Does a rapid decline in the hematological and biochemical parameters induced by interferon and ribavirin combination therapy for the hepatitis C virus predict a sustained viral response ?

1McGill University Health Centre; 2Sir Mortimer B Davis-Jewish General Hospital, McGill University, Montreal, Quebec Correspondence: Dr Constantine A Soulellis, McGill University Health Centre, 687 Pine Avenue West, Room R 6.24B, Montreal, Quebec H3A 1A1. Telephone 514-934-1934 ext 36538, fax 514-934-4452, e-mail csoulellis@hotmail.com Received for publication January 26, 2007. Accepted September 4, 2007 C Turbide, CA Soulellis, M Deschênes, N Hilzenrat. Does a rapid decline in the hematological and biochemical parameters induced by interferon and ribavirin combination therapy for the hepatitis C virus predict a sustained viral response? Can J Gastroenterol 2008;22(2):149-152.

H uman interferon (IFN) alpha-2a and 2b, in combination with ribavirin (RBV), is the current standard of care for the treatment of chronic hepatitis C virus (HCV) infection worldwide.Although these drugs are usually well tolerated, many side effects have been reported.One that is of particular clinical significance is the change in hematological parameters seen during treatment, namely, anemia, thrombocytopenia and leukopenia (1).
These cytopenias are well documented effects of IFN therapy, and many mechanisms have been proposed; for example, a rapid decline in all three major cell lines (within the first 12 h to 24 h) is often explained by sequestration of platelets and white blood cells (WBCs) in the liver and spleen (2).Another suggested mechanism is the direct inhibition of progenitor cells in the bone marrow by IFN (3).Other proposed mechanisms include capillary trapping and increased apoptosis (4-6) of all cell lines.Finally, RBV itself is known to cause hemolysis, and the addition of this drug to IFN results in an additive effect on hemoglobin decline (7).
Presently, there are little data examining the relationships between the acute hematological toxicities, decreases in alanine aminotransferase (ALT) and outcomes in the treatment of chronic HCV with standard IFN and RBV combination therapy.The objective of the present study was to determine whether the decline rates of these parameters are markers of therapeutic efficacy and, therefore, early markers of a sustained viral response (SVR) in HCV patients.

METHODS
Data from 111 patients with chronic HCV, who were treated between the years 1998 and 2001 at the McGill University Health Centre in Montreal, Quebec, were retrospectively analyzed.All patients were treated with a standard IFN and RBV combination therapy (standard IFN, three million units subcutaneously, three times per week; weight-dosed RBV, 1000 mg [patients less than 75 kg] or 1200 mg [patients 75 kg or more], both orally daily).Fifty-six patients underwent liver biopsy.All patients were considered for combination therapy when their ALT levels were greater than 1.5 times normal and were treated for a total of 48 weeks.At the time of data collection, genotype differentiation and viral load quantification were not used in Quebec, and were therefore not considered in the present study.A positive response was defined as an SVR at 24 weeks after the end of treatment.Additionally, standard IFN and RBV dosage adjustments for hematological toxicities were recorded, and no growth factors were used.
The data retrieved were as follows: hemoglobin, WBC, neutrophil, platelet, and ALT levels.Other WBC subpopulations (such as lymphocytes, eosinophils, etc) were not studied, because they are not typically associated with the hematotoxic profile of the treatment.These values were measured at baseline, and again at weeks 2, 4, 8, 12 and 24.The difference between the values at week 2 and baseline, week 4 and baseline, and so forth, up to the difference at week 24 and baseline were calculated; this was defined as the decline rate for each interval.Additionally, the decline rates for ALT were converted to per cent changes from the baseline measurement.The decline rates of each interval for ALT and each hematological parameter were then compared between the responders and nonresponders using Wilcoxon's signed rank test.This statistical test was used because the studied parameters in the subject population did not follow a normal distribution.

RESULTS
The study included a total of 111 patients (68 men and 43 women), with a mean ± SD age of 48.1±10.9years.A total of 60 patients (54%) achieved an SVR.There were no significant differences between the responder and nonresponder groups for age, sex, body mass index, degree of fibrosis (of those biopsied), baseline hematological values or ALT levels (Table 1).
No statistically significant difference in the decline rates for hemoglobin or platelets between the two groups was observed throughout the treatment duration.
A total of 16 patients required dose adjustments of standard IFN and RBV combination therapy to less than 80% of the expected dose because of excess hematological toxicity.There were five dose reductions in each group (responders and nonresponders) due to significant neutropenia and three reductions in each group due to significant anemia.This represented approximately 13% of the responders and 16% of the nonresponders (the difference was not statistically significant).
Within the first month, the decline rate that was statistically significant for the prediction of an SVR was the ALT level.At week 2, these values were 71±92 U/L and 44±85 U/L for the responders and nonresponders, respectively, (P<0.0046)(Figure 3).Calculated as a per cent decrease from baseline, this corresponded to declines of 64% and 43% for the responders and nonresponders, respectively (P<0.0003).At week 4, the decline rates were 101±96 U/L and 84±100 U/L for the responders and nonresponders, respectively (P<0.0154).As a per cent decrease from baseline, this corresponded to declines of 66% for the responders and 41% for the nonresponders (P<0.0001)(Table 2).

DISCUSSION
This was a retrospective study of 111 patients with chronic HCV who were all treated with a standard IFN and RBV combination therapy for a total of 48 weeks.Our initial hypothesis was that the biological activity of the therapy, typically leading to side effects of cytopenia and decreased ALT levels, was a surrogate marker for therapeutic efficacy.Alternatively, greater hematotoxicity and larger declines in ALT were predictors of an SVR.We also theorized that larger reductions in cell lines may have contributed to the success of the therapy.The present study demonstrated that rapid and significant declines in WBCs (within two weeks) and neutrophils (within two and four weeks) were associated with significantly lower rates of achieving an SVR.The decline rates of hemoglobin and platelets were not found to be significantly different between the responder and nonresponder groups.In our study, only 16 patients sustained dose reductions for neutropenia or anemia; these were evenly divided among responder and nonresponder groups.
Our study also demonstrated a significant difference in the decline rates of the ALT level between the responder and nonresponder groups at weeks 2 and 4 of treatment.Our data suggest that a patient undergoing standard IFN and RBV combination therapy has a greater chance of achieving an SVR if the ALT declines by approximately 65% of the baseline value within two and four weeks.
Contrary to our hypothesis, our study found that rapid declines in the WBCs and neutrophils in the initial two weeks of therapy predict nonresponsiveness.Several studies have argued that when significant treatment-induced cytopenia results in dose reductions to less than 80% of either drug, this may lead to significantly lower SVR rates (8).Therefore, in such studies, poorer outcomes may have been related to inadequate dosing.Our data demonstrate that dose adjustment was equivalent in both groups, suggesting that this was probably not a factor in predicting an SVR in our population.Additionally, the average age and degree of fibrosis, determined by the METAVIR scoring system (of those biopsied), between the two groups was not different, failing to explain the difference in declining neutrophil rates within the first month.It should be noted that approximately one-half of the patients did not undergo liver biopsy.The role of neutrophils in the elimination of HCV is unknown at the present time.It has been shown that treatment with all types of IFN and RBV results in a reduction in the absolute number of neutrophils in all treated patients (both responders and nonresponders), but does not alter neutrophil function by assays such as the C5a and formylmethionyl-leucyl-phenylalanine migration killing curves (9).In our study, there were no differences in absolute neutrophil counts between the two groups throughout the duration of therapy.This may suggest that absolute numbers of neutrophils are not important in achieving an SVR.Moreover, in a study comparing African American genotype 1 HCV patients with non-Hispanic Caucasians, a multivariate analysis showed that even significantly different baseline neutrophil values were not associated with the outcome of therapy (10).We experienced similar expected dose-related reductions in neutrophils in both groups, but our nonresponder arm experienced significantly greater decline rates within the first month of therapy.This suggests that the therapy and other factors induce a rapid change in the neutrophil population in certain individuals within the first four weeks, which ultimately lowers the chance of an SVR.From our data, it appears that neutrophil kinetics are important for the elimination of HCV.It is known that immune cells (including neutrophils) harbour HCV RNA, are replication sites for the virus and may contribute significantly to total viral load (11).We theorize that the rapid elimination of neutrophils within the first month occurs in patients with a large HCV RNA load within those neutrophils.Effectively, neutrophils may be seen as a reservoir of 'hidden' viral RNA in nonresponders.
In the present study, both responder and nonresponder groups sustained equivalent hemoglobin rates of decline and absolute nadir.In our study, a small but equal number of patients in both the responder and nonresponder groups sustained a dose reduction for anemia.This suggests that this parameter, in isolation, plays no role in predicting an SVR, which is consistent with the findings of other major studies (8).
To our knowledge, this is the first study that has demonstrated the relationship between a decline rate of ALT and the ability to achieve an SVR.The current standard of care when treating chronic HCV is to obtain a viral load measurement at weeks 4 and/or 12 of therapy (12), which is accepted as a good predictor of response.If ALT is regarded as a marker of total hepatocyte damage or death, then rapid decreases in ALT may theoretically reflect rapid resolution of the ongoing inflammatory state as the virus is eliminated (13,14).It has been established elsewhere that viral elimination patterns are biphasic, with the first rapid decline occurring within the first month.Our data demonstrate that larger decline rates of ALT within the first two and four weeks are predictors of an SVR.Therefore, decline rates of ALT may reflect this first phase of viral kinetics, and may be used as a strategy to help predict a response well before the four-or 12-week viral load quantification is performed.
The present study had some limitations.First, due to the abnormal distribution of hematological parameters and ALT levels of our patient population, we were obliged to use Wilcoxon's signed rank test in the analysis.It is possible that a larger population would have permitted the application of a multivariate analysis, which would have had more statistical power.Second, at the time of data collection, viral genotyping and viral load measurements were not available.These parameters are presently known as important predictors of the outcome of HCV treatment.This precluded us from correlating these known parameters with our findings.However, the treatment dosage and duration was equal among all study subjects and, therefore, among all represented genotypes in the present study.Third, although nearly all patients had a baseline ALT level greater than 1.5 times normal, the decline rates of ALT at weeks 2 and 4 of therapy may not be applicable for those with a baseline ALT in the normal range.
To summarize the key points, when treating a patient with chronic HCV with standard IFN and RBV combination therapy, a decline in ALT of greater than 64% from baseline at weeks 2 and 4 predicts an SVR.However, a significant decline in neutrophils and WBCs after two weeks predicts a nonresponder.Hemoglobin and platelet decline rates or nadir values were not significant in predicting an SVR.
Larger studies are necessary to investigate the role of ALT decline rates in the outcome of IFN and RBV combination therapy.Further studies are needed to explore the importance of neutrophil subpopulations and kinetics on the elimination of HCV.Additionally, the role of granulocyte-colony stimulating factor as an adjunct to therapy in cases of rapid decline within the first month should be investigated.

Figure 2 )
Figure2) Neutrophil level versus week of therapy for responders and nonresponders to interferon and ribavirin therapy

Figure 3 )Figure 1 )
Figure3) Alanine aminotransferase (ALT) level versus week of therapy for responders and nonresponders to interferon and ribavirin therapy

TABLE 1
Baseline demographics of responders and nonresponders to interferon and ribavirin combination therapy (n=111)

TABLE 2
Significant decline rates of white blood cells (WBCs), neutrophils and alanine aminotransferase (ALT) levels during interferon and ribavirin combination therapy