Jean Ulrick

The CXCR4 antagonist plerixafor corrects panleukopenia in patients with WHIM syndrome

WHIM syndrome is a rare congenital immunodeficiency disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (neutropenia because of impaired egress from the BM); most patients also have severe panleukopenia. Because WHIM syndrome is caused by mutations in the chemokine receptor CXCR4 that result in increased agonist-dependent signaling, we hypothesized that the CXCR4 antagonist plerixafor (Mozobil [Genyzme Corporation], AMD3100), might be an effective treatment. To test this, we enrolled 3 unrelated adult patients with the most common WHIM mutation, CXCR4(R334X), in a phase 1 dose-escalation study. Plerixafor increased absolute lymphocyte, monocyte, and neutrophil counts in blood to normal without significant side effects in all 3 patients. Peak responses occurred at 3-12 hours after injection and waned by 24 hours after injection which tracked the drugs pharmacokinetics. All 3 cell types increased in a dose-dependent manner with the rank order of responsiveness absolute lymphocyte > monocyte > neutrophil. These data provide the first pharmacologic evidence that panleukopenia in WHIM syndrome is caused by CXCL12-CXCR4 signaling-dependent leukocyte sequestration, and support continued study of plerixafor as mechanism-based therapy in this disease. This study is registered at http://www.clinicaltrials.gov as NCT00967785.

A phase 1 clinical trial of long-term, low-dose treatment of WHIM syndrome with the CXCR4 antagonist plerixafor

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare immunodeficiency disorder caused by gain-of-function mutations in the G protein-coupled chemokine receptor CXCR4. The CXCR4 antagonist plerixafor, which is approved by the US Food and Drug Administration (FDA) for stem cell mobilization in cancer and administered for that indication at 0.24 mg/kg, has been shown in short-term (1- to 2-week) phase 1 dose-escalation studies to correct neutropenia and other cytopenias in WHIM syndrome. However, long-term safety and long-term hematologic and clinical efficacy data are lacking. Here we report results from the first long-term clinical trial of plerixafor in any disease, in which 3 adults with WHIM syndrome self-injected 0.01 to 0.02 mg/kg (4% to 8% of the FDA-approved dose) subcutaneously twice daily for 6 months. Circulating leukocytes were durably increased throughout the trial in all patients, and this was associated with fewer infections and improvement in warts in combination with imiquimod; however, immunoglobulin levels and specific vaccine responses were not fully restored. No drug-associated side effects were observed. These results provide preliminary evidence for the safety and clinical efficacy of long-term, low-dose plerixafor in WHIM syndrome and support its continued study as mechanism-based therapy in this disease. The ClinicalTrials.gov identifier for this study is NCT00967785.

Severe congenital neutropenia resulting from G6PC3 deficiency with increased neutrophil CXCR4 expression and myelokathexis

Mutations in more than 15 genes are now known to cause severe congenital neutropenia (SCN); however, the pathologic mechanisms of most genetic defects are not fully defined. Deficiency of G6PC3, a glucose-6-phosphatase, causes a rare multisystem syndrome with SCN first described in 2009. We identified a family with 2 children with homozygous G6PC3 G260R mutations, a loss of enzymatic function, and typical syndrome features with the exception that their bone marrow biopsy pathology revealed abundant neutrophils consistent with myelokathexis. This pathologic finding is a hallmark of another type of SCN, WHIM syndrome, which is caused by gain-of-function mutations in CXCR4, a chemokine receptor and known neutrophil bone marrow retention factor. We found markedly increased CXCR4 expression on neutrophils from both our G6PC3-deficient patients and G6pc3(-/-) mice. In both patients, granulocyte colony-stimulating factor treatment normalized CXCR4 expression and neutrophil counts. In G6pc3(-/-) mice, the specific CXCR4 antagonist AMD3100 rapidly reversed neutropenia. Thus, myelokathexis associated with abnormally high neutrophil CXCR4 expression may contribute to neutropenia in G6PC3 deficiency and responds well to granulocyte colony-stimulating factor.

WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4

WHIM syndrome is a rare, autosomal dominant, immunodeficiency disorder so-named because it is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (defective neutrophil egress from the BM). Gain-of-function mutations that truncate the C-terminus of the chemokine receptor CXCR4 by 10-19 amino acids cause WHIM syndrome. We have identified a family with autosomal dominant inheritance of WHIM syndrome that is caused by a missense mutation in CXCR4, E343K (1027G → A). This mutation is also located in the C-terminal domain, a region responsible for negative regulation of the receptor. Accordingly, like CXCR4(R334X), the most common truncation mutation in WHIM syndrome, CXCR4(E343K) mediated approximately 2-fold increased signaling in calcium flux and chemotaxis assays relative to wild-type CXCR4; however, CXCR4(E343K) had a reduced effect on blocking normal receptor down-regulation from the cell surface. Therefore, in addition to truncating mutations in the C-terminal domain of CXCR4, WHIM syndrome may be caused by a single charge-changing amino acid substitution in this domain, E343K, that results in increased receptor signaling.

AMD3100 is a Potent Antagonist at CXCR4R334X, a Hyperfunctional Mutant Chemokine Receptor and Cause of WHIM Syndrome

WHIM is an acronym for a rare immunodeficiency syndrome (OMIM #193670) caused by autosomal dominant mutations truncating the C‐terminus of the chemokine receptor CXC chemokine receptor 4 (CXCR4). WHIM mutations may potentiate CXCR4 signalling, suggesting that the United States Food and Drug Administration (FDA)‐approved CXCR4 antagonist AnorMED3100 (AMD3100) (also known as Plerixafor) may be beneficial in WHIM syndrome. We have tested this at the preclinical level by comparing Chinese hamster ovary (CHO) and K562 cell lines matched for expression of recombinant wild‐type CXCR4 (CXCR4WT) and the most common WHIM variant of CXCR4 (CXCR4R334X), as well as leucocytes from a WHIM patient with the CXCR4R334X mutation versus healthy controls. We found that CXCR4R334X mediated modestly increased signalling (∼2‐fold) in all functional assays tested, but strongly resisted ligand‐dependent down‐regulation. AMD3100 was equipotent and equieffective as an antagonist at CXCR4R334X and CXCR4WT. Together, our data provide further evidence that CXCR4R334X is a gain‐of‐function mutation, and support clinical evaluation of AMD3100 as mechanism‐based treatment in patients with WHIM syndrome.

Sarcoidosis in Chronic Granulomatous Disease

In addition to increased susceptibility to infections in patients with chronic granulomatous disease (CGD), a higher incidence of sterile inflammatory disorders in these patients has been noted. However, sarcoidosis has not been reported previously in CGD. In this report, we describe two patients who have CGD and a disorder consistent with sarcoidosis on the basis of unequivocal clinical-radiographic presentations, their responses to treatment, and serum angiotensin-converting enzyme levels. Serum angiotensin-converting enzyme levels were measured in 26 other patients with CGD to establish an appropriate reference range. A possible relationship between CGD and sarcoidosis is discussed.

Skin ulcers and disseminated abscesses are characteristic of Serratia marcescens infection in older patients with chronic granulomatous disease

To the Editor: Chronic Granulomatous Disease (CGD) is a primary immune deficiency resulting from mutations in any of four subunits of phagocytic cell NADPH oxidase. CGD patients are at risk for infections with catalase-positive bacteria and fungi [1-3]. Serratia marcescens infection of bone and soft tissue is a common presentation of CGD in infancy [4-6]. In older children and adults with CGD, infection with Staphylococcus aureus, Burkholderia cepacia and Nocardia species, or fungi such as Aspergillus make up the majority of infections [3,4]. Our experience indicates S. marcescens remains a significant cause of infection in older children and adults with CGD. These infections have a pattern different from that seen in CGD infants. Osteomyelitis is less common, skin infections form poorly healing ulcers [7], and infections frequently occur at multiple sites. We describe 16 episodes of S. marcescens infections in 15 patients 8–39 years of age with CGD followed between 1999–2008 at the National Institutes of Health (Table 1). Only patients with culture documented S. marcescens were included in this report. All patients were participants in an IRB approved study of the natural history of CGD. Written informed consent specifically allowed study of infections and other complications of CGD. Table 1 CGD mutation, age at diagnosis of infection, clinical sites of infection and length of antibiotic therapy in patients presenting with S. marcescens infections Three patients had additional documented S. marcescens infection at some time prior to the index period under review for this report. One of these three patients had a history of two previous S. marcescens lung infections. Each of these prior infections antedate the index review period by at least four years. We did not include these older episodes of infection, as detailed information about those infections was deemed less reliable. During the review period, some patients had one or more infections characteristic of CGD with organisms other than S. marcescens. Only one (Patient #13, Table 1) developed two episodes of infection with S. marcescens. We conclude that they are distinct Serratia infections and not relapse of the first infection, because those two infections were three years apart. This assumption is consistent with a previous report noting that for most infections in CGD, re-infection is more common than persistent infection [8]. Sites of infection included lung parenchyma (6), vertebrae (1), lymph nodes (3), deep intra-abdominal or pelvic abscess (4), deep soft tissue abscess of limb (6), and superficial infection of skin (2). Seven patients were found to have metastatic infection at multiple sites (Table 1). None of the 6 patients presenting with pneumonia had clinically apparent secondary sites of S. marcescens infection. Conversely, 7 of the 10 episodes of S. marcescens infection that did not involve pneumonia presented with widely metastatic dissemination. The characteristics of this dissemination included more than one non-contiguous site of skin abscess, deep muscle abscess in limbs and/or abscess in internal organs. A particularly informative example was Patient #9 (Table 1) who presented with skin and soft tissue abscesses (Figure 1). Epididymitis, apparent on physical examination, prompted pelvic Magnetic Resonance Imaging that revealed a prostate abscess (Figure 2). Figures 1A-C Photograph of skin ulcers resulting from S. marcescens infection in Patient #9. Figure 2 Magnetic resonance image of the pelvis of patient #9. The two bright confluent round objects in the center of this image represent a large abscess in the prostate. Mixed infection in the same biopsy sample (S. aureus or S. anginosis in addition to the Serratia) was identified in four patients, but in all cases the predominant organism by colony count appeared to be S. marcescens. Although the significance and relative impact of these additional organisms on the clinical course was unknown, antibiotic coverage was adjusted as necessary to include coverage for treatment of these additional organisms. In all cases, the S. marcescens isolate was sensitive to a broad range of antibiotic classes including carbapenems, quinolones, cephalosporins, aminoglycosides, extended-spectrum beta-lactams and trimethoprim-sulfamethoxazole. There was a tendency to include or use a quinolone as primary treatment in most cases. If a second antibiotic was used it tended to be either an intravenous carbapenem or intravenous high dose trimethoprim-sulfamethoxazole. In all of the cases these Serratia infections occurred in a setting where standard long term infection prophylaxis had included trimethoprim-sulfamethoxazole. Compliance with this long term prophylaxis was variable. Notably, 10 of 16 of the Serratia isolates were sensitive to this agent. The majority of patients received intravenous antibiotic therapy with two agents for at least a portion of the treatment period. One patient was treated with three intravenous antibiotics and one patient was treated with only one intravenous antibiotic (levofloxacin). Two patients received oral quinolone (ciprofloxacin or levofloxacin) therapy only (lymph node infection in one case and superficial abscess in the other; both without metastatic infection). Duration of therapy varied according to severity of the infection (range 14–180 days; mean 81 days; median 55 days). All infections were successfully eradicated; and all patients survived his or her infection. Our current report represents the largest aggregate detailed description of Serratia infections in older children and adults with CGD. Previous reports describe either single patients or small case series [4-7], primarily in infants and young children. In other publications, the incidence of Serratia infection is reported incidental to an overall report of a registry of CGD patients without detailed description of the characteristics of the Serratia infections [3]. The important findings of our study reporting the details of 16 episodes of S. marcescens infections in 15 older children and adults with CGD are: 1. Serratia osteomyelitis was uncommon compared to that reported in infants with CGD; 2. Serratia infections of skin form large poorly healing ulcers; 3. Serratia pneumonia in our series was not associated with clinically apparent infection to extrapulmonary sites; 4. Seven of the ten (70%) non-pulmonary infections with Serratia in our series were associated with metastatic spread of infection to multiple sites. As clinically indicated by history, exam or laboratory findings, this group of patients often may require additional imaging studies (CT, MRI or PET scan) to demonstrate the presence of cryptic sites of infection. Finally, we note that all the infections in our series were eventually eradicated; indicating that full recovery from Serratia infection in CGD is an expected outcome with recognition of the variable presentation of this disease and with proper management.