype.[5, 11, 12] More recently, germline heterozygous IKZF1 GOF variants have been found to cause enhanced T helper 2 (Th₂) and plasma cell (PC) differentiation, as well as immune dysregulation characterized by autoimmunity, allergies, and lymphoproliferative diseases.[13]

While most of IKAROS-associated disease manifestations can be managed long-term through prophylactic and symptomatic therapeutic approaches, some of the more aggressive cases required additional interventions to control the disease. This is particularly clear with the most severe phenotypes, as with IKZF1 DN cases, where most of the patients required hematopoietic cell transplantation (HCT) to improve survival and quality of life.

2. Results

Expanded clinical features and HCT experience in IKAROS GOF patients

We report seven patients from two unrelated families carrying IKZF1 GOF mutations, with two of the patients requiring HCT due to progressive and uncontrolled disease (Figure 1). Besides the clinical features previously described to be associated to the IKZF1 GOF allelic variant including allergic/atopic manifestations (e.g., asthma, rhinitis, dermatitis, food and drug allergies, and eosinophilic esophagitis); autoimmune/inflammatory diseases (e.g., Evans syndrome, celiac disease, autoimmune hepatitis, type 1 diabetes mellitus, Hashimoto thyroiditis, colitis, leukocytoclastic vasculitis, alopecia and vitiligo) and plasma cell proliferation (e.g., in lymph nodes, bone marrow and the gastrointestinal tract), other severe and life-threatening complications manifested in the patients herein described. In this cohort, patients also presented with invasive fungal infections (e.g., histoplasmosis), other immune dysregulation/autoimmune diseases (e.g., Crohn’s disease, sarcoidosis, eosinophilic granulomatosis with polyangiitis, and a history of multiple sclerosis), and most noticeable malignant (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma) and non-malignant (e.g., monoclonal gammopathy of uncertain significance, myelodysplastic syndrome) hematologic disorders. Clinical and laboratory data of all seven patients included in this report are summarized below and in Table 1. Patients who underwent HCT (Patient 1/Family 1 and Patient 2/Family2) are described in detail.

Figure 1:

Pedigrees of two families with IKZF1 gain-of-function (GOF) variants

Family members who are male or female are each represented by a square or a circle, respectively. Affected/mutation positive individuals are depicted in black; mutation negative/untested individuals (?)in white. WT, wild-type allele; Mut, mutated allele.

Table 1.

Characteristics of the patients with IKZF1 GOF mutation receiving HCT

	Patient 1	Patient 2
IKZF1 mutation	IKZF1(NM_006060):c.547C>T (p.R183C)	IKZF1(NM_006060):c.548G>A (p.R183H)
Onset of symptoms	Childhood	Adulthood
Age at HCT, yr/sex	40/F	62/M
Hematologic disease	IgA lambda gammopathy of undetermined significance (MGUS) progressed to smoldering multiple myeloma Polyclonal polytypic plasmacytosis	DLBCL ALK-negative ALCL
Inflammatory/autoimmune diseases	Crohn’s disease Enteropathic arthritis Alopecia universalis	EGPA Multiple sclerosis Erythema annulare centrifugum
Allergic diseases	Atopic dermatitis Asthma Allergic rhino-conjunctivitis Eosinophilic esophagitis Multiple drug allergies	Exercise-induced urticaria Drug allergy
Infections
Bacterial	Pneumonia Recurrent sinusitis	Pseudomonas sepsis Recurrent sinusitis
Viral	Warts (HPV) JC viruria (asymptomatic)	COVID-19 with organizing pneumonia JC viruria (asymptomatic) BK viruria (asymptomatic)
Opportunistic infection	No	No
Affected cell lineage
CD19+ lymphocyte count, Abs (cells/mm3) (%)	9 (0.5) Reference range 61–321 (3.3–19.3)	7 (1.5) Reference range 61–321 (3.3–19.3)
CD4+ lymphocyte count, Abs (cells/mm3) (%)	1451 (82) Reference range 359–1565 (31.9–62.2)	298 (66.2) Reference range 359–1565 (31.9–62.2)
CD8+ lymphocyte count, Abs (cells/mm3) (%)	207 (11.7) Reference range 178–853 (11.2–34.8)	55 (12.3) Reference range 178–853 (11.2–34.8)
CD16+ or CD56+ lymphocyte count, Abs (cells/mm3) (%)	76 (4.3) Reference range 126–729 (6.2–34.6)	78 (17.3) Reference range 126–729 (6.2–34.6)
Immunoglobulin levels at baseline
IgG (mg/dL)	941# (Reference range 700–1600)	1012 (Reference range 540–1822)
IgA (mg/dL)	1706 (Reference range 70–400)	298 (Reference range 101–645)
IgM (mg/dL)	12 (Reference range 40–230)	113 (Reference range 22–240)
IgE (IU/mL)	36.9 (Reference range 0–90)	50 (Reference range 0–100)
HCT details
HCT comorbidity index*	4	11
Donor HLA match and relatedness	10/10 URD	10/10 URD
Graft type	T-replete BM	T-replete PB
Graft dose (cells/kg)	TNC: 5.86×108 CD3+: 6.7×107 CD34+: 7.85×106	TNC: 13.79×108 CD3+: 39.54×107 CD34+: 12.42×106
Conditioning (RIC)	Pentostatin Low-dose cyclophosphamide Busulfan (total target AUC 37.8mg × h/L)	Equine antithymocyte globulin Pentostatin Low-dose cyclophosphamide Busulfan (total target AUC 37.8mg × h/L)
GVHD prophylaxis	High-dose PTCy Sirolimus days +5 to +90	High-dose PTCy MMF days +5 to +25 Tacrolimus days +5 to +41 (stopped early upon GVHD treatment with systemic steroids)
HCT complications	Acute GVHD, skin only, max grade I Demodex folliculitis BK-associated cystitis Autoimmune hypothyroidism	Acute GVHD, skin only, max grade II Demodex folliculitis Giardiasis Otochondritis COP Pulmonary aspergillosis with bronchopulmonary hemorrhage
Outcome/ Follow up	At 4 yrs post-HCT: IgA lambda gammopathy regressed back to MGUS levels with bone marrow showing polytypic plasma cells of normal number and morphology Remission of Crohn’s disease and eosinophilic esophagitis Off IgGRT Off all systemic immunosuppression No chronic GVHD Karnofsky performance score 100%	At 2.5 yrs post-HCT: Continued complete remission of Alk-negative ALCL and DLBCL Off all systemic immunosuppression, without multiple sclerosis symptoms No chronic GVHD Karnofsky performance score 100%

^#On IgG replacement therapy;

^*Scores of 3 or greater are associated with increased transplant-related mortality

Abbreviations: ALCL, anaplastic large cell lymphoma; Abs, absolute count; BM, bone marrow; COP, cryptogenic organizing pneumonia; DLBCL, diffuse large B cell lymphoma; EGPA, eosinophilic granulomatosis with polyangiitis; GVHD, graft-versus-host disease; HCT, hematopoietic cell transplantation; HPV, human papillomavirus; IgGRT, Immunoglobulin replacement therapy; MGUS, monoclonal gammopathy of undetermined significance; MMF, mycophenolate mofetil; PB, peripheral blood; PTCy, post-transplantation cyclophosphamide; RIC, reduced intensity conditioning; TNC, total nucleated cells; URD, unrelated donor.

Patient 1 is a 45-year-old North American Caucasian female carrying IKZF1 GOF mutation [IKZF1(NM_006060):c.547C>T (p.R183C)] diagnosed at age 39, with disease manifestations including CVID, refractory Crohn’s disease with enteropathic arthritis, eosinophilic esophagitis, polyclonal plasmacytosis, and atopy. Starting in childhood, she presented with atopic dermatitis (well-controlled in adulthood), moderate persistent asthma (also under control on budesonide-formoterol and prednisone), allergic rhino-conjunctivitis (positive skin testing for cat, dust mites, trees, grasses, weeds requiring allergen immunotherapy for 4–5 years with good results), and eosinophilic esophagitis managed with swallowed fluticasone and proton pump inhibitors, complicated by strictures requiring dilation twice. At age 32 she was diagnosed with stricturing Crohn’s disease which was only partially responsive to treatment with multiple medications, including infliximab, adalimumab, 6-mercaptopurine, and vedolizumab with systemic steroids. She ultimately required a total abdominal colectomy and ileostomy at age 37. Subsequently, she stabilized on tofacitinib twice daily, prednisone, and dietary limitations, albeit with continued intermittent flares and radiologic and endoscopic evidence of active disease. Her infectious history included two pneumonias, recurrent sinusitis requiring endoscopic surgery, and finger warts that previously resolved following topical medication. At the age of 39 years she was found to be hypogammaglobulinemic, diagnosed with CVID and started on IgG replacement therapy (IgGRT); since then she presented with almost no infections. Furthermore, she also presented with IgA lambda monoclonal gammopathy of undetermined significance (MGUS), characterized by polytypic plasmacytosis with elevated IgA (1500 mg/dL) and 20% plasma cells in the bone marrow, but less than ten percent of the cells were lambda predominant. Subsequently, her MGUS progressed to smoldering multiple myeloma. Family history was remarkable for the [IKZF1(NM_006060):c.547C>T (p.R183C)] variant being detected in one of her two children and one of her two brothers, both also diagnosed with hypogammaglobulinemia, food allergy, allergic rhinitis, and asthma. Her mother is healthy, while her father had esophageal carcinoma, asthma, was frequently sick, and passed away from lung carcinoma (no samples available for testing) (Figure 1).

Patient 1 underwent HCT at age 41, less than 2 years after her IKAROS GOF disease diagnosis. She received reduced intensity conditioning (RIC) with pentostatin, hyperfractionated cyclophosphamide, and busulfan and received a T-cell-replete matched unrelated donor (MUD) bone marrow graft. Graft-versus-host disease (GVHD) prophylaxis consisted of high-dose post-transplantation cyclophosphamide (PTCy) and sirolimus through day +90. She engrafted uneventfully on post-HCT day +13 and has demonstrated full donor chimerism in T and myeloid cells, as well as B and NK cells. Her post-HCT course was complicated by toxic erythema of chemotherapy, mild steroid-responsive acute GVHD (skin only, maximum Grade I) along with Demodex folliculitis and minimally symptomatic BK-associated cystitis. Routine follow up upper and lower endoscopy nine months post-HCT revealed complete remission of eosinophilic esophagitis and Crohn’s disease off immunosuppression. Fifteen months post-HCT she was diagnosed with autoimmune hypothyroidism. Otherwise, Patient 1 has had one ambulatory lower respiratory tract infection but no other complications of HCT, including no evidence of chronic GVHD. She remains on azithromycin prophylaxis given her underlying bronchiectasis but has demonstrated robust vaccine responses and no longer requires IgGRT. Her most recent bone marrow biopsy, 3 years post-HCT, showed normal numbers of polytypic plasma cells, while with persistent but improving MGUS in the peripheral blood.

Patient 2 is a 64-year-old North American Caucasian man carrying an IKZF1 GOF mutation [IKZF1(NM_006060):c.548G>A (p.R183H)] diagnosed at age 58. The patient became symptomatic in his late forties when he was diagnosed with multiple sclerosis after developing lower limb numbness, double vision and white matter lesions on MRI. He also presented with exercise-induced urticaria and paroxysmal atrial fibrillation, leading to cardioembolic stroke and seizures. In his fifties, he was diagnosed with ANCA-negative eosinophilic granulomatosis with polyangiitis (EGPA) in the setting of pulmonary hemorrhage and a skin biopsy showing vasculitis. Treatments included methotrexate, steroids, and mepolizumab. He additionally required chronic low-dose corticosteroid therapy for interstitial lung disease and organizing pneumonia following COVID-19 infection. At age 60, he was diagnosed with ALK-negative anaplastic large cell lymphoma (ALCL) involving the skin and treated with targeted radiation. Shortly thereafter, a liver biopsy revealed an Epstein-Barr virus (EBV) negative DLBCL (stage IV) germinal-center B cell-like (GCB) subtype. Patient 2 was treated with 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) for DLBCL and achieved a complete response. Following his DLBCL treatment, a bone marrow biopsy revealed no lymphoma or B cells, but a small increase of polyclonal plasma cells (CD138+) accounting for 5% of the bone marrow cellularity and distributed in perivascular aggregates was detected. Importantly, mutation [IKZF1(NM_006060):c.548G>A (p.R183H)] was detected at a 50% of variant allele frequency (VAF) on bone marrow genetic sequencing. Subsequently, germline origin was confirmed via Sanger sequencing of gDNA extracted from Patient 2 non-hematopoietic tissue (i.e., nail clippings), and in three of his surviving siblings. In the meantime, his ALK-negative ALCL relapsed, and he was treated with radiation and brentuximab, attaining complete remission prior to HCT. Ultimately, he received HCT at age 62, around 3 months after the diagnosis of germline IKZF1 GOF mutation was confirmed and 1.5 years after his initial lymphoma diagnosis.

Patient 2 received reduced intensity conditioning consisting of equine anti-thymocyte globulin (ATG), pentostatin, hyperfractionated cyclophosphamide, and busulfan, followed by a T-cell-replete matched unrelated peripheral blood stem cell graft. GVHD prophylaxis included high-dose PTCy, mycophenolate mofetil (MMF), and tacrolimus. He achieved neutrophil engraftment on day +17 with full donor T and myeloid chimerism by day +34. His post-HCT course was complicated by skin-only acute GVHD, maximum grade II, treated with systemic steroids, along with giardiasis, Demodex folliculitis, and otic soft tissue infection, all responsive to standard therapy. Five months post-HCT, he was hospitalized with pulmonary hemorrhage concerning for EGPA flare and diagnosed with pulmonary aspergillosis, responsive to systemic steroid and antifungal therapy. Seven months after HCT, he had a relatively mild course of COVID-19 in the setting of monoclonal antibody therapy. However, he developed cryptogenic organizing pneumonia requiring prolonged systemic steroid therapy until 22 months post-HCT. Despite this, Patient 2 remained active and functional. Currently, thirty months after HCT, the patient is in continued complete remission. He has had no recurrence of multiple sclerosis symptoms and remains off systemic immunosuppression for the past eight months with no chronic GVHD or other complications of HCT, with continued full donor chimerism.

Upon Patient 2’s IKZF1 mutation detection, his extensive family history and genetics were interrogated. Patient 2 is the 9^th of 10 siblings born to non-consanguineous parents. His father passed away at the age of 79 years due to a stroke, and his mother died at the age of 38 years due to multiple myeloma. Three of his siblings are also deceased: one died as a toddler due to unknown causes (10^th sibling), one died in her late forties and was diagnosed with immune thrombocytopenia (ITP; 1^st sibling), and another died in his sixties and was diagnosed with ITP, sarcoidosis, and Hodgkin lymphoma (5^th sibling). No biologic material was available for genetic testing on either of the parents or the three deceased siblings. Three of Patient 2’s surviving siblings who also carry the [IKZF1(NM_006060):c.548G>A (p.R183H)] variant were diagnosed with sarcoidosis, myelodysplastic syndrome, and renal cell carcinoma (2^nd sibling); sarcoidosis, ITP, and Hodgkin lymphoma (8^th sibling), and basal cell carcinoma and squamous cell carcinoma (6^th sibling) (Figure 1). Of note, basal cell carcinomas and squamous cell carcinomas were also diagnosed in three other IKZF1 mutation negative siblings. Patient 2 himself has had prostate cancer and multiple skin cancers.

3. Discussion

Different clinical phenotypes have been observed in patients carrying germline heterozygous IKZF1 mutations, collectively known as IKAROS-associated diseases. While all complications fall within the primary immunodeficiency/inborn errors of immunity spectrum, the clinical characteristics associated with IKZF1 GOF variants differ from those previously identified in IKAROS HI, DN, and DD patients. Moreover, the effects of IKZF1 GOF variants seem mostly opposed to those from IKZF1 DN mutations: e.g., while IKZF1 DN variants usually present with no B cells, no plasma cells, agammaglobulinemia and eosinopenia, resulting in a “protective” status to immune dysregulation and allergy, IKZF1 GOF variants are associated with normal/high B cells, plasma cell proliferation, normo/hypergammaglobulinemia, high IgE and eosinophils, all resulting in increased immune dysregulation and allergy susceptibility. In this report, we expand the range of immune dysregulation, autoimmune, allergic, and in particular malignant or lymphoproliferative manifestations that IKZF1 GOF mutation carriers can exhibit. Moreover, we also show how HCT was able to rescue the phenotype in the two transplanted patients. Patient 1, carrying the IKAROS R183C GOF variant, presented with manifestations of refractory inflammation (Crohn’s disease), notable allergic diseases, and abnormal plasma cell proliferation (both smoldering multiple myeloma and polyclonal plasmacytosis). Patient 2, carrying the IKAROS R183H GOF variant, was diagnosed with autoimmune vasculitis as well as two separate hematologic malignancies. Patient 1 and Patient 2 exhibited abnormally low B cell counts, which contrast with the findings reported by Hoshino et al. where normal or elevated B cell number were observed[13]. Notably, by the time of their immune evaluation both patients were on systemic immunomodulatory medications, and Patient 2 had also received rituximab and brentuximab, introducing limitations to the interpretation of their immunologic evaluation.

While no formal clinical guidelines exist for the treatment of patients with IKAROS-associated diseases, the severity and/or therapeutic unresponsiveness of Patient 1 and Patient 2’s clinical conditions prompted the decision to explore HCT as a therapeutic and curative option for IKAROS GOF disease, as has been previously shown for other forms of IKAROS-associated diseases [11]. After undergoing RIC HCT, both patients successfully achieved full donor chimerism post-HCT with evolving phenotype reversal and remain off immunosuppression at 4 and 2.5 years post-HCT, respectively.

In a murine leukemia model of allo-HCT, Ikzf1 DN mutations were associated with increased mortality and GVHD severity but no improved graft-vs-tumor effect [14]. Interestingly, increased peripheral blood IKAROS expression was also associated with increased risk of moderate/severe chronic GVHD in a cohort of HCT recipients with acute leukemia [15]. However, in the two IKZF1 GOF patients herein reported, HCT was well tolerated with no severe acute GVHD or any chronic GVHD and in remission from their hematologic malignancies. Of note, a similarly benign post-HCT outcome has also been described for HCT recipients with IKZF1 DN mutations [11]. Whether these apparently discrepant results may reflect intrinsic species-related differences between murine and human HCT in Ikzf1/IKZF1-mutated individuals, the use of high dose PTCy as GVHD prophylaxis in the transplant setting, the IKAROS expression level in the graft, or other yet to be determined factors, still has to be elucidated. As of last follow up, both IKZF1 GOF transplanted patients remain off systemic immunosuppression, with stable full donor chimerism and appropriate immune reconstitution.

4. Conclusions

In this report we expand the IKAROS GOF-associated disease phenotypes in seven patients and describe our experience with HCT in two high risk individuals with complex phenotypes. Our observations, although limited, highlight positive outcomes of HCT following RIC with full engraftment of donor cells and no severe GVHD complication. These results suggest that HCT is a well-tolerated and curative option in patients with IKZF1 GOF mutations and severe clinical manifestations.

Highlights.

• IKAROS GOF disease phenotype expanded to include hematologic malignancies.

• Myeloablation is not needed for engraftment in HCT recipients with IKAROS GOF disease.

• HCT is a well-tolerated curative approach in IKAROS GOF patients with severe clinical manifestations.