Baseline Thrombopoietin Level is Associated with Platelet Count Improvement in Thrombocytopenic Chronic Hepatitis C Patients After Direct-Acting Antiviral Agent Therapy

in C (DAA). Thrombopoietin (TPO), a thrombopoietic cytokine, is involved in megakaryocyte maturation and platelet production. 18 TPO is primarily produced in the liver and its level is affected by liver brosis stage and platelet turnover. 3, 13–15,19 This partly accounts for thrombocytopenia in CLD patients. 3 TPO mimetics have been shown to improve platelet count in thrombocytopenic CLD patients. 20 Data on the relationship between TPO and rapid thrombocytopenia improvement after DAA therapy has not been well reported. Hence, this study investigated the association between baseline (BL) TPO and platelet count improvement after HCV clearance.


Background
Thrombocytopenia (de ned as < 150 × 10 3 /µL) in patients with chronic liver disease (CLD) is relatively common, especially in chronic hepatitis C (CHC)-infected patients. The prevalence is about 6% in CLD patients, 24% in CHC patients, and up to 78% in cirrhotic patients. [1][2][3] Sustained virologic response (SVR), achieved by interferon-based therapy, for CHC-related advanced liver brosis improves thrombocytopenia after a long-term follow-up. 4,5 This was thought to be associated with an improved brosis stage and portal hypertension. 4,5 Reportedly, thrombocytopenia rapidly improves in CHC patients receiving directacting antiviral agents (DAA). [6][7][8][9][10] However, this improvement, within short-term follow-up, is less likely due to changes in the brosis stage. 11 It suggests that factors other than brosis may play important roles, such as hypersplenism, thrombopoietic cytokines, antiplatelet antibody, or direct effect from hepatitis C virus (HCV) via suppressing hematopoiesis. 3,12−17 Thrombopoietin (TPO), a thrombopoietic cytokine, is involved in megakaryocyte maturation and platelet production. 18 TPO is primarily produced in the liver and its level is affected by liver brosis stage and platelet turnover. 3,[13][14][15]19 This partly accounts for thrombocytopenia in CLD patients. 3 TPO mimetics have been shown to improve platelet count in thrombocytopenic CLD patients. 20 Data on the relationship between TPO and rapid thrombocytopenia improvement after DAA therapy has not been well reported. Hence, this study investigated the association between baseline (BL) TPO and platelet count improvement after HCV clearance.

Patient selection
CHC-infected patients with BL thrombocytopenia (platelet count < 150 × 10 3 /µL), who underwent DAA treatment with SVR at Dalin Tzu Chi Hospital from June 2016 to February 2019, were screened in this retrospective study. All patients had been positive for anti-hepatitis C antibody for more than 6 months and had detectable serum HCV RNA levels at the time of entry into the study. Treatment duration and regimen were based on guidelines. [21][22][23][24] Patients without SVR, patients with concurrent DAA and anticancer therapy, patients who passed away before 12 weeks after end of treatment (P12), patients with treatment interruption, patients lost to follow-up before P12, patients with incomplete medical records, or patients without informed consent were excluded. A total of 104 patients were enrolled in the study (Fig. 1 Clinical monitoring For all patients, blood tests, including serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, creatinine, hemoglobin, white blood cell, and platelet counts, and HCV RNA, were checked at the hepato-gastrointestinal outpatient clinic at BL, end of treatment (EOT), and P12. We de ned signi cant platelet count improvement as a > 10% platelet count improvement ratio at P12, compared to that at BL. The platelet count improvement ratio was de ned as [platelet count (EOT or P12)-platelet count (BL)]/platelet count (BL).
Abdominal sonography was also conducted before, during, or after DAA treatment as recommended. 21,24 Hepatic brosis was evaluated using the noninvasive brosis-4 (FIB-4) test. 22 Advanced brosis was de ned as FIB-4 > 3.25. 22 Fatty liver diagnosis was based on abdominal ultrasound results, including hepatorenal echogenicity contrast, liver brightness, deep attenuation, and vessel blurring. 25 Splenomegaly was diagnosed when the spleen length of the long axis was more than 11 cm. 26 All ultrasonographic images were stored as photographs.
Other clinical factors, including chronic hepatitis B status, hepatocellular carcinoma (HCC), and alcoholism, were recorded by chart review. Alcoholism was de ned as alcohol consumption of more than 40 g/day. 27 HCC was diagnosed either by biopsy or by imaging in the setting of liver cirrhosis. 23 Chronic hepatitis B was diagnosed if a patient had seropositivity for the hepatitis B surface antigen for at least 6 months.
HCV quanti cation and genotyping Serum HCV RNA was quanti ed at BL, EOT, and P12 using the COBAS AmpliPrep /COBAS TaqMan HCV Test, v2.0 (Roche Diagnostics, Rotkreuz, Switzerland), with a lower limit of quanti cation of 15 IU/mL. HCV genotyping was performed using the COBAS HCV GT (Roche Diagnostics).
Thrombopoietin assay TPO was measured in BL serum samples with a sandwich enzyme-linked immunosorbent assay (ELISA; FineTest, Wuhan, China), following the manufacturer' s recommendations. 28 Samples were stored at − 40 °C until analyzed. The lower detection limit of the TPO assay was 18.75 pg/mL. Additionally, quality control samples were included in each assay.

Statistical analyses
The commercial statistical software package (SPSS for Windows, version 22) was used for all statistical analyses. Continuous variables were presented as median and range. The chi-square or the Fisher's exact test was used for nominal variables. Continuous variables were compared using the Student's t-test or the Mann-Whitney U test, when applicable for two independent groups. The Spearman's rank correlation coe cient was used to examine factors associated with platelet count improvement. A receiver operating characteristic (ROC) curve was used to calculate the TPO predictive value for patients with signi cant platelet improvement. P < 0.05 was considered signi cant in all analyses.

Discussion
This study showed that overall platelet counts were signi cantly increased in thrombocytopenic CHC patients after successful DAA treatment. BL TPO is positively associated with signi cant platelet count improvement and platelet count improvement ratio. BL TPO level > 120 pg/ml can predict signi cant platelet count improvement.
BL TPO can be positively associated with signi cant platelet count improvement and is correlated with platelet count improvement ratio at EOT and P12 (ρ = 0.282, P = 0.004 and ρ = 0.502, P < 0.001 at EOT and P12, respectively). This association may be related to the splenic size and brosis stage. Previous studies have demonstrated that a higher TPO is related to a smaller spleen size 29,30 and less hepatic brosis. 13-15, 19,31 In our study population, it was shown that those without splenomegaly had signi cantly higher TPO levels. However, analysis of the TPO levels between non-advanced and advanced brosis patient groups showed no signi cant difference. TPO alone may not truly re ect synthesis function of the liver because it is produced by the liver at a constant rate and removed from circulation by TPO receptors on megakaryocytes and platelets. 32,33 Platelet turnover also affects TPO level. 3, 34 We did not check platelet turnover; therefore, the low TPO level might be attributed to either poor liver function or high platelet turnover. Another likely reason is that we classi ed non-advanced or advanced hepatic brosis by the FIB4 score; some patients in the advanced brosis group may actually have non-advanced hepatic brosis. The actual reason requires further studies checking platelet turnover and using liver biopsy as a classi cation method for TPO level analysis in different brosis stages.
Though we knew baseline TPO was positively correlated to the platelet count change, the cut-off value was unclear. Therefore, we used ROC test and found the baseline TPO level > 120 pg/ml was associated with signi cant platelet count improvement. The sensitivity is 88.6%, and speci city is 71.7%, with accuracy being 78.8%. Hence, when thrombocytopenia is observed in untreated CHC patients, this cut-off value may be helpful in predicting who would have signi cant improvement after DAA therapy.
In the current study, BL TPO levels were not correlated with BL platelet count (ρ = 0.054, P = 0.587). This result was also corroborated by other studies. 19,29,30 It is reasonable to see this phenomenon because absolute platelet count is affected by TPO, spleen size, autoimmune status, megakaryocyte mass in bone marrow, and the aforementioned viral effect. 16,29 We also found that the TPO level (32-3944 pg/mL) in patients was more variable, compared to the normal population (81.3-237.7 or 19.25-377.75 pg/ml). 29,35 This suggests that the ability of our patients to produce or destroy TPO differed signi cantly. Although some patients, with liver disease, had higher TPO levels, the elevation was mostly inadequate, compared with thrombocytopenic patients due to other diseases such as aplastic anemia or chemotherapy-induced thrombocytopenia. 31 In patients with early stage liver brosis, TPO production may increase to partly compensate for the decreased platelet count; those with more advanced brosis or liver cirrhosis produce relatively and inappropriately low TPO. 13-15, 19,31 Moreover, other factors such as hypersplenism, may offset the positive effect of elevated TPO. Congested spleen contains a large platelet mass, which would bind to, internalize, and destroy TPO. 30,36 We found that those with splenomegaly had signi cantly lower TPO than those without splenomegaly. A negative association between TPO level and spleen size was also reported by other studies, 29,30 but there was a study revealing no correlation between them. 19 These differences may be due to the relatively small number of cases in these studies and patient selection criteria.
This study found that the only factor associated with signi cant platelet count improvement was high BL TPO. Other BL characteristics were not related to such changes after DAA therapy. This was partly different from our previous study. 8 Our previous study showed that moderate to severe fatty liver and low BL platelet count could predict signi cant platelet count improvement. The most likely reasons were that some differences existed in the previous study, compared to ndings of the present study, including more case numbers (n = 249), more cancer patients (n = 19 vs. n = 0, P = 0.001), viable HCC patients (n = 14 vs. n = 0, P = 0.013), more advanced hepatic brosis patients (n = 213 vs. n = 72, P = 0.001), and more moderate to severe fatty liver patients (n = 24 vs. n = 5, P = 0.2).
There are some limitations of our study. First, we did not perform biopsy of the liver in all patients to check in ammation and brosis severity. Instead, we used noninvasive indices such as the FIB-4 test, to identify patients with advanced brosis; this is a relatively reliable method. 22 Second, we did not perform bone marrow biopsy in this study to exclude possible indolent hematological disorders in our patients.
Bone marrow biopsy is an invasive procedure, which could not be performed except with a clinically strong indication. Third, we did not check for platelet turnover. However, hypersplenism is the main contributor to platelet turnover in patients with splenomegaly, 34 which were included in our study. In patients without splenomegaly, more evidence is required to clarify the association between platelet count and turnover, TPO level, and liver brosis severity. Fourth, other thrombopoietic cytokines such as interleukin-6, -11, and-3, were not evaluated. Their importance among these thrombocytopenic patients remains unknown. Therefore, further studies are needed.

Conclusion
Successful DAA therapy for CHC patients results in signi cant platelet count improvement. BL TPO level is positively correlated with signi cant platelet count improvement and platelet count improvement ratio.
The BL TPO cutoff value of 120 pg/ml can predict signi cant platelet count improvement among CHCrelated thrombocytopenic patients.
content, nal approval of the version to be published, agreement to be accountable for all aspects of the work. All authors approved the nal version of the article. Flow diagram used in building this study cohort.

Figure 2
Overall platelet count change (median with con dence interval) at BL, EOT, and P12. The median (range) platelet count was 110. Receiver operating characteristic (ROC) curve of thrombopoietin (TPO) stratifying CHC-related thrombocytopenic patients, with signi cant platelet count improvement. Area under the curve for TPO in these patients was 0.765 (95% con dence interval, 0.670-0.861).