Immunohistochemical study of Ki-67 (MIB-1), p53 protein, p21WAF1, and p27KIP1 expression in benign, atypical, and anaplastic meningiomas

Histologic grading of meningiomas has prognostic and clinical therapeutic implications. Meningiomas were histologically classified into 3 different World Health Organization grades. Grade II, an atypical meningioma, was defined by major and various minor histologic criteria. However, these histologic criteria sometimes are not fulfilled, and other criteria are necessary. We studied and analyzed the immunohistochemical expression of MIB-1, p53, p21WAF1, p27KIP1 proteins in 146 cases of meningiomas, including 109 benign, 27 atypical, and 10 anaplastic meningiomas. Most of the benign meningiomas expressed low MIB-1 labeling index (mean, 1.5%), and fewer cases had p53 protein expression. In contrast, the anaplastic meningiomas had a high labeling index of MIB-1 (mean, 19.5%) and always expressed p53 protein, with a mean labeling index of 6.3%. The atypical meningiomas had MIB-1 and p53 labeling indexes in the range between benign and anaplastic meningiomas, with mean labeling indexes of 8.1% and 3.5%, respectively. These expressions were statistically significant among benign, atypical, and anaplastic meningiomas (P <.001). We conclude that the immunohistochemistry of MIB-1 and p53 protein will be valuable in discriminating atypical meningiomas from benign or anaplastic meningiomas, at least in histologically borderline cases. In addition, we also found direct correlation of p21 and inverse correlation of p27 expressions in meningiomas with increasing histologic grade and proliferative index.     

Expression of cell cycle regulatory proteins in the multistep process of oesophageal carcinogenesis: stepwise over-expression of cyclin E and p53, reduction of p21(WAF1/CIP1) and dysregulation of cyclin D1 and p27(KIP1)

AIMS: Cell cycle regulatory proteins were analysed by immunohistochemistry in order to clarify how their expression changes with the degree of atypia as oesophageal surface squamous epithelium progresses from normal mucosa, through reactive change, low-grade dysplasia, and high-grade dysplasia to mucosal invasive carcinoma. METHODS AND RESULTS: Immunostaining for cyclin D1, cyclin E, p21, p27, p53 and Ki67 proteins was performed using 22 normal mucosa, 17 reactive change, 22 low-grade dysplasia, 15 high-grade dysplasia and 22 mucosal invasive carcinoma specimens. Normal mucosa, low-grade dysplasia and high-grade dysplasia samples were taken from patients without any oesophageal invasive carcinoma by endoscopic biopsy or endoscopic mucosal resection, and reactive change and mucosal invasive carcinoma were obtained from oesophagectomy material. Stepwise over-expression of cyclin E (P < 0.0001) and p53 (P < 0.0001), reduction of p21 (P=0.0189) and dysregulation of cyclin D1 and p27 were observed in the multistep process of oesophageal carcinogenesis. Significant differences in expression of p27 (P < 0.0001), p53 (P=0.0299) and Ki67 (P=0.0101) were observed between reactive change and low-grade dysplasia. Furthermore, expression of cyclin D1, cyclin E, p27 and p53 in mucosal invasive carcinoma were significantly different from those in high-grade dysplasia (P=0.0079, P=0.0237, P=0.0042 and P= 0.0299, respectively). CONCLUSIONS: Cell cycle regulatory proteins, cyclin E, p53 and p21 show stepwise over-expression or reduction with progression of oesophageal carcinogenesis, correlating with the increased cell proliferation observed with Ki67 labelling. We conclude that immunohistochemical analysis for p27, p53 and Ki67 is practically useful for the discrimination between low-grade dysplasia and reactive change. Cyclin D1, cyclin E, p27 and p53 help to distinguish high-grade dysplasia from mucosal invasive carcinoma.     

非霍奇金淋巴瘤p27Kip1、p21WAF1及cyclin E表达的意义

目的 :探讨在非霍奇金淋巴瘤 (NHL)中 p2 7Kip1及其同族抑癌基因 p2 1WAF1、细胞周期素E(cyclinE)的表达意义。 方法 :应用免疫组化S P法检测 40例NHL患者受累淋巴结中 p2 7Kip1、p2 1WAF1、cyclinE蛋白表达情况 ,同时结合临床病理资料进行分析。结果 :40例NHL中 ,p2 7、p2 1、cyclinE蛋白阳性表达分别为 2 0例 (72 5 % )、8例 (2 0 % )、2 6例 (6 5 % )。 3种蛋白在NHL中的阳性表达与患者的性别、年龄及免疫分型无明显相关性 (P >0 0 5 ) ,但均与组织分化程度明显相关 (P <0 0 5 )。p2 7与cy clinE的表达水平间存在显著负相关 (r =- 0 2 7,P <0 0 1)。结论 :NHL患者淋巴组织中存在较高比例的 p2 7、p2 1蛋白阴性表达及较高比例的cyclinE阳性表达 ,说明在NHL的发生发展过程中 ,p2 7及其相关因素cyclinE ,同族基因 p2 1蛋白水平的异常变化起着重要作用 ,可作为NHL诊断标记物及预后指标。     

Antibody selection in immunohistochemical detection of cyclin D1 in mantle cell lymphoma

An assessment in Nordic immunohistochemical Quality Control (NordiQC) revealed that only 23% participant laboratories performed optimal staining for detection of cyclin D1 (CyD1) in mantle cell lymphoma (MCL). False-negative results were secondary to suboptimal protocols. We compared the 5 anti-CyD1 antibodies (monoclonal SP4, P2D11F11, and DCS-6 and polyclonal CP236 and 06-137) used in the Scandinavian laboratories. Evaluated were 31 MCLs, 16 other malignant lymphomas, and 19 samples of normal tissues. Sensitivity was as follows: CP236, 100%; SP4, 95%; P2D11F11, 90%; DCS-6, 84%; and 06-137, 53%. SP4 produced the strongest staining. Correlation of CyD1 with the proliferative index was best with polyclonal antibodies CP236 and 06-137. The use of heat-induced epitope retrieval in an alkaline buffer such as 1/10 mmol/L of Tris-EDTA buffer, pH 9, seemed mandatory. For the optimal detection of CyD1 expression, both SP4 and CP236 antibodies should be available in the laboratory.     

Immunohistochemical analysis of c-erbB-2, Bcl-2, p53, p21WAF1/Cip1, p63 and Ki-67 expression in hydatidiform moles

Hydatidiform moles (HM) are characterized by an abnormal proliferating trophoblast with a potential for a malignant transformation. Similar to other human tumors, trophoblastic pathogenesis is likely a multistep process involving several molecular and genetic alterations. The study was performed to investigate the expression patterns of c-erbB-2 and Bcl-2 oncoproteins, p53, p21^WAF1/CIP1 and p63 tumor suppressor proteins and Ki-67 cell proliferation marker in HM.We conducted a retrospective study of 220 gestational products, including 39 hydropic abortions (HA), 41 partial HM (PHM) and 140 complete HM (CHM). The expression of c-erbB-2, Bcl-2, p53, p21^WAF1/CIP1, p63 and Ki-67 was investigated by immunohistochemistry on archival tissues. c-erbB-2 expression was observed in three PHM and 10 CHM. Bcl-2 immunostaining was significantly higher in PHM (61%) and CHM (70.7%) compared with HA (7.7%, p?=? 0.001 and p?<? 0.0001, respectively). p53 expression was stronger in CHM (73.6%) compared with PHM (24.4%, p < 0.0001) and HA (12.8%, p < 0.0001). p21^WAF1/CIP1 staining was observed as well in molar and non-molar gestations (p?>? 0.05). p63 immunoexpression was significantly described in CHM (85.7%) and PHM (78%) compared with HA (10.2%, p < 0.0001 and p = 0.0001, respectively). Ki-67 was significantly expressed in CHM (72.1%) compared with HA (46.2%, p = 0.005).Altered expression of Bcl-2, p53, p63 and Ki-67 reflects the HM pathological development. Immunohistochemical analysis is beneficial to recognize the HM molecular and pathogenic mechanisms. Furthermore, it could serve as a useful adjunct to conventional methods for refining HM diagnosis.     

An immunohistochemical analysis of cyclin D1, p53, and p21waf1/cip1 proteins in tumors originating from the follicular epithelium of the thyroid gland

This study aimed at clarifying the factors closely related to the tumor progression of thyroid neoplasms. We examined the immunoreactivity of cyclin D1, p53, and p21waf1/cip1 proteins in 179 thyroid tumors originating from the follicular epithelium using an immunohistochemical technique. Cyclin D1 positivity was frequent in well-differentiated thyroid carcinomas (39/122 cases), but it was rarely seen in follicular adenomas (1/33 cases), (p < 0.05). Positivity for p53 was more frequent in poorly differentiated carcinomas (7/19 cases) and undifferentiated carcinomas (4/5 cases) than in well-differentiated carcinomas (14/122 cases) (p < 0,05, respectively). P21waf1/cip1 positivity was more frequent in well-differentiated thyroid carcinomas (43/122 cases) than in follicular adenomas (4/33 cases) (p < 0.05). Regarding the relationships of these proteins, co-positivity for cyclin D1 and p53 was observed more often in poorly differentiated carcinomas (5/7 cases) than in well-differentiated carcinomas (7/39 cases) (p < 0.05). Most cases with cyclin D1 positivity did not show p21waf1/cip1 expression in poorly differentiated carcinomas (6/7 cases). Three cases examined showed co-positivity of p53 and p21waf1/cip1. Our results suggest that cyclin D1 is invoved in thyroid oncogenesis. Moreover, p53 might be closely related to the development of poorly differentiated carcinomas and undifferentiated carcinomas originating from well-differentiated carcinomas.     

The significance of immunohistochemical expression of Ki-67, p53, p21, and p16 in meningiomas tissue arrays

Although histologic grading of meningiomas has prognostic and clinical implications, it is difficult in some cases to predict the outcome of patients. There have been several efforts to evaluate the use of different immunohistochemical markers for predicting meningioma prognosis. We analyzed the immunohistochemical expression of Ki-67, p53, p21, p16, and PTEN proteins in 130 meningiomas (64 benign, 39 atypical, and 27 malignant meningiomas) using tissue microarray. The tumors were graded according to the World Health Organization classification. There was a statistically significant correlation between the expression of Ki-67, p53, p21, p16, and the grade of meningiomas (p0.001). By ordinal logistic regression, p53 and Ki-67 were significantly associated with grade, and an increase of 1% in the labeling index of these markers resulted in an increase in the risk of raising the grade by 2.17 and 1.49, respectively. Histological grade, p53, Ki-67 labeling indices, and overexpression of p16 were strongly associated with decreased event-free survival in univariate analysis. In contrast, multivariate analysis revealed that only tumor grade is an independent factor for predicting meningioma recurrence. We conclude that the Ki-67 and p53 labeling indices are useful additional tools in discriminating atypical from benign or anaplastic meningiomas, especially in histological borderline cases.     

Expression of cyclin dependent kinase inhibitor p21WAF1 alone and in combination with p27KIP1 shows prognostic value in gastric carcinoma.

Evidence suggests that multiple molecular events, including alteration of cell cycle regulators are involved in the development and progression of gastric carcinoma. Recently, it has been reported that the expression of p21 and p27, integrating the effects of one or more cell cycle regulators, consequently, acting as a single indicator of several possible cell cycle gene alterations is associated with tumor suppression. In the present study, we studied the immunohistochemical expression of p21 and p27 in gastrectomy specimens from 84 patients with gastric adenocarcinoma, and analysed its correlation to clinicopathologic data, including patients survival. Loss of p21 and p27 expression was noted in 45 (53.6%) and 44 (52.4%) of the 84 gastric carcinoma tissues, respectively. The expression of p21 was significantly correlated with histological type (p= 0.005), recurrence (p=0.002) and death (p=0.002) after surgery, and p27 expression (p=0.001). Kaplan-Meier survival plots showed p21 negative group (p= 0.0014) or both p21 and p27 negative group (p=0.0048) was significantly poorer in overall survival than both p21 and p27 positive or one of both positive group. Our results suggest that the status of p21 and p27 expression in immunohistochemical stain may be a useful prognostic marker of gastric carcinoma.     

Expression of cell-cycle regulatory proteins cyclin D1, cyclin E, and their inhibitor p21 WAF1/CIP1 in gastric cancer.

The expression and prognostic role of cyclin D1, cyclin E, and p21 (WAF1/CIP1) were immunohistochemically investigated in 413 curatively resected gastric carcinomas. p21 was expressed in 65.4 per cent (n=270), cyclin D1 in 23.7 per cent (n=98), and cyclin E in 13.6 per cent ( n=56) of the tumours. The expression of p21, cyclin D1, and cyclin E was positively associated with the papillary or tubular type of the WHO classification, as well as with the intestinal type according to the Lauren classification. No significant correlation could be found between the expression of p21, cyclin D1 and cyclin E and the parameters pT category, lymph node involvement, and blood vessel and lymphatic vessel invasion. Concerning survival, no prognostic impact of p21, cyclin D1, and cyclin E expression could be verified, even when different subgroups of patients were analysed separately according to the pT and pN category as well as to the Lauren classification. The present data suggest that neither cyclin D1, cyclin E nor their inhibitor p21 can predict the survival of gastric cancer patients, nor is their immunohistochemical detection a suitable tool for identifying subgroups of patients who may be at higher risk.     

Expression profiling of colorectal carcinomas using tissue microarrays: cell cycle regulatory proteins p21, p27, and p53 as immunohistochemical prognostic markers in univariate and multivariate analysis.

With the rapidly growing understanding of tumor biology, molecular staging of cancer is expected to improve prognostication. This would be particularly important for cancers amenable to adjuvant treatment, such as colorectal carcinomas. To generate data for this, the tissue microarray technique may prove useful. Tissue microarrays were constructed with triplicate cores (0.6 mm diameter) from the invasive margins of a consecutive single-institution series of 184 colorectal carcinomas. Immunostaining for p53, p21, p27, Ecadherin, and beta-catenin was scored. Tumor cell proliferation was assessed by mitotic indices and Ki-67 labeling, apoptosis by quantification of apoptotic bodies. Reduced nuclear immunostaining for p21 (<10%) and p27 (< or =50%) and reduced membranous expression of Ecadherin were significantly associated with a poorer clinical course by univariate analysis. beta-catenin immunostaining had no prognostic impact. Mitotic and apoptotic indices as well as Ki-67 labeling below the median were indicators of poor prognosis. Complete absence of p53 nuclear staining was a significant adverse prognostic factor. By Cox regression, p53 = 0%, p53 = 0%, in combination with p27 < or = 50%, the mitotic index and the combined mitotic and apoptotic index added prognostic information to UICC stage. The authors found that growth pattern, lymphohistiocytic response, lymphatic permeation, and venous spread, too, each was a strong prognosticator in addition to UICC stage. The results support that tissue microarrays are a useful tool for screening immunohistochemical markers for prognostic use. An immunopanel of p21, p27, and p53 could be useful for prognostication in colorectal carcinoma in addition to UICC stage.     

非霍奇金淋巴瘤中McM7、p53和Ki-67的表达

目的 探讨McM7、p53和Ki-67在非霍奇金淋巴瘤（NHL）中表达的意义及相互关系。方法 应用组织芯片和免疫组化S-P法检测McM7、p53和Ki-67在9例反应性增生淋巴结、175例NHL组织中的表达。结果 NHL各组中McM7标记指数（1abelling index,LI）均高于Ki-67 LI;惰性组中McM7 LI和Ki-67 LI低于侵袭性组和高度侵袭性组,差异有显著性（P＜0．05）。NHL中p53表达的阳性率为23．4％,p53在惰性组、侵袭性组及高度侵袭性组之间的表达差异无显著性（P＞0．05）。Ki-67、p53和McM7三者在NHL中的表达呈平行关系（P＜0．01）。结论 McM7是反映细胞增殖的良好指标,作用优于Ki-67,其表达指数与NHL的组织分型、细胞增殖及恶性程度有关。p53基因突变在大多数NHL的发生、发展中可能并不是一个主要的分子事件。     

Expression of cell cycle regulators p57(KIP2), cyclin D1, and cyclin E in epithelial ovarian tumors and survival

The search for new prognostic indicators is especially important in the diagnosis and treatment of ovarian cancer because clinicopathologic criteria currently used to predict survival are largely inadequate. We examined 2 groups of patients with epithelial ovarian cancer, 1 group of long-term survivors (>5 years), and 1 group of short-term survivors (<2 years) for levels of expression of the cell cycle regulators p57(KIP2), cyclin D1, and cyclin E and their relationship with survival. Our findings show that p57(KIP2) is not associated with prognosis, in contrast to p27(KIP1) expression, which is previously shown to be positively associated with long-term survival in univariate analysis (P =.001). Cyclin E expression, in contrast to cyclin D1 expression, is marginally associated with short-term survival in univariate analysis for a group of 53 women. Among the short-term survivors, 15 (65%) of 23 were positive for cyclin E expression, compared with only 11 (37%) of 30 long-term survivors (P = 0.054). This association remained significant (P =.04) in a logistic regression analysis adjusted simultaneously for performance status and extent of residual disease, the 2 strongest predictors of survival in our study. We also found a significant difference in the frequency of the cyclin E staining pattern between nonserous and serous ovarian tumor subtypes (P =.0002). Immunostaining for levels of cyclin E and p27(KIP1) expression may have potential as prognostic markers in the management of ovarian cancer.     

Expression of cyclin D1, Ki-67 and PCNA in non-small cell lung cancer: prognostic significance and comparison with p53 and bcl-2

Uncontrolled cell proliferation is the hallmark of malignant tumours. Thus, the proliferative potential of tumour cells is an important prognostic factor. However, evaluation of the prognostic significance of the expression of proteins involved in regulation of cell proliferation remains controversial. In the present study, expression of Ki-67, PCNA and cyclin D1 was estimated in a group of 89 surgically resected non-small cell lung carcinomas using immunohistochemistry. The results were compared with expression of bcl-2 and p53 and with clinicopathological parameters including patients' survival. Ki-67 and PCNA were found to be moderately and highly expressed in 39% and 44% of the tumours, respectively. There was a strong correlation between Ki67 and PCNA expression. Forty five of 88 tumours (51%) showed overexpression of cyclin D1. Surprisingly, cyclin D1 was mainly localized in the cytoplasm and only a small group of tumours (9/88, 10%) showed nuclear staining as well. Bcl-2 and p53 expression was observed in 69% and 30% of the tumours, respectively. All these markers were found to be independent of clinicopathological parameters, except for Ki-67 and bcl-2 expression, which was associated with squamous cell carcinomas. It is concluded that none of the markers that were studied can be used as an independent prognostic factor, whereas the following combinations of markers may have favourable prognostic value: p53 positivity and low Ki-67 expression, p53 positivity and lack of cyclin D1 expression, bcl-2 positivity and low Ki-67 expression, and lack of cyclin D1 expression and low Ki-67 expression.     

Correlation between human papilloma virus infection in cervical lesions and expression of p53, p21 proteins and Ki-67

BACKGROUND: Human papilloma virus (HPV) is the most important factor in the oncogenic mechanism of cervical tumor. Furthermore, in a separate multi-stage process, abnormality in cell cycle kinetics has been demonstrated. In order to elucidate the oncogenic mechanism, we examined the relationship between cervical carcinoma and HPV infection, and also investigated the expression of p53 and p21 proteins as well as the cell proliferation capability by detecting Ki-67, and analyzed the correlations of these factors. MATERIALS AND METHODS: We studied the biopsy specimens from 107 patients of chronic cervicitis, cervical intraepithelial neoplasia and squamous cell carcinoma (SCC). HPV DNA was detected by the hybrid capture method. Immunostaining by LSAB procedures were performed using antibodies to p53 protein, p21 and MIB-1. The PCR-denaturing gradient gel electrophoresis (DGGE) method was used to search for mutation in exons 5, 6, 7 and 8 of p53. RESULTS: Of 107 cases studied, high-oncogenic HPV was detected in 80 cases (74.8%) with a particularly high prevalence in SCC. No correlation was observed between HPV infection and expression of p53, p21 or Ki-67. The degree of positivity of Ki-67 expression tended to be higher with disease progression. Cases strongly positive (2+) for p53 and p21 proteins were weakly positive for Ki-67, and cases positive (1+) or negative for p53 and p21 were strongly positive for Ki-67. CONCLUSION: In oncogenesis of cervical carcinoma, p53 protein, p21 protein and HPV may act separately as independent factors in some cases, and there is a strong possibility that other factors are involved.     

Protein expression of p53, p21 (WAF1/CIP1), bcl-2, Bax, cyclin D1 and pRb in human colon carcinomas

Tumour growth is regulated by a balance between proliferation, growth arrest and programmed cell death (apoptosis). Until recently, the majority of the studies dealing with oncogenesis has been focused on the regulation of cell proliferation. There is now growing understanding that control of growth arrest and apoptosis play key roles in the development of human cancer and in cancer treatment. Some of the more heavily studied proteins of importance for the control of growth arrest and apoptosis are p53, p21, bcl-2 and bax. Alterations in the p53 protein may lead to malignant transformation and defect therapy response, most likely as a result of defective p53-dependent apoptosis. In addition, p21 (WAF1/CIP1) is involved in cell-cycle arrest and probably in induction of p53-dependent apoptosis. Proteins belonging to the bcl-2 family are also important for normal apoptosis. Overexpression of bcl-2 protein is thought to reduce the apoptotic capacity, while bax protein seems to be necessary for induction of apoptosis. In this study, we have immunostained tissues from 93 primary colon carcinomas and have examined the expression of p53, p21 (WAF1/CIP1), bcl-2 bax, pRb and cyclin D1 for evaluation of their roles in colon-cancer progression. A highly significant association between p53 accumulation and downregulation of p21 (WAF1/CIP1) was seen. We also found a strong association between reduced/absent p21 and the development of metastases and death due to cancer disease. Cyclin D1, bcl-2 and bax protein failed to have independent prognostic impacts. Bcl-2 and bax protein levels showed an inverse relationship. The results of the present study indicate that reduced p21 protein levels play an important role in progression of colon cancer. We concluded that evaluation of p21 expression in primary colon carcinomas at the time of surgery might be a valuable tool in defining patients with a high risk of developing metastases. Received: 22 June 1999 / Accepted: 24 September 1999     

Prognostic value of immunohistochemical staining of p53, bcl-2, and Ki-67 in small cell lung cancer

Small cell lung cancer (SCLC) is one of the most fatal cancers in humans and many factors are known to be related to its poor prognosis. Immunohistochemical (IHC) stainings were done on SCLC specimens in order to investigate the prognostic value of the apoptosis-related gene expression and the tumor proliferative maker, and the relationships among these IHC results and patients clinical characteristics, chemoresponsiveness, and survival were analyzed. The medical records of 107 patients were reviewed retrospectively. IHC stainings for p53, bcl-2 and Ki-67 expressions were performed in the 66 paraffin-embedded biopsy samples. Sixty-six out of the 107 patients were evaluable for response rate and survival. The overall response rate was 75% (95% Confidence Interval=74-76%) and the median survival time was 14 months. The median survival time of limited stage was 16 months and that of extensive stage was 10 months. The prevalence of p53, bcl-2 and Ki-67 expression was 62%, 70%, and 49%, respectively. There were no correlations among the immunoreactivities of p53, bcl-2 and Ki-67 with clinical stage, chemoresponsiveness or overall survival. The clinical stage was the only prognostic factor influencing survival. The expression rates of p53, bcl-2, and Ki-67 were relatively high in SCLC without any prognostic significance. The exact clinical role of these markers should be defined through further investigations.     

Possible prognostic significance of p53, cyclin D1 and Ki-67 in the second primary malignancy of patients with double primary malignancies

Patients with two types of primary cancers are rare. In this study, we investigated the expression of p53, cyclin D1, and Ki-67 in the second primary malignancy. Tissue samples were obtained from the second primary cancer site of 43 patients who met the diagnostic criteria for double primary cancer. p53, cyclin D1 and Ki-67 were determined using immunohistochemistry. Categorical variables were compared using the Chi-squared test; correlation between data scores and histology was calculated using the Spearman’s rank-order correlation. The expression rates of p53, cyclin D1 and Ki-67 in the second primary malignancy site were 60.5%, 30.2% and 65.1% respectively. p53 expression showed statistically significant association with tumor occurrence interval, pathological grading and nodal metastasis (p < 0.05). Positive correlation was detected between the expression of cyclin D1 and Ki-67 and the expression of p53 (r = 0.313, p = 0.041; r = 0.319, p = 0.037, respectively). High-expressing p53 or cyclin D second primary malignancies were associated with decreased overall survival (p = 0.040 and p = 0.043, respectively). Ki-67 expression levels did not exhibit statistically significant differences in survival. In conclusion, elevated protein expression of p53, cyclin D1 and Ki-67 in the second primary malignancy is an indicator of more aggressive malignant behavior of the secondary tumor. These markers may have prognostic value in the clinical setting.