Acquired cyclic neutropenia associated with cocaine-induced anti-neutrophil cytoplasmic antibodies binding to human neutrophil elastase
Authors: Francesca Schieppati, Gina Gregorini, Amber M. Hummel, Mariella D'Adda, Erika Borlenghi, Cinzia Lamorgese, Ulrich Specks, Giuseppe Rossi
Journal: American Journal of Hematology (2018)

Life-threatening agranulocytosis (AGC) is a recognized complication of cocaine use.1 Neutropenia usually improves with the drug withdrawal,2 but little information is available about the long-term sequelae of this condition. A particular subtype of anti-nuclear cytoplasmic antibodies (ANCAs) reacting with human neutrophil elastase (HNE) has been characterized in cocaine abusers3; however, it has not been described in patients manifesting AGC. We evaluated the presence of HNE-ANCA in 3 previously healthy individuals who developed cyclical recurrent episodes of symptomatic AGC after cocaine exposure, to determine if a immune mechanism may sustain a cyclic hematopoiesis, similar to the mechanism of elastase mutation in congenital cyclic neutropenia (CyN). We also assessed the efficacy of immunosuppressive therapy (IST) in inducing clinical remission from AGC in these patients. The clinical data were collected at patient's admission at the Division of Hematology of the Hospital of Brescia, and during follow-up. To rule-out a primitive neoplastic or systemic autoimmune disorder, multimodality ANCA testing was performed in the laboratory of Dr Ulrich Specks in the Thoracic Disease Research Unit, Mayo Clinic, Rochester (for details see online Supporting Information), due to ANCA positivity at the routine laboratory workup. For HNE-ANCA determination, functional expression of recombinant HNE (rHNE) in the human mast cell line HMC-1 (HMC-1/HNE) provided a HMC-1/HNE substrate for indirect immunofluorescence (IIF).3 Three different enzyme-linked immunosorbent assay (ELISA) methods were used to confirm HNE-ANCA positivity, using rHNE as substrate: a standard capture ELISA using polyclonal sheep anti-HNE,3 an anti-c-myc capture ELISA,4 and nickel capture ELISA.5 From July 2007 to January 2010, 3 consecutive patients were admitted to our Department due to acute AGC. All patients showed myeloid maturational arrest at the bone marrow examination. Surprisingly, all patients showed high titers of ANCA, without any clinical sign of vasculitis or systemic immune disorder. Moreover, 2 patients exhibited an activated partial thromboplastin time (aPTT) prolongation and tested positive for circulating lupus anti-coagulant (LAC), with no history of previous thrombosis. Case 1: A 45-year old woman presented to the emergency room due to high fever and oral mucosa ulceration and an absolute neutrophil count (ANC) of 0 × 109/L. Antibiotics, prednisone, and granulocyte-colony stimulating factor (G-CSF) were administered, with normalization of ANC. A year later, a relapse of AGC associated to c-ANCA/PR3-ANCA positivity occurred, and retreatment with prednisone and G-CSF led to a new remission. A month later, the patient was readmitted due to fever, AGC and multiple oral ulcers, with high titer ANCA; the patient disclosed cocaine use before hospitalization. A course of high-dose intravenous immunoglobulin (IVIG) (400 mg/kg for 5 days) associated with G-CSF for 7 days was administered, resulting in recovery of neutrophil counts. A readmission a month later was prompted by AGC and Pseusomonas aeruginosa cutaneous infection. Another course of IVIG led to recovery of ANC once again. The patient denied recurrent cocaine use. For 1 year since initiation of IST she remained in remission from cycling AGC (Figure 1A). Case 2: A 34-year old man, with a history of cocaine and marijuana abuse, was admitted for fever and pneumonia, associated to isolated AGC (ANC 0.02 × 109/L). G-CSF treatment led to rapid recovery of neutrophil counts. Three months later the patient relapsed with perianal ulcerative lesions associated with AGC, responsive to a new course of G-CSF. Three months later, the patient started developing severe cyclic neutropenia, with monthly drops of neutrophil counts and stomatitis. A strong positivity for c-ANCA was demonstrated by IIF, with no specific ANCA subtype. LAC was also positive without thrombosis. At his seventh episode of cyclic AGC, the patient was tested for urinary cocaine metabolites, resulted negative, and the patient denied any use of cocaine in the past year. He then received a course of high-dose IVIG, leading to full recovery of neutrophil counts. The response to IST with high-dose IVIG lasted 13 months, until the patient developed a new episode of AGC after cocaine re-exposure, confirmed by urinary tests. He was treated with 2 consecutive cycles of high-dose IVIG, with long-lasting remission at 12 months and no further AGC episodes (Figure 1B). Case 3: A previously healthy 41-year old woman presented to the emergency room with persistent tonsillitis and fever, associated to ANC 0.01 × 109/L and LAC at high titer. Neutropenia was responsive to G-CSF. She eventually relapsed 9 months later, with c-ANCA positivity, with no specific ANCA subtypes. She was treated with high-dose of prednisone for 4 days, and then rapidly tapered, with recovery. She successively developed recurrent, monthly episodes of AGC, with clinical and hematological response to corticosteroids and G-CSF at each episode. The patient disclosed only 1 cocaine exposure a year before. Indeed, urinary tests during AGC were negative. A course of high-dose IVIG was administered, with a response duration of 2 months. A second course of IVIG was given at relapse. Therefore, an immunosuppressive dose of prednisone was started for 2 months, with slow tapering. Neutrophil counts never dropped during the treatment. Two months after the end of corticosteroids the patient relapsed, and started azathioprine, obtaining long-lasting remission. She only relapsed once due to poor adherence to the immunosuppressive treatment, but she then responded again to a short course of IVIG (Figure 1C). Given the peculiarity of these 3 cases, a more detailed ANCA study was conducted (see online Supporting Information for details), and all patients resulted positive for HNE-ANCA. AGC after cocaine exposure has been considered by many authors as secondary to the cocaine adulterant levamisole,1 although this was not confirmed by in vitro studies. Due to its very brief half-life, we were not able to detect levamisole in our patients’ specimens. We have previously identified HNE-ANCA as the most prevalent ANCA specificity among cocaine users and a diagnostic marker differentiating cocaine-induced midline destructive lesions from primary ANCA-associated vasculitis, particularly granulomatosis with polyangiitis.3 However, the association of cocaine-induced AGC with HNE-ANCA has not been described to date. We observed HNE-ANCA in 3 patients with cyclical AGC after cocaine exposure, with the target of this ANCA subtype being neutrophil elastase. A heterozygous mutation in the ELANE gene that encodes for neutrophil elastase causes CyN, leading to cycling AGC by a mechanism that is still partially unclear.6 In our study, all patients showed a cycling recurrence of AGC distant in time from the previous cocaine exposure, and all were positive for HNE-ANCA, suggesting a potential pathogenetic link between HNE-ANCA and acquired cyclic neutropenia. Since HNE-ANCA were demonstrated also during clinical remission, it may be hypothesized that cocaine exposure, rather than being directly toxic on myeloid precursors, stimulates the immune system to produce HNE-ANCA in predisposed individuals. This hypothesis is also suggested by the clinical benefit our patients obtained from immune-suppressive therapy. To the best of our knowledge, this study provides the first evidence of an association between cocaine-induced AGC and HNE-ANCA. The longer follow-up of the exposed patients allowed the demonstration of the self-sustained recurrence of AGC showing a clinical picture suggestive of acquired cyclic neutropenia, in spite of abstention from cocaine exposure. The present study highlights the need for clinical awareness of the potential link between cocaine abuse, the development of HNE-ANCA and a severe disease like cyclic neutropenia. The authors would like to thank all doctors and nurses who provided patients’ care at the Departments of Hematology and Nephrology, ASST Spedali Civili di Brescia, Brescia, Italy. The authors declare that they have no conflicts of interest with the contents of this article. All authors read and approved the final version of the article. Designed research, provided patient care, wrote and edited the article: Schieppati, Rossi, Gregorini Provided patient care: D'Adda, Borlenghi Processed the samples: Lamorgese Conducted and coordinated the experiments and edited the article: Specks Conducted the experiments: Hummel Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.