Clinica chimica acta; international journal of clinical chemistry · 2026
Quantification of plasma mutant calreticulin for diagnosis and therapeutic response assessment of peginterferon-α in myeloproliferative neoplasms: A proof-of-concept study.
СтатьяНазвание и аннотация: Перевод в очереди
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Tian Jingjing, Tang Xuemei, Zhou Weiyan, Yang Can, Zhang Qingyun, Wu Quanli, Ma Xiaotong, Tang Gusheng, Guan Ming, Wu Zhiyuan
Аннотация
BACKGROUND: Calreticulin (CALR) exon 9 mutations are drivers in essential thrombocythemia (ET) and primary myelofibrosis (PMF). These mutations lead to extracellular secretion of mutant CALR, but a quantitative plasma assay to leverage this for management is lacking.
OBJECTIVE: To develop and validate a quantitative method based on homemade monoclonal antibody (McAb) targeting mutant CALR for diagnosing ET and PMF, and evaluating therapeutic response in PEG-IFN-α-treated ET patients.
METHODS: We generated a hybridoma clone (Cal26B11) producing anti-mutant CALR McAb and confirmed its reactivity via Western blot, immunofluorescence, and immunohistochemistry. Using Cal26B11 as the capture antibody, we developed a double-antibody sandwich ELISA (DAS-ELISA) to quantify plasma CALR mutant (pCALRmut). We assessed its diagnostic performance in 135 subjects (53 CALR type-1, and 37 type-2 mutant MPN patients, 11 non-CALR-mutated MPN patients, 9 non-MPN hematological malignancies, and 25 healthy donors) and conducted a 2-year follow-up of 49 CALR-mutant ET patients to evaluate its correlation with therapeutic response.
RESULTS: Cal26B11 McAb showed high affinity (Kd = 5.90 × 10-10 M) and specificity for mutant CALR proteins. The established DAS-ELISA demonstrated good linearity (6.25-200 ng/ml) and sensitivity (LOQ: 7.78 ng/mL for type-1; 9.82 ng/ml for type-2 mutants). Using a cut-off of 15.98 ng/mL, the assay discriminated CALR-mutant from wild-type cases with 96.67 % sensitivity and 100 % specificity. Baseline pCALRmut concentrations rose step-wise from ET to pre-fibrotic MF and overt MF (P < 0.0001). Although cross-sectional pCALRmut-VAF was weak, strong within-individual correlations during follow-up highlight its utility for serial monitoring. In patients treated with PEG-IFN-α, pCALRmut decreased in 76.5 % of cases, with a median reduction of 23.04 %, accompanied by a medium-to-long term complete hematological response (CHR) rate of approximately 75 %. Consistently, patients achieving CHR exhibited significantly lower pCALRmut levels than non-CHRs after PEG-IFN-α treatment, demonstrating a correlation between its dynamic reduction and treatment response.
CONCLUSIONS: The Cal26B11-based DAS-ELISA provides a reliable, non-invasive tool for detecting CALR mutations and shows promise for monitoring disease dynamics and treatment response, supporting its potential as a complementary biomarker. These preliminary findings warrant validation in larger prospective studies.
OBJECTIVE: To develop and validate a quantitative method based on homemade monoclonal antibody (McAb) targeting mutant CALR for diagnosing ET and PMF, and evaluating therapeutic response in PEG-IFN-α-treated ET patients.
METHODS: We generated a hybridoma clone (Cal26B11) producing anti-mutant CALR McAb and confirmed its reactivity via Western blot, immunofluorescence, and immunohistochemistry. Using Cal26B11 as the capture antibody, we developed a double-antibody sandwich ELISA (DAS-ELISA) to quantify plasma CALR mutant (pCALRmut). We assessed its diagnostic performance in 135 subjects (53 CALR type-1, and 37 type-2 mutant MPN patients, 11 non-CALR-mutated MPN patients, 9 non-MPN hematological malignancies, and 25 healthy donors) and conducted a 2-year follow-up of 49 CALR-mutant ET patients to evaluate its correlation with therapeutic response.
RESULTS: Cal26B11 McAb showed high affinity (Kd = 5.90 × 10-10 M) and specificity for mutant CALR proteins. The established DAS-ELISA demonstrated good linearity (6.25-200 ng/ml) and sensitivity (LOQ: 7.78 ng/mL for type-1; 9.82 ng/ml for type-2 mutants). Using a cut-off of 15.98 ng/mL, the assay discriminated CALR-mutant from wild-type cases with 96.67 % sensitivity and 100 % specificity. Baseline pCALRmut concentrations rose step-wise from ET to pre-fibrotic MF and overt MF (P < 0.0001). Although cross-sectional pCALRmut-VAF was weak, strong within-individual correlations during follow-up highlight its utility for serial monitoring. In patients treated with PEG-IFN-α, pCALRmut decreased in 76.5 % of cases, with a median reduction of 23.04 %, accompanied by a medium-to-long term complete hematological response (CHR) rate of approximately 75 %. Consistently, patients achieving CHR exhibited significantly lower pCALRmut levels than non-CHRs after PEG-IFN-α treatment, demonstrating a correlation between its dynamic reduction and treatment response.
CONCLUSIONS: The Cal26B11-based DAS-ELISA provides a reliable, non-invasive tool for detecting CALR mutations and shows promise for monitoring disease dynamics and treatment response, supporting its potential as a complementary biomarker. These preliminary findings warrant validation in larger prospective studies.