Abstract
BACKGROUND. In 2011, we discovered the TEMPI syndrome. TEMPI is a rare monoclonal gammopathy of clinical significance (MGCS) with patients displaying five hallmark features of disease: (1) Telangiectasias (primarily involving the upper torso and face), (2) elevated serum Erythropoietin (EPO) levels and erythrocytosis, (3) Monoclonal gammopathy, (4) Perinephric fluid collections, and (5) microscopic Intrapulmonary shunting.
To date, we have identified 52 patients world-wide. TEMPI is an acquired syndrome, and there does not appear to be any geographic, racial, or gender predisposition.
The dramatically elevated serum EPO is striking, with EPO levels often exceeding 500-times the upper limit of normal. These EPO levels are much higher than those seen in EPO-secreting tumors, suggesting a severe dysregulation of the body's hypoxia sensing pathway and of EPO production. High EPO leads to uncontrolled red blood cell production such that patients present with marked erythrocytosis. Patients are commonly misdiagnosed with polycythemia vera and often start programs of therapeutic phlebotomy.
The use of plasma cell directed therapies in several patients (e.g., bortezomib, lenalidomide, daratumumab, teclistamab, autologous transplant) have been used to eradicate the TEMPI MGUS. Remarkably, elimination of the MGUS leads to complete resolution of the other TEMPI syndrome symptoms. This clinical finding has confirmed the monoclonal antibody as the pathologic driver of the disease.
HYPOTHESIS. The TEMPI syndrome is an acquired and reversible disease, driven by the auto-reactive monoclonal gammopathy (MGCS). It stands to reason that the TEMPI syndrome may have a remote pathogenic trigger (e.g., a virus, bacteria, fungi), and that the resulting immune response has some degree of molecular mimicry between the antibody epitope on the pathogen and a partially cross-reactive human membrane protein. We suspect that the antibody has a low affinity to the human target given the very high serum concentration of monoclonal antibody required to drive the other features of the TEMPI syndrome such as EPO production.
EXPERIMENTS. To date, bone marrow aspirates have been successfully collected from 11 patients with the TEMPI syndrome. Plasma cells were isolated, RNA was purified and long read RNA sequencing was performed to identify the exact heavy and light chain sequence of the monoclonal antibody (mAb). These sequences were cloned into expression vectors, permitting the in vitro production of unique, pure, recombinant, TEMPI patient mAbs. These TEMPI mAbs were assayed in vivo and also used to probe pathogen arrays and a membrane proteome arrays.
RESULTS. (1) TEMPI mAbs were injected into wild-type mice to mimic the same serum monoclonal antibody concentration as seen in patients. The TEMPI mAbs increased the serum EPO concentration as well as increasing the hemoglobin and hematocrit. (2) TEMPI mAbs were used to screen a focused pathogen array. This implicated a potential previous exposure to toxoplasmosis as a trigger in two TEMPI syndrome patients. (3) Two TEMPI mAbs were used for flow cytometric screening of a membrane proteome array of ~6000 human membrane proteins. This has generated a candidate list of potential TEMPI syndrome membrane protein auto-antigens.
Ongoing experiments are underway to confirm the antibody target of the TEMPI syndrome monoclonal antibodies. Ultimately, we hope to understand how an extracellular monoclonal antibody can mimic hypoxia and lead to the dysregulated production of EPO.
DISCUSSION. Having followed rare patients with TEMPI syndrome for more than a decade, we are poised to identify the host antigen target of the TEMPI patient monoclonal antibodies. If successful, this will reveal a novel antibody-antigen interaction capable of driving hypoxia independent erythropoietin production.
The TEMPI syndrome is a model of MGCS, where identification of the antibody target may shed light on disease pathogenesis. Our work demonstrates a straightforward pipeline from bone marrow biopsy to recombinant antibody production where patient-specific mAbs can be readily generated for in vitro diagnostic and research approaches.
The TEMPI syndrome has also been a model of international hematology collaboration. In a disease with ~50 patients world-wide, sample collection and research has depended on the generosity and thoughtfulness of colleagues and patients across the globe.
