ALBERT

Description: The mutation rate (μ) is likely to be a critical parameter in leukemogenesis, but has not previously been measured in human myeloid cells. Using the PIG-A gene as a sentinel, we have recently measured the mutation rate in B-lymphoblastoid cell lines (BLCLs) and shown that it is elevated in cell lines derived from lymphoid malignancies. Here we have measured μ in normal human CD34+ cells derived from cord blood samples that have been transduced with an AML-ETO fusion gene, growing under the influence of cytokines. PIG-A is an X-linked gene essential for the synthesis of glycosylphosphatidylinositol (GPI). Due to X-inactivation in females and hemizygosity in males, a single mutation can produce the GPI (−) phenotype. From patients with PNH, it is known that a broad spectrum of mutations can inactivate PIG-A. Mutants do not express GPI-linked surface proteins such as CD55 or CD59 and do not bind to the FLAER reagent (which binds GPI directly), but do express transmembrane proteins (e.g. CD45). To measure the rate of new mutations in vitro, we first flow-sorted each culture to collect CD59(+) cells, in order to exclude pre-existing mutants. Then we expanded the collected cells over 3 weeks; using cell counts, we calculated population doublings in culture (d), which ranged from 5.1 to 7.4. Cells were then stained sequentially with biotinylated FLAER, murine anti-CD55 and anti-CD59 antibodies, rabbit anti-mouse PE and streptavidin-PE secondary conjugates, and CD45-FITC. Live cells were identified by forward and side scatter, propidium iodide exclusion, and expression of CD45. The mutant frequency (f) was calculated as the number of GPI (−) cells [which do not bind anti-CD55, CD59 or FLAER] divided by the total number of cells analyzed. An average of 1.2 million cells were analyzed to detect rare mutants. The mutation rate was calculated by the formula μ = f/d. Among 5 cultures derived from 2 different cord blood samples, the mean mutation rate was 12.4 × 10−7 (range 5.1 to 21.4 × 10−7) per cell division. This value is consistent with mathematical models of the mutation rates in human populations as well as our previous experimental determination of μ in BLCLs. We also analyzed 6 cell lines in which p53 had been disrupted by various methods, including shRNA, a dominant-negative p53 mutant, and introduction of HPV E6/E7. Overall, disruption of p53 resulted in a 30% increase in the mutation rate (p=.02). We conclude that spontaneous mutations in human myeloid cultures are rare but measurable and that disruption of p53 results in a modest increase in μ. We predict that the ability to measure this critical parameter in human hematopoietic cells will facilitate the investigation of the role of specific oncogene and tumor suppressor mutations in inducing hypermutability.

Description: In neoplasms, the presence of chromosomal abnormalities and point mutations is suggestive of genomic instability, but could also be a consequence of selection. While genomic instability may increase the chances that a malignant population acquires adaptive mutations, extremely high mutation rates may not be compatible with cell survival. To investigate the role of genomic instability in lymphoid neoplasms, we have applied a new method for the quantification of the human mutation rate, using the PIG-A gene as a sentinel. PIG-A is essential for the biosynthesis of glycosylphosphatidylinositol (GPI) and is mutated in blood cells of patients with Paroxysmal Nocturnal Hemoglobinuria. A broad range of mutations can produce the GPI (−) phenotype, and because PIG-A is on the X-chromosome, the effect of a single mutation is detectable. Since a host of proteins require GPI for attachment to the cell surface, rare mutants are readily detected by flow cytometry. We have previously shown that PIG-A mutations arise spontaneously in normal donors, and we determined that the mutation rate in normal B cell lines ranges from 2 to 29 per 107 cell divisions. Here we analyzed cell lines derived from: a transformed low grade lymphoma harboring a t(14;18) translocation; a mantle cell lymphoma harboring a t(11;14) translocation; a marginal zone lymphoma; and T cell ALL. Cells were first stained with an antibody specific for CD59 (a representative GPI-linked protein) and pre-existing GPI (−) cells were eliminated from the population by flow cytometric sorting, by gating on the upper 50th percentile of the distribution curve. The collected GPI (+) cells were then returned to culture and the number of cell divisions (d) determined by cell counts. After 3–4 weeks, the frequency (f) of new mutants arising in culture was determined by flow cytometric analysis of a large number of cells (median 2.3 x 106). Cells were stained simultaneously with antibodies specific for at least 3 GPI-linked proteins (e.g. CD48, CD52, CD55, and CD59) as well as a transmembrane protein (e.g. HLA-DR or CD45) to identify live cells. FLAER and proaerolysin-- which bind to GPI-- were used to confirm the phenotype. The frequency of mutants was determined by the number of GPI(−) cells divided by the number of GPI(+) cells analyzed, and the mutation rate (μ) was calculated with the formula μ = f ÷ d. We demonstrated a high mutation rate in 3 out of the 4 cells lines: 1750 x 10−7 (transformed lymphoma), 335 x 10−7 (mantle cell lymphoma), 112 x 10−7 (T cell ALL). Of note, the mutation rate was normal (4 x 10−7) in the marginal zone lymphoma—consistent with this being an indolent neoplasm. These data support the hypothesis that an elevated mutation rate is part and parcel of aggressive neoplasms and demonstrate that a 2-log elevation in this parameter is compatible with cell survival. With this model, it may be possible to predict the development of mutations that confer chemotherapy drug resistance.

Description: Abstract 4408 PNH is a hemolytic disorder resulting from dysregulation of complement on the rbc, and is associated with immune mediated marrow failure and marked hypercoagulability. Thrombosis can be prevented with anticoagulation or the complement inhibitor eculizumab, and it often involves the hepatic, portal, and splenic veins. This can result in an increase in portal pressure and complications that we term the “Thrombosis-Splenomegaly-Thrombocytopenia” syndrome (TST). TST is frequently associated with abdominal pain, and thrombocytopenia may be simultaneously exacerbated by marrow failure. Particularly, thrombocytopenia can complicate efforts at anticoagulation, leaving the patient exposed to the risk of further events. In some patients thromboses can reversed with tPA, but others will not be candidates because they have presented late after their thromboses. Ablating splenic function would be desirable: however, with a high risk of perioperative thrombosis, surgical splenectomy may be hazardous, particularly in patients who have already had thrombosis, and will confer a risk of sepsis. Here we report on 4 patients with PNH and late-presenting TST. We referred these 4 patients for selective splenic artery embolization (SSAE), which involves cannulating branches of the splenic artery– beyond the hilum– and introducing gelfoam and microcoils. We planned a multi-session stepwise approach: we started with the inferior branches of the splenic artery, to decrease the risk of pleural effusions. We planned to infarct no more than 1/3 of the spleen at any one time and to allow weeks to months for recovery. We routinely administered vaccinations and discontinued anticoagulation temporarily, and patients were given prophylactic antibiotics, fluids, analgesics, and antipyretics, and they were observed in the hospital with a back-up surgical team. Prior to the procedure, the median platelet count was 17 and the median spleen size was 22 cm. Patients 1–4 were treated with 3,2,1, and 3 procedures respectively, which resulted in a significant reduction in spleen volume in all 4 patients. The post procedure platelet counts were respectively 123, 12, 44, and 90, which represented a significant increase for all patients except patient 2, who remained thrombocytopenic; since there was evidence of bone marrow failure, she underwent a successful unrelated SCT. Patients 1,2, and 4 all had had abdominal pain before the procedure, which very significantly improved after recovery from the procedure, which we attribute to decreased venous return from the spleen into the portal circulation. Patients 1–3 were treated before eculizumab was available and patient 1, 3, and 4 are now on it. Patient 4 is a special case in that she had been on eculizumab for several months prior to the SSAE, but had had only a partial reduction in the red cell transfusion requirement; C3d deposition on rbcs was documented by flow cytometry, and it was thought that extravascular hemolysis was limiting her response to eculizumab, as has been described. Of note, her hemoglobin began to rise after the embolization procedure, concurrent with the increase in the platelet count, and a significant further reduction in the red cell transfusion requirement, suggesting that partially reducing splenic function markedly reduced C3-mediated extravascular hemolysis. Of the 9 procedures performed on these 4 patients, only one was complicated by a clinically significant left sided pleural effusion, which was drained by thorascope. All patients are doing well between 3.5 and 11 years after their procedures. We conclude that SSAE, when performed by an experienced interventional radiologist, is relatively safe in patients with PNH and TST, it produces sustained correction of hypersplenism without the risk associated with surgery or the asplenic state, and can be of benefit to patients on eculizumab who have hypersplenism. Pt Age at diagnosis Lowest plt count pre-SSAE Spleen size (cm) pre-SSAE Abdominal pain pre-SSAE Bone marrow Plt count post SSAE Spleen size post SSAE Abdominal pain after recovery from SSAE Complications of SSAE Follow up (yrs) 1 30 42 21 + Hypercellular Megakaryocyte aggregates 123 12 No Abd pain and fever 10 2 18 19 19 + Hypocellular Megas reduced 12 12 No Pain, pleural effusion SCT 3 27 14 36 – Erythroid hyperplasia Megas adequate 44 NA No Abd pain 11 4 26

Description: Abstract 4239 Paroxysmal Nocturnal Hemoglobinuria (PNH) is associated with clonal expansion of stem cells with an acquired somatic PIG-A mutation. The PIG-A gene is essential for the biosynthesis of glycosylphosphatidylinositol (GPI), and mature blood cells derived from the PNH stem cell clone exhibit a loss of all proteins that require this structure for attachment to the cell surface, notably the complement inhibitors CD55 and CD59. The loss of these proteins on the surface of red cells is responsible for hemolysis, and thrombosis may be the consequence of the loss of these proteins from the surface of platelets: both hemolysis and thrombosis can be attenuated by anti-complement therapy. Thrombosis can occur, however, despite anticomplement therapy and despite anticoagulation and has been historically the most important determinant of death in patients with PNH. Some patients will present with thrombosis and some patients will not be candidates for anticoagulation or anticomplement therapy: therefore treatment of thrombosis remains an important part of the management of PNH patients. Thrombolysis with tissue plasminogen activator (tPA) in PNH has been reported in small series or case reports, generally with encouraging outcomes. Here we report what we believe to be the largest series on the outcome of the use of tPA. Of 38 patients with PNH who had at least one thrombotic event, 13 were thought to have had a thrombus sufficiently recent to be amenable to fibrinolysis; of these, 4 patients were regarded as ineligible on account of active hemorrhage or high risk of hemorrhage. Of the 9 eligible patients who received tPA, all of whom had potentially life-threatening thromboses, 3 also required tPA on subsequent hospitalizations, and the results of a total of 15 hospitalizations during which tPA infusions were given are reported here. tPA was given in the ICU by systemic infusion through a peripheral vein at a dose of 1 mg/kg delivered over 24 hours, with anticoagulants withheld temporarily during this time. Response was monitored by follow-up imaging, and most patients required several 24 hour infusions. Platelets were given for thrombocytopenia and FFP was given to reverse oral anticoagulation or when low circulating plasminogen was documented. On all 15 occasions a radiologically documented response was obtained, including reversal of thrombosis in hepatic veins, portal veins, the IVC, cerebral dural venous sinuses, and an intrahepatic portocaval shunt. Among the 15 courses of tPA, serious hemorrhagic complications developed in 3 cases. At last follow-up visit, of the 9 patients treated, 3 have expired, one patient (who has been non-compliant with post treatment anticoagulation and anticomplement therapy) was in good clinical condition despite extensive residual occlusions, and 5 others were in good to excellent condition in terms of clinical and radiological outcome. The only patient in whom tPA may have contributed to a fatal outcome also had complications of ‘heparin induced thrombocytopenia with thrombosis’ (HITT), which we diagnosed in a milder form in 3 additional patients. The other two fatalities were associated with bowel edema (probably due to progressive small vessel thrombosis) in one case, and a progressive concurrent myeloproliferative disorder associated with a JAK2 mutation in the other case. On the other hand, we feel tPA must be credited as having been immediately life-saving in 2 patients who had been moribund with Budd-Chiari syndrome, and in one who had impending renal failure associated with an IVC thrombosis. Given the high incidence of HITT, we favor the use of direct thrombin inhibitors or fondaparinux rather than heparin products in patients with PNH. Given the high mortality and morbidity associated with thrombosis in PNH patients, and given the excellent radiographic responses, we conclude that, in spite of the risk of hemorrhage, thrombolysis is strongly indicated to reverse intra-abdominal and intracranial thromboses. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1262 PNH is characterized by circulating blood cells that are deficient in the surface expression of proteins that require glycosylphosphatidylinositol (GPI). The loss of the GPI-linked complement inhibitors CD55 and CD59 on the red cell results in complement mediated hemolysis, and this defect on the surface of platelets is likely responsible for the marked hypercoagulable state seen in PNH. The loss of GPI-linked proteins is due to the clonal expansion of a stem cell with an acquired somatic mutation in the PIG-A gene, which is responsible for an early step in the production of GPI. In PNH, as in aplastic anemia, there is a decreased stem cell and erythroid progenitor pool, there are oligoclonal T cell expansions, there is an HLA allele association, and cytopenias can occur, which respond to immunosuppression. Normal individuals harbor occult populations of cells with the PNH phenotype and genotype, and there is evidence that PIG-A mutations are growth neutral in animals. These findings are all supportive of the immune escape model, which posits that the abnormal environment associated with marrow injury represents the “second hit” which selects in favor of the PNH clone. However, there is a long-standing interest in finding second genetic hits. Indeed, others have reported rearrangements of HMGA2 in PNH; we have reported a series where 24% of patients had a cytogenetic abnormality and another series where 3 out of 29 patients with PNH had the JAK2V617F mutation and an MPN/PNH overlap syndrome. Here we have investigated a series of 17 patients with PNH to see whether we would find mutations in TET2, which is commonly mutated in myeloid disorders. We separated granulocytes from patients with at least 40% GPI-negative granulocytes, extracted DNA, which was subjected to whole genome amplification, followed by bi-directional sequencing using a dye terminator approach. In 11 out of 17 patients, we identified the 5284A〉G, I1762V variant, with an allele frequency of 41% (95% confidence interval 25% to 58%) compared with 22% in the NCBI dbSNP database. In 4 of the patients we identified the 5162T〉G, L1721W variant, with an allele frequency of 12%, which was not significantly different from the NCBI dbSNP database (9%). In 3 patients, we identified the 1088C〉T, P363L variant, with an allele frequency of 9%, which also was not significantly different from the database (3%). However, one remarkable patient was heterozygous for all three of these SNPs– and was also heterozygous for a previously undescribed nonsense mutation, 2697T〉A, Y899X. This mutation was seen in 3 separate sequencing reactions, and the alleles were present in approximately a 1:1 ratio. This mutation occurs at the 3' end of exon 3, a region where chain terminating mutations have been previously reported. Of note, it has been recently reported that heterozygous disruption of Tet2 in a mouse model results in an increase in the stem cell compartment as determined by a competitive repopulation assay, suggesting that this patient's TET2 mutation may have partly contributed to the advantage that the PNH clone demonstrated. Because 100% of her granulocytes were GPI–negative, it was not possible to be certain which abnormality came first. This patient's diagnosis of severe hemolytic PNH had been first confirmed 13 years ago, and she has a prior history of intra-abdominal thromboses and a DVT. She is now doing well on both coumadin and eculizumab. Unlike patients with a concurrent JAK2 mutation, she does not have features of an MPN, but her blood counts are higher than most of the other patients in this series, with a WBC of 7.7 compared with a median of 3.7 for the other patients, a platelet count of 321 compared with a median of 131, and a reticulocyte count of 247,000 compared with a median of 132,000. In conclusion, inactivating TET2 mutations, like activating JAK2 mutations, may contribute to the expansion of PNH clones in a subset of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of the hematopoietic stem cell (HSC). Somatic mutations in thePIG-A gene result in the deficiency of several glycosylphosphatidylinositol-linked proteins from the surface of blood cells. This explains intravascular hemolysis but does not explain the mechanism of bone marrow failure that is almost invariably seen in PNH. In view of the close relationship between PNH and idiopathic aplastic anemia (IAA), it has been suggested that the 2 disorders might have a similar cellular pathogenesis, namely, that autoreactive T-cell clones are targeting HSCs. In this paper, we searched for abnormally expanded T-cell clones by size analysis of the complementarity-determining region 3 (CDR3) in the beta variable chain (BV) messenger RNA (mRNA) of the T-cell receptor (TCR) in 19 patients with PNH, in 7 multitransfused patients with hemoglobinopathy. and in 11 age-matched healthy individuals. We found a significantly higher degree of skewness in the TCR BV repertoire of patients with PNH, compared with controls (R2 values 0.82 vs 0.91,P 

Description: Abstract 3994 Myeloma cells can harbor ∼ 35 mutations, whereas according to Loeb's model, if 〉 2 oncogenic mutations are required for the development of a malignancy, then an elevation in the mutation rate would be required for these mutations to occur in the same cell. Indeed, there is evidence that hypermutability in myeloma may be a result of abnormal homologous recombination or activation induced cytidine deaminase. An alternative model is that successive rounds of clonal selection in an expanding pre-malignant population could result in an accumulation of oncogenic mutations. Using our method for the detection of rare cells that have an acquired somatic mutation of the PIG-A gene, we have previously demonstrated hypermutability in some but not all lymphoma and myeloma cell lines, and we have recently shown that samples of ex vivo blasts from ∼ 50% of patients with ALL demonstrate hypermutability. Here we have hypothesized that genomic instability could be identified in myeloma. PIG-A encodes an enzyme that is necessary for the biosynthesis of glycosylphosphatidylinositol (GPI) and is particularly promising as a sentinel gene for spontaneous mutations because it is X-linked, and thus a single mutation can produce the mutant phenotype. PIG-A is mutated in Paroxysmal Nocturnal Hemoglobinuria (PNH), and it is known from this condition that a broad spectrum of inactivating mutations can produce the PNH phenotype, which is a loss of all GPI-linked surface proteins. PIG-A mutations do not affect transmembrane proteins and are growth-neutral under almost all circumstances. The PNH phenotype can be readily detected by flow cytometry, using antibodies specific for GPI-linked proteins (e.g., CD48, CD55, and CD59), as well the FLAER reagent, which binds directly to GPI. In order to quantitate the frequency of myeloma cells with the PNH phenotype, we analyzed thawed ficolled samples from patients with a heavy burden of myeloma cells in the marrow. Cells were stained sequentially with FLAER-Alexa 488, then with a mixture of murine anti-CD48, anti-CD55, and anti-CD59 antibodies, followed by FITC-conjugated rabbit anti-mouse immunoglobulin, followed by a PE-conjugated antibody specific for a myeloma-specific transmembrane protein–either CD38 or CD138. Live myeloma cells were identified by forward/side scatter and propidium iodide exclusion and expression of CD38 or CD138. Using this approach, in previous studies we have seen that cells with the PNH phenotype have a low FITC/FLAER fluorescence, defined as 〈 4% of the level of the GPI (+) cell population, after gating for cells with at least 10% of the PE fluorescence of the GPI (+) population. For a negative control, we analyzed 2 non-malignant B-lymphoblastoid cell lines (BLCLs) from normal donors, and for a positive control, we analyzed the mantle cell lymphoma cell line HBL2A (in this case using CD45-PE to identify transmembrane proteins). The normal BLCLs demonstrated a frequency of PNH cells of 6.3 × 10−6 and 18.4 × 10−6, which is in the range that we have previously reported for BLCLs and granulocytes from normal individuals. These values are also similar to the frequency of spontaneously arising phenotypic variants in normal donors using other genes. In contrast, as we have previously reported, the mantle cell line demonstrated a markedly higher frequency of cells with the PNH phenotype– 1034 × 10−6. Of the involved marrow samples analyzed, there were at least 2 distinct groups. One group, representing 14 of the 20 samples (70%), demonstrated a mutant frequency that is comparable to non-malignant cell populations, with a median value of 9.5 × 10−6 (range 2.4 to 37 × 10−6). The remaining 6 samples (30%) demonstrated a markedly increased frequency of PNH cells, with a median value of 90 × 10−6 (range 73 to 11,763 × 10−6). Most of the samples analyzed were obtained from patients who had received prior therapy, but one of the samples demonstrating a very high frequency of PNH cells (1314 × 10−6) was derived from a patient who had not had prior therapy and was known to have had an abnormality of p53 based on FISH. This data demonstrates that an increase in inactivating mutations—as determined by this assay– is not essential for the development of myeloma, but it does seem to be a common feature of this condition. This flow-based assay could be applied at the time of diagnosis to facilitate investigations as to whether hypermutability correlates with outcome in patients with myeloma. Disclosures: No relevant conflicts of interest to declare.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the presence in the patient's hematopoietic system of a large cell population with a mutation in the X-linked PIG-A gene. Although this abnormal cell population is often found to be monoclonal, it is not unusual that 2 or even several PIG-A mutant clones coexist in the same patient. Therefore, it has been suggested that the PIG-A gene may be hypermutable in PNH. By a method we have recently developed for measuring the intrinsic rate of somatic mutations (μ) in humans, in which PIG-A itself is used as a sentinel gene, we have found that in 5 patients with PNH, μ ranged from 1.24 × 10–7 to 11.2 × 10–7, against a normal range of 2.4 × 10–7 to 29.6 × 10–7 mutations per cell division. We conclude that genetic instability of the PIG-A gene is not a factor in the pathogenesis of PNH.

Description: Paroxysmal Nocturnal Hemoglobinuria is a stem cell disorder characterized by an acquired somatic mutation in the PIG-A gene. This results in a loss of GPI-linked complement inhibitors, CD55 and CD59, on the surface of red blood cells. Untreated patients experience chronic intravascular hemolysis and hemoglobinuria mediated by complement, as well as acute attacks. The lack of these complement inhibitors on platelets may lead to their activation and may explain the marked hypercoagulable state in this disorder. Eculizumab, a humanized monoclonal antibody targeting the C5 component of complement, prevents the formation of the C5b-9 complex and results in a major reduction in red cell transfusion requirements, a marked decrease in thrombotic events, and an improvement in quality of life. It was recognized soon after the introduction of this therapy that patients would often maintain an elevation in the reticulocyte count despite marked improvements in the LDH, and that some patients would have only a partial normalization of the hemoglobin--in some cases accompanied by an increase in bilirubin levels. It is now understood that in patients treated with C5 inhibition, C3 degradation products can accumulate on the red cell surface, resulting in opsonization and extravascular hemolysis. Based on this model, it would be expected that treated patients with PNH would now resemble those with other forms of extravascular hemolysis, with some of the same complications. Indeed, a rising ferritin has been reported in some patients, but until now, there have been only a few case reports of gallstone formation, and only one report commented on whether the stones were comprised of bilirubin. Here we review a set of 51 patients with PNH (31 women and 20 men) treated with eculizumab for a total of 280 patient years. There have been 7 cases of symptomatic cholelithiasis requiring surgery (5 women and 2 men), as well as one woman with a radiologically abnormal gallbladder removed at the time of a hemicolectomy for colon cancer. In four of these cases we have photographs of the surgical specimen: 3 patients had dark stones and/or debris, and one patient had stones with a mixed bilirubin/cholesterol appearance. For patients who had cholecystectomies at outside institutions, the pathology report indicated small bilirubin stones in one case, there was a verbal report of dark gallstones in another, and in two cases the nature of the stones is not known. Based on a Kaplan-Meier curve, the risk of gallstones leading to a cholecystectomy after 10 years of treatment was estimated to be 22.5% (95% CI 10%-46%) and did not differ based on gender. We then considered that markers of hemolysis might predict gallstone disease. The median bilirubin was only slightly higher in those with gallstones (2.1 vs 1.6 mg/dl, p = 0.06, one sided rank sum test), the median reticulocyte count was somewhat higher (303,500 versus 206,000 per μl, p=0.04, one sided rank sum test), and the median ferritin levels were approximately the same in both groups (361 versus 291, normal 10-143 ng/ml p=0.16). The median age at the time of the event or last follow-up was 46 years in both groups. All of the patients have recovered after their surgery. However, one of these patients had a complicated intraoperative course due to significant varices surrounding the gallbladder associated with cavernous transformation of the portal vein, due to prior thrombotic complications of PNH. This patient had presented with severe symptoms of cholelithiasis, including bacteremia from a biliary source. Another patient required a cholecystostomy tube due to severe gallbladder wall inflammation several weeks before the operation was performed. Another patient developed biochemical evidence of pancreatitis shortly before the procedure. We believe that bilirubin stones, a predictable consequence of extravascular hemolysis, represent, in addition to meningococcal infection and iron overload, a potentially significant side effect of inhibition of C5 in patients with PNH. This should be discussed with patients prior to initiation of therapy, and physicians should have a low threshold for obtaining appropriate imaging if a patient on a C5 inhibitor experiences complaints or laboratory evidence suggestive of gallstone disease. Disclosures No relevant conflicts of interest to declare.