Impact of tumor size on outcome after stereotactic body radiation therapy for inoperable hepatocellular carcinoma.

Stereotactic body radiation therapy (SBRT) for inoperable hepatocellular carcinoma (HCC) offers excellent local control rates. This study retrospectively analyzed the influence of different tumor size on treatment outcomes after SBRT.Between December 2008 and February 2014, 141 HCC patients were treated with Cyberknife SBRT. Patients were divided into 3 groups namely small tumors (≤4 cm), intermediate-sized (>4-<10 cm), and large (≥10 cm) tumors. Treatment outcomes, prognoses, and safety at each tumor size were compared and analyzed.A total of 52 patients with small tumors, 55 with intermediate tumors, and 34 patients with large tumors were retrospectively analyzed with a median follow-up of 16 months. Objective responses were achieved at 96.15%, 90.90%, and 76.47% for small, intermediate, and large tumors, respectively (P ≤ .0001) and the 3-year local control rates were 97.85%, 71.99%, and 82.14%, respectively (P = .0035). The 3-year overall survival rates were 50.26%, 45.29%, and 33.38% for small, intermediate, and large tumors, respectively (P = .3757). No significant differences were found in overall-survival, intra-hepatic recurrence free survival, disease-progression free survival, or distant metastasis-free survival.SBRT offers the best effective local control rate and response rate for small HCCs. However, tumor size did not significantly affect the overall survival rate, intra-hepatic recurrence free rate, or disease-progression free rate.

Perihepatic nodes detected by point-of-care ultrasound in acute hepatitis and acute-on-chronic liver disease.

AIM: To study the manifestations of perihepatic lymph nodes during the episode of acute hepatitis flare by point-of-care ultrasonography. METHODS: One hundred and seventy-six patients with an episode of acute hepatitis flare (ALT value > 5 × upper normal limit) were enrolled retrospectively. Diagnosis of etiology of the acute hepatitis flare was based on chart records and serological and virological assays. The patients were categorized into two groups (viral origin and non-viral origin) and further defined into ten subgroups according to the etiologies. An ultrasonograpy was performed within 2 h to 72 h (median, 8 h). The maximum size of each noticeable lymph node was measured. Correlation between clinical parameters and nodal manifestations was analyzed RESULTS: Enlarged lymph nodes (width ≥ 5mm) were noticeable in 110 (62.5%) patients, mostly in acute on chronic hepatitis B (54.5%). The viral group had a higher prevalence rate (89/110 = 80.9%) and larger nodal size (median, 7 mm) than those of the non-viral group (21/66 = 31.8%; median, 0 mm) (P < 0.001 for both). Meanwhile, there were significant differences in the nodal size between acute and chronic viral groups (P < 0.01), and between acute hepatitis A and non-hepatitis A viral groups (P < 0.001). In logistical regression analysis, the nodal width still showed strong significance in multivariate analysis (P < 0.0001) to stratify the two groups. The area under the curve of ROC was 0.805, with a sensitivity of 80.9%, a specificity of 68.2%, positive predictive value of 80.92%, negative predictive value of 68.18%, and an accuracy of 76.14%. CONCLUSION: Point-of-care ultrasonography to detect perihepatic nodal change is valuable for clarifying the etiologies in an episode of acute hepatitis flare.

Manifestations of perihepatic lymph nodes in acute flare of chronic hepatitis B: association with HBeAg status and with HBeAg seroconversion.

UNLABELLED: It has been observed that enlargement of perihepatic lymph nodes may be seen in patients with chronic hepatitis B, particularly during acute flares of CHB. We hypothesized that there may be a correlation between the nodal change patterns in CHB patients with acute flare and HBeAg status. Perihepatic lymph node sizes of 87 patients with acute flares of CHB were documented, with a median follow up of 43 months. Patients were separated into 3 groups, HBeAg-positive with HBe seroconversion (group 1), HBeAg-positive without HBe seroconversion (group 2), and HBeAg-negative (group 3). Group 1 has the highest incidence of enlarged lymph nodes (92.3%) compared with group 2 (75.8%) and group 3 (46.8%) (p = 0.003). And if nodal width at acute flare was > 8mm and interval change of nodal width was >3mm, the incidence of HBeAg seroconversion will be 75% (p<0.001). CONCLUSION: Larger perihepatic lymph nodes are seen in CHB acute flare patients with positive HBeAg and the magnitude of nodal width change may predict HBeAg seroconversion at recovery.

The early on-treatment perihepatic lymph node response predicts sustained viral response of anti-hepatitis C virus therapy.

BACKGROUND AND AIMS: In chronic hepatitis C, the change of perihepatic lymph nodal size after antiviral therapy could be a marker of virologic response. Whether the on-treatment nodal manifestations predict virologic responses is unknown. METHODS: Patients (n=88) with biopsy-proven chronic hepatitis C received standard doses of bi-therapy for 24 weeks; sequential changes of the perihepatic lymph nodes were evaluated prospectively by ultrasound. Pretreatment and on-treatment factors were analyzed and correlated with sustained virologic response, focusing on early on-treatment nodal changes (12 weeks). RESULTS: Perihepatic lymph nodes were prevalent in 75% of the patients; 72 patients (81.8%) achieved sustained viral response. Before treatment, no factor was significantly associated with the nodal prevalence or size. The pretreatment nodal width (mean 5.3 vs. 3.6 mm; P=0.023) and the on-treatment nodal manifestations including a reduction in nodal width at 12 weeks of antiviral treatment (median; 1.05 vs. 0 mm, P=0.029) and a reduction of nodal volume at the end of treatment (24 weeks; median 0.62 vs. -0.01 ml, P=0.015) were significantly correlated with the sustained virologic response. A reduction of nodal width greater than 2.5 mm at 12 weeks always predicts sustained virologic response (100 vs. 77%; P=0.019). CONCLUSION: Results confirm the high prevalence of perihepatic lymphadenopathy in patients with chronic hepatitis C. The use of the nodal width measurement in routine ultrasound follow-up may be a simpler early predictor of sustained virologic response during standard bi-therapy.

Hepatitis B virus (HBV) core antigen-specific regulatory T cells confer sustained remission to anti-HBV therapy in chronic hepatitis B with acute exacerbation.

Acute exacerbations (AEs) of chronic hepatitis B (CH-B) are thought to be the result of breakdown of immune tolerance on the natural history of chronic hepatitis B virus (HBV) infection. Immune tolerance to HBV maintained in CH-B patients without hepatitis is under the control of the host's forkhead box p3-expressing regulatory T cells (Tregs). Its breakdown mimics the occurrence of autoimmune diseases. Severe AEs may lead to liver decompensation and mortalities. Consequently, AEs are currently the major therapeutic targets in patient treatment. In this study, we employed the SYFPEITHI scoring system to identify epitopes on HBV core antigen (HBcAg) for the construction of human leukocyte antigen class II tetramers to measure HBcAg-specific Treg frequencies (Tregf). Upregulation of Treg gene profiling accompanied by increased HBcAg-specific Tregf was detected in AE patients with sustained remission (SR) to anti-HBV therapy. Depletion of Tregs from peripheral blood mononuclear cells enhanced proliferation to HBcAg. HBcAg-specific Treg clones inhibited the killing capacity of cytotoxic T lymphocyte clones in an antigen-independent manner. A greater posttherapy increase in HBcAg-specific Tregf correlated with a higher SR rate to anti-HBV therapy. These results suggest that HBcAg-specific Tregs function as suppressor effectors and confer SR to anti-HBV therapy.

Prediction of mortality after emergent transjugular intrahepatic portosystemic shunt placement: use of APACHE II, Child-Pugh and MELD scores in Asian patients with refractory variceal hemorrhage.

OBJECTIVE: This study was designed to determine if existing methods of grading liver function that have been developed in non-Asian patients with cirrhosis can be used to predict mortality in Asian patients treated for refractory variceal hemorrhage by the use of the transjugular intrahepatic portosystemic shunt (TIPS) procedure. MATERIALS AND METHODS: Data for 107 consecutive patients who underwent an emergency TIPS procedure were retrospectively analyzed. Acute physiology and chronic health evaluation (APACHE II), Child-Pugh and model for end-stage liver disease (MELD) scores were calculated. Survival analyses were performed to evaluate the ability of the various models to predict 30-day, 60-day and 360-day mortality. The ability of stratified APACHE II, Child-Pugh, and MELD scores to predict survival was assessed by the use of Kaplan-Meier analysis with the log-rank test. RESULTS: No patient died during the TIPS procedure, but 82 patients died during the follow-up period. Thirty patients died within 30 days after the TIPS procedure; 37 patients died within 60 days and 53 patients died within 360 days. Univariate analysis indicated that hepatorenal syndrome, use of inotropic agents and mechanical ventilation were associated with elevated 30-day mortality (p < 0.05). Multivariate analysis showed that a Child-Pugh score > 11 or an MELD score > 20 predicted increased risk of death at 30, 60 and 360 days (p < 0.05). APACHE II scores could only predict mortality at 360 days (p < 0.05). CONCLUSION: A Child-Pugh score > 11 or an MELD score > 20 are predictive of mortality in Asian patients with refractory variceal hemorrhage treated with the TIPS procedure. An APACHE II score is not predictive of early mortality in this patient population.

Acute hepatitis E virus infection in Taiwan 2002-2006 revisited: PCR shows frequent co-infection with multiple hepatitis viruses.

Sporadic cases of acute hepatitis E virus (HEV) infection with production of anti-HEV IgM have been reported occasionally in Taiwan despite no reported outbreaks in the past. This study was undertaken to determine whether serological markers correlated with virus detection. From 2002 to 2006, 72 reported cases of acute hepatitis E seropositive for anti-HEV IgM in Taiwan were enrolled for investigation. Acute phase serum samples were collected for detection of HEV RNA, HBV DNA, HCV RNA, and GBV-C RNA by PCR. The results showed that viral sequences of HEV, HBV, HCV and GBV-C were detected in 54 (75%), 21 (29.2%), 9 (12.5%), and 22 (30.6%) of cases, respectively. Acute hepatitis A co-infection was excluded in all patients because none were seropositive for anti-HAV IgM and, nine patients (12.5%) did not seroconvert to anti-HEV IgG. These results suggest that serum markers did not correlate completely with viremia in the diagnosis of acute HEV infection. Multiple viruses may co-infect with acute hepatitis E virus in Taiwan. Detection of hepatitis E viremia together with seropositivity for anti-HEV IgM and followed by seroconversion to anti-HEV IgG should be included in the diagnostic criteria for HEV infection.

Wnt-1 protein as a prognostic biomarker for hepatitis B-related and hepatitis C-related hepatocellular carcinoma after surgery.

BACKGROUND: Up-regulation of Wnt-1 protein has been reported in hepatitis B virus (HBV)-related and hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) tissues and cell lines. It is known to play a fundamental role in signaling cancer progression, whereas its prognostic role in HCC remains unexplored. METHODS: As a prognostic biomarker, this study analyzed Wnt-1 protein expression in 63 histology-verified HCC patients receiving curative resection. In each paired tumor and nontumor specimen, Wnt-1 levels were semiquantitatively measured by Western blotting and expressed by tumor/nontumor ratio. The data were further correlated with quantitative real-time PCR as well as with beta-catenin and E-cadherin expression by immunohistochemistry. Cumulative tumor recurrence-free survival curves were constructed using the Kaplan-Meier method and compared by the log-rank test. RESULTS: The results showed that 26 (group I) and 37 (group II) HCC patients had an expression ratio of Wnt-1 > or =1.5 and <1.5, respectively. The amount of Wnt-1 estimated by tumor/nontumor ratio correlated with the results by quantitative real-time PCR. High tumor Wnt-1 expression correlated with enhanced nuclear beta-catenin accumulation, diminished membranous E-cadherin expression, and increased tumor recurrence after curative tumor resection. CONCLUSIONS: These results suggest that Wnt-1 may be used as a predisposing risk factor for HCC recurrence. The use of tumor Wnt-1 as prognostic biomarker may identify patients with HBV- and/or HCV-related HCC patients with a high risk of tumor recurrence who may then benefit from further intensive therapy after surgery.

Lamivudine monotherapy for chronic hepatitis B infection with acute exacerbation revisited.

BACKGROUND: Acute exacerbation (AE) of chronic hepatitis B virus (HBV) infection in cancer chemotherapy patients and in organ transplant recipients receiving immunosuppressants may cause catastrophe and high mortality. Hence, immediate treatment with nucleoside analogues for such patients has become a consensus. Anti-HBV therapeutic trials in Asia have shown that AE of chronic hepatitis B (CH-B) may result in increased sustained remission (SR) rate with lamivudine monotherapy. Nonetheless, AE episodes in CH-B patients may evolve uneventfully and lead to spontaneous remission. Thus, the policy of immediate anti-HBV therapy for AE patients reaches an impasse. Once treatment is initiated, life long HBV suppression may be necessary. OBJECTIVE: To determine whether lamivudine monotherapy during an AE of CH-B results in an increase in SR compared with no therapy. METHODS: A cohort of 154 CH-B patients seropositive for hepatitis B e antigen with AE formed the study group. This included 102 cases receiving a nationwide therapeutic trial of 18-month lamivudine monotherapy that were compared with 52 cases with no therapy. All were observed for at least 30 months, which encompassed the 18-month on treatment period and a 12-month posttreatment follow-up. RESULTS: No significant increase was observed in the SR rate in the lamivudine treatment group compared with the spontaneous remission rate in the untreated patients (P=0.782, Fisher's exact test). CONCLUSION: AE does not increase the SR rate during 18-month lamivudine monotherapy. Immediate lamivudine therapy for AE patients is not justified as mandatory. The policy should be only applied to AE patients with impending liver failure.

Chronic autoimmune hepatitis with Epstein-Barr virus superinfection: a case report and review of literature.

A 66-year-old female presented with acute illness of severe hepatic dysfunction. She had a past history of chronic hepatitis of low disease activity. After admission and clinical investigation including liver biopsy, it showed an underlying chronic liver disease suggestive of autoimmune hepatitis (AIH) with early liver cirrhosis. Together with other clinical features, this patient was diagnosed as definite AIH type 1 by using the IAIHG (International Autoimmune Hepatitis Group) criteria. During this episode, superinfection by Epstein-Barr virus (EBV) was evidenced by positive PCR (polymerase chain reaction) test, and serial changes of EBV VCA IgM and IgG tests. Severe hepatic impairment was evidenced by markedly elevated AST level 3090 IU/L, high bilirubin level 26.4 mg/dL, and presence of ascites. The patient gradually recovered and liver function improved in agreement with the decline of EBV VCA titers. Immunosuppressive therapy resulted in further improvement of the aminotransferases levels. This is an unusual case of EBV superinfection on pre-existing AIH with early cirrhosis, which caused enhancement of the autoimmune disease process and resulted in severe hepatic decompensation and jaundice. We herein describe the case and briefly review the literature.

Randomized trial of low-pressure carbon dioxide-elicited pneumoperitoneum versus abdominal wall lifting for laparoscopic cholecystectomy.

BACKGROUND: Two alternative surgical techniques for elective laparoscopic cholecystectomy (LC), low-pressure insufflation of the peritoneal cavity and abdominal wall lifting (AWL), have been developed over time to minimize the disadvantages associated with CO2-elicited pneumoperitoneum. To the best of our knowledge, the 2 methods have seldom been compared as regards their relative advantages and disadvantages. METHODS: Eighty patients scheduled for elective LC were randomized into either a low-pressure (8 mmHg) CO2 insufflation method (LPLC) group, or a gasless technique using a subcutaneous abdominal wall lifting device (GLC group). The duration of the surgical procedure, the surgical results including level of postoperative pain, and perioperative cardiopulmonary function changes experienced by the members of both groups were compared. RESULTS: Laparoscopic surgery was completed for all but 1 patient from each group due to an inadequate surgical-site exposure. There was no mortality for study participants, and no major complications were noted for members of either group. The LPLC group evidenced a shorter surgical duration as compared to the GLC group (77 +/- 28 minutes vs. 98 +/- 27 minutes, respectively; p < 0.01) and a lower incidence of postoperative shoulder pain (2/38 vs. 8/39, respectively; p < 0.05), although significant differences in intraoperative pulmonary function were noted (an increased PaCO2, Pet CO2 and peak-airway pressure and decreased arterial blood pH; p < 0.01) for the LPLC group compared to the GLC group. CONCLUSION: Both alternative methods for this type of surgery appeared feasible and safe for LC. Low-pressure CO2 pneumoperitoneum had a shorter surgical duration and less postoperative shoulder pain compared to the GLC technique, but did not feature any other advantage over the AWL technique with regard to impact on cardiopulmonary function.

Multiple molecular markers as predictors of colorectal cancer in patients with normal perioperative serum carcinoembryonic antigen levels.

PURPOSE: In this study, a high-sensitivity colorimetric membrane array method was used to detect circulating tumor cells (CTC) in the peripheral blood of colorectal cancer (CRC) patients with normal perioperative serum carcinoembryonic antigen (CEA) levels. This membrane array method was evaluated as a potential diagnostic and postoperative surveillance tool. STUDY DESIGN: Membrane arrays consisting of a panel of mRNA markers that include human telomerase reverse transcriptase, cytokeratin-19, cytokeratin-20, and CEA mRNA were used to detect CTCs in the peripheral blood of 157 postoperative CRC patients with normal perioperative serum CEA levels and in 80 healthy individuals. Digoxigenin-labeled cDNA were amplified by reverse transcription-PCR from the peripheral blood samples, which were then hybridized to the membrane array. The sensitivity, specificity, and accuracy of membrane arrays for the detection of CTCs were then calculated. RESULTS: Using the four markers in combination, expression of any three markers or all the four markers in this panel was significantly correlated with the clinicopathologic characteristics, including depth of tumor invasion, lymph node metastasis, tumor-node-metastasis stage, and postoperative relapse (all P < 0.05). The interval between the detection of all four positive molecular markers and subsequent elevated CEA ranged from 3 to 8 months (median 6 months). The expression of all four mRNA markers was an independent predictor for postoperative relapse. CRC patients with all four mRNA markers expression showed a significantly poorer survival rate than those with less than four positive markers. CONCLUSIONS: The constructed membrane array method was helpful in the early prediction of postoperative relapse in CRC patients with normal perioperative serum CEA levels.

Significant association of HLA-DQ5 with autoimmune hepatitis in Taiwan.

Genetic predisposition is known to be an important etiopathogenic factor of autoimmune hepatitis (AIH). HLA antigens associated with AIH have been well studied in Western countries and Japan, but there is no HLA typing data of AIH patients in Taiwan. We therefore investigated HLA phenotypes and their association with AIH patients and compared the results with those of normal subjects and patients with chronic liver disease. Group 1 consisted of 22 AIH patients. All were born in Taiwan with no history of blood transfusion. Group 2 consisted of 19 chronic liver disease patients. Group 3 consisted of 81 unrelated healthy subjects who were normal blood donors. All three groups were tested for HLA phenotypes (HLAA, B, C, DR, DQ) using the polymerase chain reaction-sequence specific probe method. The statistical method used was Fisher's exact test. We found that HLA-DQ5 was significantly more frequent in the AIH group compared to the control group (RR, 2.03; p = 0.034). Low frequency of A1 (n = 2/22), B8 (n = 1/22) and DR3 (n = 0/22) were noted compared to results from the West; only HLA-DR4 showed a higher rate in our AIH patients (n = 8/22). This is a preliminary report of our study of HLA antigens in AIH patients. Further investigation to characterize AIH patients into HLA allelic subgroups is being done.

HBcAg-specific CD4+CD25+ regulatory T cells modulate immune tolerance and acute exacerbation on the natural history of chronic hepatitis B virus infection.

Acute exacerbations (AEs) of chronic hepatitis B (CH-B) are accompanied by increased T cell responses to hepatitis B core and e antigens (HBcAg/HBeAg). Why patients are immunotolerant (IT) to the virus and why AEs occur spontaneously on the immunoactive phase remain unclear. The role of HBcAg-specific CD4(+)CD25(+) regulatory T (T(reg)) cells in AE and IT phases was investigated in this study. The SYFPEITHI scoring system was employed to predict MHC class II-restricted epitope peptides on HBcAg overlapping with HBeAg that were used for T(reg)-cell cloning and for the construction of MHC class II tetramers to measure T(reg) cell frequencies (T(reg) f). The results showed that HBcAg-specific T(reg) f declined during AE accompanied by increased HBcAg peptide-specific cytotoxic T lymphocyte frequencies. Predominant Foxp3-expressing T(reg) cell clones were generated from patients on the immune tolerance phase, while the majority of Th1 clones were obtained from patients on the immunoactive phase. T(reg) cells from liver and peripheral blood of CH-B patients express CD152 and PD1 antigens that exhibit suppression on PBMCs proliferation to HBcAg. These data suggest that HBcAg peptide-specific T(reg) cells modulate the IT phase, and that their decline may account for the spontaneous AEs on the natural history of chronic hepatitis B virus infection.

Type 1 autoimmune hepatitis in Taiwan: diagnosis using the revised criteria of the International Autoimmune Hepatitis Group.

Autoimmune hepatitis (AIH) is rare in Asian countries compared to the West, and an exceptionally low prevalence was noted previously in Taiwan. Using the revised criteria of the IAIHG, 48 cases of AIH patients were diagnosed. All patients were consecutively diagnosed over a period of 5 years. Detailed medical histories including disease onset, hepatitis B and C, alcohol, drugs, blood transfusion, and family history of autoimmune disease were recorded. Clinical manifestations, result of steroid therapy, outcome, and survival rate were investigated and analyzed. Clinical data on AIH patients with cirrhosis and without cirrhosis were compared and analyzed for their outcome. The statistical methods used were Fisher's exact test, Wilcoxon rank sum test, and Kaplan-Meier curve. Forty-eight patients were diagnosed as AIH type 1, with a median age of 58 years and a female:male ratio of 37:11. The most common clinical features at presentation were fatigue, jaundice, and anorexia. Ninety-eight percent of patients were ANA positive, and most of the patients showed elevated values of AST, ALT, serum globulin, and bilirubin. A substantial proportion of patients presented with poor liver function at entry and 35% of patients had liver cirrhosis, with relatively prolonged PT (P=0.001) and poorer outcome (P=0.005) compared to the noncirrhotics. As a whole there was a favorable treatment response and the overall survival rate was 85%. We conclude that the incidence of AIH in Taiwan is much higher than previously presumed and AIH type 1 is the predominant type of the disease. Although a substantial proportion of AIH patients presented with poor hepatic function at entry, as a whole there was a favorable clinical outcome.

Nonresponse to 18-month lamivudine monotherapy in chronic hepatitis B patients with dual genotype B and C infection and acute exacerbation.

Molecular epidemiologic studies have indicated the possible existence of mixed infection of different hepatitis B virus (HBV) genotypes in chronic hepatitis B (CH-B) carriers, but the effect of dual HBV genotype B and C infection on the efficacy of lamivudine therapy remains unclear. We report four CH-B patients with dual HBV genotype B and C infection and acute exacerbation who received lamivudine monotherapy for about 18 months. None of them had achieved a sustained response at the end of the 18-month trial of treatment.

Transcatheter arterial embolization for hemorrhage caused by injury of the hepatic artery.

BACKGROUND: The aims of the study were to compare (i) the effects of transcatheter arterial embolization on initial hemostasis and the control of rebleeding in the treatment of hemorrhage due to hepatic artery injury; and (ii) the outcomes of embolization by different locations. METHODS: Subjects were 32 patients with suspected hepatic artery injury who were transferred to Chi-Mei Foundation Medical Center for hepatic angiography and embolization. The causes of arterial injury included liver trauma (n = 15) and iatrogenic injury (n = 17). The sites of embolization were classified into four groups: group 1 (n = 8) was classified as 'combined outlet, target and inlet control' with embolization of the vascular lesion (target) and hepatic artery distal (outlet) and proximal (inlet) to the vascular lesion simultaneously; group 2 (n = 11) as 'combined target and inlet control'; group 3 (n = 8) as 'combined outlet and inlet control'; group 4 (n = 5) as 'inlet control' only. RESULTS: Successful initial hemostasis was achieved in 30 of the 32 patients (93.8%), with two failures, both of which were caused by liver injury and occurred in subjects in group 4. Rebleeding was seen in three patients who had successful initial hemostasis: two of them in group 4 (66.7%) and one in group 1 (12.5%). All rebleedings were successfully managed by repeat embolization. Abscess formation was found in two group 1 patients, and both were successfully managed by percutaneous drainage. CONCLUSIONS: Transcatheter arterial embolization is an effective method for hemostasis in hepatic artery hemorrhage for both patients with liver trauma and patients with iatrogenic injuries to the hepatic artery. Based on this experience, embolization of the vascular lesion and/or the arterial lumen distal to the vascular lesion combined with inlet control is recommended for preventing recurrent hemorrhage, but studies with larger sample sizes will be required to validate this conclusion.