Page 191 - 2019_06-Haematologica-web

Anti-spacer idiotope profiles in iTTP patients
1, TTP-73 and I-9 idiotopes using the 3 newly developed anti-idiotypic antibodies. In a second step, we stratified the patients according to their anti-spacer idiotope profile.
The 151 iTTP plasma samples were all collected during an acute iTTP episode (see the Online Supplementary Methods for details). All patients presented with severe ADAMTS13 deficiency (< 10% activity) and detectable anti-ADAMTS13 IgG titers. Anti-ADAMTS13 IgG titers ranged from 16 to ≥100 IU/mL (median: 87 IU/mL) (Figure 4). Of the 151 iTTP patients, 34% (52 out of 151) were positive for antibodies with the anti-spacer II-1 idiotope (recognized by anti-idiotypic antibody 17H9) (Figure 4A, red dots) with median anti-spacer II-1 idiotope levels of 47 ng/mL (Figure 4A, red squares). Twenty-five percent (37 out of 151) of the patients were positive for antibodies with anti-spacer TTP73 idiotope (recognized by anti-idio- typic antibody 9G12) (Figure 4B, green dots) with median anti-spacer TTP73 idiotope levels of 174 ng/mL (Figure 4B, green squares). Forty-two percent (63 out of 151) of the patients were positive for antibodies with anti-spacer I-9 idiotope (recognized by anti-idiotypic antibody 7D10) (Figure 4C, orange dots) with median anti-spacer I-9 idiotope levels of 57 ng/mL (Figure 4C, orange squares).
We next stratified the acute iTTP patients according to their anti-spacer idiotope profile (Figure 5). The 8 possible profiles correspond to the presence of either 1, 2, 3 or none of the 3 anti-spacer idiotopes. All 8 anti-spacer idiotope profiles were identified in the iTTP patient cohort (n=151) (Figure 5). In 28% (42 out of 151) of the patients, only one particular idiotope could be detected in the plasma, with 8% (12 out of 151) having the II-1 idiotope (profile 1), 4% (6 out of 151) having the TTP73 idiotope (profile 2), and 16% (24 out of 151) having the I-9 idiotope (profile 3). In 19% (28 out of 151) of the patients, 2 idiotopes were iden- tified in their antibody repertoire, with 5% (7 out of 151) having II-1 and TTP73 idiotopes (profile 4), 10% (15 out of 151) having II-1 and I-9 idiotopes (profile 5), and 4% (6 out of 151) having I-9 and TTP73 idiotopes (profile 6). In 12% (18 out of 151) of the patients, all 3 idiotopes were present in their antibody repertoire (profile 7). In 42% (63 out of 151) of the patients, none of the 3 idiotopes were detected (profile 8).
In conclusion, using the 3 developed anti-idiotypic anti- bodies, we here for the first time unraveled the specific II- 1, TTP73, and I-9 idiotope profiles in iTTP patients and showed that 58% of the patients had antibodies with II-1, TTP73, and I-9 idiotopes in their plasma, and this in dif- ferent combinations, while 42% of the patients were neg- ative for these idiotopes.
Anti-spacer idiotope profiles and their possible link with disease severity
We next investigated whether the identified anti-spacer idiotope profiles (Figure 5) could be linked with disease severity, although the number of patients per profile group was rather low and we only screened for the presence or absence of 3 anti-spacer idiotopes. As a measure of disease severity, we studied disease outcome and applied treat- ment strategy. This part of the study was performed on the 95 patients of the French Reference Center for Thrombotic Microangiopathy, as detailed information on laboratory, clinical and outcome parameters were avail- able for these patients (Online Supplementary Table S2).
We first analyzed whether the anti-spacer idiotope pro- files could be linked with disease outcome. Disease out-
come was previously identified in the patients at time of diagnosis by determining a score defined by Benhamou et al.39 This score (either 1, 2, 3 or 4) is a risk score for early death in TTP based on three factors related to clinical and biological presentation (age, high LDH levels, and cerebral involvement). A score of ≥3 has a positive predictive value for mortality (patients at risk of 30-day mortality after
A
B
C
Figure 4. Total anti-ADAMTS13 IgG titers and anti-spacer II-1, TTP73 and I-9 idiotope concentrations in acute immune-mediated thrombotic thrombocy- topenic purpura (iTTP) patients. (A-C) Total anti-ADAMTS13 IgG titers (IU/mL) were determined via TECHNOZYM® except for three samples (ID 124, 128 and 130) which were determined via an in house developed anti-ADAMTS13 IgG ELISA (§ in Online Supplementary Table S1). Anti-spacer II-1 (red square) (A), TTP73 (green square) (B), and I-9 (orange square) (C) idiotope concentrations were determined by adding patient plasma to coated murine anti-idiotypic anti- body 17H9 (A), 9G12 (B) or 7D10 (C). Bound human autoantibodies were detected using a mixture of anti-human IgG1-4-HRP. A dilution series of human anti-spacer autoantibodies II-1 (A), TTP73 (B) or I-9 (C) was used as a calibra- tion curve to determine idiotope concentrations (ng/mL). Median is represent- ed for total anti-ADAMTS13 IgG titers and II-1, TTP73 and I-9 idiotope concen- trations.
haematologica | 2019; 104(6)
1273