Increased Incidence of Clonal Hematopoiesis in Lung Transplant Recipients Involves DNA Damage Response Genes

Lung transplant recipients receive lifelong immunosuppression that includes a calcineurin inhibitor (tacrolimus or cyclosporine), an anti-proliferative agent (mycophenolate or azathioprine) and corticosteroids. They are also known to be at increased risk for a broad spectrum of adverse hematologic events. This includes relatively common complications such as neutropenia and other cytopenias, which occur in over half of patients, as well as rarer events such as de novo hematopoietic malignancy. Certain patterns of clonal hematopoiesis, the expansion of unique somatic clones in hematopoietic stem cells, are known to be associated with cytotoxic therapy and the development of hematologic malignancies. We hypothesized that the stress of transplantation and subsequent immunosuppression provides an environment whereby a unique pattern of clonal hematopoiesis emerges. To evaluate this hypothesis, we designed a custom panel of 59 genes using an error-corrected sequencing assay capable of detecting variant allele frequency (VAF) as low as 0.01. We characterized the overall burden and distribution of clonal hematopoiesis and compared differences among lung transplant recipients (n=73) and age-matched healthy controls (n=19). Clonal hematopoiesis was identified in 51/73 (69.9%) of lung transplant recipients. This was significantly higher than the frequency in the age-matched healthy controls (6/19,31.6% p=0.0071). Additionally, 27/51 (52.9%) of patients with clonal hematopoiesis had multiple variants. The increase in clonal hematopoiesis was mainly due to mutations in DNA damage response (DDR) genes (ATM, PPM1D, SRCAP or TP53), with 29/73 (39.7%) of lung transplant recipients carrying one or more mutation compared with 1/19 (5.3%) of age-matched healthy controls (5.3%, p=0.029). No significant difference in the frequency of clonal hematopoiesis due to non-DDR genes was also observed (48% vs. 26.3%, p=0.121). We first evaluated the relationship between clonal hematopoiesis and lung transplant indication. This is relevant, since interstitial lung disease (ILD), a common indication for lung transplantation, is associated with telomeropathies which are also linked to bone marrow failure and clonal hematopoiesis. However, the frequency of DDR clonal hematopoiesis in ILD patients (17/39, 44%) was similar to that observed in patients with COPD (9/20, 45%). We next investigated the association of immunosuppression with clonal hematopoiesis. Overall, 30/48 patients on mycophenolate (MPA) and 13/19 patients on azathioprine (AZA) had at least 1 clonal hematopoiesis mutation. We found no significant difference in overall clonal hematopoiesis frequency among patients on MPA vs. AZA. However, the frequency of DDR clonal hematopoiesis was significantly higher in patients on AZA (OR 4.04, 95% CI 1.22-13.38, p=0.022) than in patients on MPA. Moreover, when we assessed clonal hematopoiesis burden via Poisson regression, it was increased in patients receiving AZA (1.68, 95% CI 1.08-2.59, p=0.020) when compared to patients on MPA. All lung transplant recipients were maintained on tacrolimus but one, so associations with type of calcineurin inhibitor could not be assessed. Finally, we assessed clonal hematopoiesis in recipients who developed neutropenia (n=24) and found no significant association. To the best of our knowledge, we report the first evidence of increased clonal hematopoiesis in a solid organ transplant population. Our data indicate that azathioprine therapy is associated with the expansion of hematopoietic clones carrying variants in DDR genes. However, azathioprine therapy often represents a failure of MPA therapy, typically due to either hematologic or gastrointestinal toxicity. Therefore, the association noted might reflect MPA intolerance rather than azathioprine therapy. Nevertheless, prior studies show that treatment with genotoxic agents, such as chemotherapy or radiation therapy, provide a fitness advantage to hematopoietic stem/progenitor cells carrying DDR gene variants. Whether azathioprine vs. MPA therapy confers a similar fitness advantage is currently under investigation. Further investigation also is warranted to determine if the presence of clonal hematopoiesis influences lung transplant outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

AB0858 SUCCESSFUL TERM PREGNANCY IN A PATIENT WITH GRANULOMATOSIS WITH POLYANGIITIS AFTER UNDERGOING RITUXIMAB THERAPY - CASE REPORT

Background:Granulomatosis with polyangiitis (GWP) is a relatively rare disease with the lack of experience in the management of pregnancy currently. Potentially negative factors of pregnancy prognosis are both disease-related and teratogenic effects of specific vasculitis therapy [1].Objectives:To describe a clinical case of Successful term pregnancy in a patient with Granulomatosis with polyangiitis after undergoing rituximab therapy.Methods:19 years-old woman was admitted to the rheumatology department of Clinical Regional Hospital After Mechnicov in 2013 with the debut of Granulomatosis with polyangiitis. Her disease course included recurrent rhinitis and sinusitis, Granulomatosis of the eye orbits. Positive ANCA titers were present in the disease onset. In the preceding 7 years she had been treated with corticosteroids, cyclophospan (discontinuated in 2017 because of lack of efficiency), than azathioprine. Aseptic necrosis of the both femoral head was estimated on fifth disease year. In 2018 azathioprine therapy had been discontinued owing to the disease progression and biological agents were prescribed – rituximab with positive effect. The disease remission was achived by rituximab therapy, the patient was warned about the need for contraception. The last dose of rituximab was introduced in February 2020. However, in March 2020, the patient reported pregnancy and therapy was discontinued. At the onset of pregnancy she was treated with 6 mg oral methylprednisolone. Daily and this dose was stable till all pregnancy period. Fetal growth assessment, congenital abnormalities screening test, and laboratory tests for gestational diabetes and preeclampsia were unremarkable during all gestational trimesters. The ANCA titers remained negative and renal function was normal and there was no flare during all pregnancy period.Results:At 38 weeks’ gestation a spontataneous labor started without induction. A 3270-g healthy boy delivered with APGAR score of 9 at 1 minute and 5 minutes. Postpartum the disease remained in remission, and the patient was maintained on corticosteroids. The infant was healthy, with normal development.Conclusion:Thus, the use of biological agents therapy in patients with GWP shows possible ways to safe reproductive potential with disease remission achievement.References:[1]Daher A, Sauvetre G, Girszyn N, Verspyck E, Levesque H, Le Besnerais M. Granulomatosis with polyangiitis and pregnancy: A case report and review of the literature. Obstetric Medicine. 2020;13(2):76-82. doi:10.1177/1753495X18822581Disclosure of Interests:None declared

Blood CD3-(CD56 or 16)+ natural killer cell distributions are heterogeneous in healthy adults and suppressed by azathioprine in patients with ANCA-associated vasculitides

Background Cytotoxic Natural Killer (NK) cells are increasingly recognized as a powerful tool to induce targeted cell death in cancer and autoimmune diseases. Still, basic blood NK cell parameters are poorly defined. The aims of this study were 1) to establish reference values of NK cell counts and percentages in healthy adults; 2) to describe these parameters in the prototype autoimmune disease group ANCA-associated vasculitis (AAV); and 3) to investigate whether NK cell counts and percentages may be used as activity biomarkers in the care of AAV patients, as suggested by a preceding study. Methods CD3-(CD56 or 16)+ NK cell counts and percentages were determined in 120 healthy adults. Lymphocyte subset and clinical data from two German vasculitis centers were analyzed retrospectively (in total 407 measurements, including 201/49/157 measurements from 64/16/39 patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), respectively). Results CD3-(CD56 or 16)+ NK cell counts and percentages in healthy adults were highly variable, not Gaussian distributed and independent of age and sex. NK cell percentages ranged from 1.9 to 37.9% of lymphocytes, and were significantly more dispersed in AAV (0.3 to 57.6%), while the median percentage was not different between AAV and healthy donors. In contrast, median NK cell counts were significantly lower in AAV compared to healthy donors. Sub-group analyses revealed that NK cell counts were low independent of AAV entity and disease activity. Azathioprine therapy was associated with significantly lower NK cell counts and percentages compared to non-azathioprine therapies. In 13.6% of azathioprine-treated patients, percentages were </= 1% which may be interpreted as temporary NK cell deficiency. NK cell counts and percentages could not separate active from inactive AAV. Conclusions NK cell counts and percentages in blood are heterogeneous and can presently not be recommended as biomarker in clinical care of AAV patients. Azathioprine treatment was associated with significantly low NK cells. These findings may be relevant for the development of drugs that aim at exploiting NK cell cytotoxicity and may help to identify patients at risk to develop malignant or infectious co-morbidities.

Severe systemic scleroderma with multiple organ involvement in a 45-years-old patient

Systemic scleroderma has inflammation and fibrosis playing a crucial role and eventually leading to severe functional failure and damage of multiple organs. Three primary mechanisms are mainly involved in the pathogenesis of scleroderma: vascular anomalies, excess fibrosis, and autoimmune phenomenon. The present case study shows a severe cardiac dysfunction with low cardiac output syndrome (LCOS) with cardiac, hepatic and renal impairment, peripheral ischemia, neurologic symptoms associated or even aggravated by a probable severe toxicity to azathioprine therapy. Currently there is no cure for systemic sclerosis, the aims of treatment are- as recommended also by EULAR: to relieve symptoms, to prevent the condition from progressing, as much as possible, to detect and treat complications early and to minimize any disability.

An unusual case of reactivated latent pulmonary cryptococcal infection in a patient after short‐term steroid and azathioprine therapy: a case report

Background Cryptococcus is one of the major fungal pathogens infecting the lungs. Pulmonary cryptococcal infection is generally considered a community-acquired condition caused by inhalation of dust contaminated with fungal cells from the environment. Here, we report a case developing pulmonary cryptococcosis 3 months after hospital admission, which has rarely been reported before. Case presentation A 73-year-old female patient who was previously immunocompetent experienced persistent dry cough for 2 weeks, 3 months after admission. Chest computed tomography (CT) showed a new solitary pulmonary nodule developed in the upper lobe of the left lung. Staining and culture of expectorated sputum smears were negative for bacteria, acid-fast bacilli, or fungus. The patient then underwent biopsy of the lesion. Histopathology findings and a positive serum cryptococcal antigen titer (1:8) indicated pulmonary cryptococcosis. Daily intravenous 400 mg fluconazole was administered initially followed by oral fluconazole therapy. Follow-up chest CT after 3 months of antifungal therapy showed complete disappearance of the pulmonary nodule. Respiratory symptoms of the patient also resolved. A complete investigation excluded the possibility of a patient-to-patient transmission or primarily acquiring the infection from the hospital environment. Based on the patient’s history of exposure to pigeons before admission and recent steroid and azathioprine use after admission for the treatment of myasthenic crisis, reactivation of a latent pulmonary cryptococcal infection acquired before admission, in this case, is impressed. Conclusions Although rarely reported, pulmonary cryptococcal infection should be included in the differential diagnosis of hospitalized patients with respiratory symptoms, especially in those with predisposing risk factors. Chest image studies and further surgical biopsy are needed for confirmation.

Tuberculous perihepatic abscess and neurosarcoidosis: report of 2 uncommon manifestations of 2 common granulomatous diseases in 1 patient

Infections caused by pathogens of the Mycobacterium tuberculosis complex, i. e., tuberculosis (TB), and the non-infectious, autoimmune disease sarcoidosis are among the most common granulomatous diseases worldwide. Typically, the lung is the primary site of infection and manifestation, respectively which makes the two diseases important differential diagnoses. Both diseases can affect virtually all organ systems, albeit with significantly lower incidence. Case presentation We report the case of a 50-year-old Indian man presenting with a tuberculous perihepatic abscess and a systemic inflammatory response after being diagnosed with neurosarcoidosis presenting as a single granuloma in the frontal lobe with lymphadenopathy in 2014. On day of admission the patient presented with right upper abdominal pain and fever for two weeks. With increased inflammatory parameters in serum and after finding of external CT images, a perihepatic abscess was suspected. This encapsulated cave was drained percutaneously under CT control. A high concentration of acid-fast rods was detected using ZN, PCR was positive for M. tuberculosis. Several samples of sputum and urine were microscopically negative but yielded growth of Mycobacteria after four weeks. Discussion This is a case presenting with two different granulomatous diseases, each of which manifested itself in an atypical form. The tuberculous liver abscess might either be explained as a flare-up of latent tuberculosis under azathioprine therapy or as a reinfection acquired during one of several visits in the high-prevalence country India. In addition, it must be discussed whether the cerebral granuloma in 2014 could have been an early stage of tuberculous granuloma. Sensitivity of ZN staining is significantly reduced in cerebral samples, and negative PCR-results might be due to low germ load or methodical issues, e. g., decreased sensitivity in formalin fixated samples.

Immunomodulation with Azathioprine therapy in Rasmussen syndrome: A multimodal evaluation

Objective:To verify safety and efficacy of the corticosteroid-sparing drug Azathioprine (AZA) in Rasmussen syndrome (RS), we retrospectively analyzed a cohort of RS patients recruited in a single pediatric neuroscience center.Methods:We compared outcomes in 30 RS patients who received AZA with 23 patients who were not treated with this drug. We used a multimodal approach to correlate therapy with clinical features (seizures, epilepsia partialis continua [EPC], hemiparesis) and neuroimaging markers of progressive brain atrophy.Results:AZA was well tolerated; only one patient discontinued treatment due to pancytopenia. In 27/30 AZA patients, all of whom were corticosteroid responders, corticosteroid therapy could be weaned or reduced without worsening of seizures in 89%. AZA patients had a lower prevalence of EPC (42% vs. 67% in controls) and hemiparesis (64% vs. 92%, respectively). Cox regression showed for the AZA group compared to controls a delayed time to: 1) EPC (of about 2 years, Exp(B)=0.295, 95%CI[0.108, 0.807];p=0.017), 2) hemiparesis (about one year, Exp(B)=0.315, 95%CI[0.137, 0.724];p=0.007), and 3) surgery (about 2 years, Exp(B)=2.068, 95%CI[1.012, 4.227];p=0.046). However, there were no group differences in cognitive decline over time (IQ change per year) or in hemispheric grey matter atrophy on serial MRI scans.Conclusion:AZA treatment appears to slow clinical progression of Rasmussen syndrome in steroid responders; this will give most advantage in patients in the early stages of the disease in whom surgical decision-making may require further time.Classification of Evidence:This study provides Class III evidence that for pediatric RS patients AZA is well tolerated and slows hemiparesis and appearance of EPC.