Cervical Carcinoma Tissues Research Articles

HHV-6 antigen and viral DNA detected in cervical cells from archived tissue using histochemical staining and hybridization

Background: Human herpesvirus type 6 (HHV-6), an ubiquitous virus, is the causative agent for exanthem subitum. The virus is frequently associated with lymphoproliferative disorders and other diseases. Recently, we have reported the frequent presence of HHV-6 in oral carcinoma and the present study extends the observation to cervical carcinoma. Objective: To examine the presence of HHV-6 in cervical carcinoma. Study design: Formalin-fixed, paraffin-embedded cervical carcinoma tissues were examined for the presence of HHV-6 by immunohistochemistry using two monoclonal antibodies that react to HHV-6-encoded p41/38 and gp 116/64/54. In situ hybridization with variant-specific probes were used to type the HHV-6 DNA sequences present. Results: A total of 14/26 (53.9%) carcinoma tissue specimens and 5/8 (62.5%) normal tissue specimens were positive for viral antigens. In situ hybridization studies revealed the presence of HHV-6 DNA sequences in 10/26 (38.5%) carcinoma tissue specimens and 1/8 (12.5%) normal tissue specimens. In the normal tissue, the HHV-6 was present in the endocervical ciliated columnar-epithelial cells and some cells in the subepithelial mucosa but in the carcinoma, the transformed cells were positive for the virus. Conclusion: HHV-6 viral proteins and DNA were found in more than one third of the cervical tissue examined suggesting possible viral expression in these tumours. The significance of the distribution and role of the HHV-6 in cervical tissue remains unclear. Since HHV-6 has an oncogenic potential, the virus may cooperate with other transforming agents for the progression of the disease.

Presence of Oncogenic HPV DNAs in Cervical Carcinoma Tissues and Pelvic Lymph Nodes Associating with Proliferating Cell Nuclear Antigen Expression

The presence of oncogenic HPV DNAs (HPV-16/18) in cervical carcinomas and their normal and metastatic pelvic lymph nodes and the expression patterns of proliferating cell nuclear antigen (PCNA) in cervical carcinomas were retrospectively studied to elucidate the possible roles of them in malignant transformation and progression of the disease. HPV-16/18 DNAs were detected by polymerase chain reaction using HPV E6 type-specific primers in 79 patients with cervical cancer: 31 patients who had pelvic lymph node metastasis (group I) and 48 patients without pelvic lymph node metastasis (group II) who were proven by pathologic examination of surgical specimens. HPV-16 or -18 DNAs were detectable in cervical carcinoma tissues in 60 patients from 79 cervical cancer patients (75.9%; HPV-16 was 67.1% and HPV-18 was 8.9%). HPV DNAs were amplified from metastatic pelvic lymph nodes in 13 patients of group I (42%) and from nonmetastatic lymph nodes in 7 group I patients (22.5%). Recurrence was identified in 9 group I patients (29.0%) in 3 years of follow-up. HPV DNAs were amplified from nonmetastatic lymph nodes in 11 group II patients (22.9%). Two group II patients, who had HPV-16 DNA by PCR in nonmetastatic nodes, were recurrent. PCNA was overexpressed in 66.7% of HPV-16- or -18-positive cervical cancers and 16.7% of HPV-16- or -18-negative cervical cancers. However, the expression levels of PCNA in cervical cancers were not influenced by the presence of oncogenic HPV DNA or pathologic metastasis in the pelvic lymph nodes. In conclusion, HPV DNA could be amplified from some metastatic and nonmetastatic pelvic lymph nodes and the detectability of oncogenic HPV DNA in pelvic lymph nodes may represent the poor outcome in the treatment of disease. The expression of PCNA protein which was associated with presence of oncogenic HPV DNAs in cervical cancers, suggesting activation of S phase of cell cycle, may contribute to the malignant progression by HPV-16 or -18.

Purification and Characterization of Hexosaminidase from Human Uterine Cervical Carcinoma

Normal human uterine cervical tissue and uterine cervical carcinoma tissue were collected and subjected to fractionation of hexosaminidase isoenzymes Hex A, Hex B, and Hex I using DEAE-cellulose anion-exchange chromatography. Hex A was found to be the major isoenzyme in control tissues, whereas Hex B was the major isoenzyme in carcinoma tissues. These two major isoenzyme fractions were first purified using heparin–Sepharose 4B affinity chromatography and then subjected to further purification on ϵ-ACMA–Sepharose 4B. The purified isoenzymes were found to be homogeneous by polyacrylamide gel electrophoresis. The Hex A and Hex B isoenzymes obtained from control and carcinoma patients were characterized. Hex A from control and carcinoma patients exhibited more or less similar properties. However, the Hex B isoenzyme from carcinoma patients exhibited some characteristic variations in pH, temperature, substrate concentration optima, and isoelectric point compared with the control. From these observations it may be inferred that altered properties of the Hex B isoenzyme fraction from carcinoma patients may be the reason for the elevated activity of hexosaminidase in carcinoma of the uterine cervix.

Simultaneous Detection by Consensus Multiplex PCR of High- and Low-Risk and Other Types of Human Papilloma Virus in Clinical Samples

A consensus multiplex PCR (CM-PCR) technique was developed to detect high-risk (HPV 16/18), low-risk (HPV 6/11), and over 40 other types of human papillomavirus (HPV), separately but simultaneously, by mixing three pairs of consensus primers in the same PCR mixture, for gene amplification. Simultaneous detection of three groups of HPV DNA provides valuable information for clinical practice and this procedure is simple and convenient for routine laboratory examinations. We detected HPV DNA sequences in plasmid HPV DNA and DNA extracted from tissues of condyloma acuminata and cervical carcinoma and from exfoliated cells of the lower genital tract of healthy Chinese women living in the People's Republic of China. We confirmed that this simple, convenient, and cost-beneficial CM-PCR technique is reliable for the detection of HPV DNA sequences.

Loss of heterozygosity on the short arm of chromosome 17 in uterine cervical carcinomas

DNA samples from 26 cervical carcinoma and normal tissue pairs were studied by restriction fragment length polymorphism (RFLP) analysis to determine the frequency of loss of heterozygosity (LOH) on 17p. Allelic loss in the p13.1 region of chromosome 17, known to contain the TP53 locus, was not detected in any of 10 informative cases. Instead, LOH was detected on 17p13.3 in eight (40%) of 20 informative cases with at least one of two 17p13.3 markers. Examination of the intragenic region of p53 in the same samples using polymerase chain reaction (PCR)-RFLP analysis showed no LOH in the gene (none of 16 informative individuals).

Lipid-Soluble Antioxidants: β-Carotene and α-Tocopherol Levels in Breast and Gynecologic Cancers

Free radical-induced damage is etiologically implicated in many chronic diseases including cancer. Epidemiologic data suggest an association between increased dietary intake of nutrients that are high in antioxidant vitamins and protection against the incidence of some human cancers. The purpose of this study was (a) to determine whether specific tissue antioxidants (β-carotene and α-tocopherol) and any differences in their levels were measurable in randomly selected human breast and gynecologic malignant neoplasms and nonneoplastic tissue samples obtained from the same patient and (b) to establish normal ranges of these two antioxidant levels in human female reproductive tract tissues. Tissue samples were excised immediately from surgical specimens and released by staff pathologists from a spectrum of human female cancers. Neoplastic and adjacent nonneoplastic tissues samples were obtained from the same patient. Normal reproductive tract tissue samples were obtained from women undergoing hysterectomy for benign gynecologic conditions. Breast carcinoma and adjacent nonmalignant tissue specimens were obtained from women undergoing mastectomy. The concentrations of β-carotene and α-tocopherol were measured by high-pressure liquid chromatography. In the same patient, β-carotene levels were significantly lower in the cervical ( P < 0.01) and endometrial ( P < 0.005) carcinoma tissues than the levels detectable in adjacent nonneoplastic sites. In contrast, β-carotene levels were higher in the ovarian ( P < 0.05), breast ( P < 0.005), and vulva ( P < 0.05) carcinoma tissues. The α-tocopherol concentrations were significantly higher in the cancer tissues of cervix ( P < 0.01) and endometrium ( P < 0.001) than those in adjacent noninvolved tissue sites. The tissue concentrations of α-tocopherol in malignant and adjacent normal sites in breast, ovary, and vulva were comparable. For the first time, the ranges for β-carotene and α-tocopherol levels in the normal female reproductive tract tissues were also established. The present findings of contrasting tissue levels of the antioxidants (β-carotene and α-tocopherol) in breast, cervix, endometrium, ovary, and vulva cancers and in nonneoplastic tissues of the same patient suggest an organ-specific and heterogenous distribution. These antioxidants appear to be essential nutritional requirements of the human female reproductive tract and breast and are implicated in the pathophysiology and carcinogenesis of these human organs. The findings require further study of the role of these antioxidant nutrients in epithelial cell proliferation, maturation, and differentiation.

Human Papillomavirus Detection in Cervical Carcinoma Tissues and Paraaortic Lymph Nodes by the Polymerase Chain Reaction

Tissues of 45 cervical cancers and paraaortic lymph nodes from the same patients were evaluated by polymerase chain reaction with L1 consensus primers and hybridization with type-specific oligomer probes of HPV-6, -11, -16, -18, -31, -33, -35, and -45 for the detection and classification of subtypes of human papillomavirus (HPV). The clinicohistologic findings of surgical specimens from radical hysterectomy were compared with the results of HPV detection to use as a possible prognostic marker for the early detection of paraaortic nodes involvement. Metastasis to paraaortic nodes in cervical cancer, suggesting extrapelvic involvement of tumor, is clinically important to predict prognosis. The HPV-16 DNA was most prevalent in cervical cancers (76%; 34/45). Five tumors were positive for HPV-18, two tumors each were positive for HPV-31 or -33, one tumor was hybridized to HPV-45, and one woman had an unidentified type of HPV. No HPV was detected in three cases of cervical cancer. The histologic types of the cervical cancers were correlated with the HPV types. Of the 39 tissue specimens of squamous carcinomas analyzed, only 2 (5%) showed HPV-18, in contrast to 30 (77%) of 39 squamous carcinomas having HPV-16. Of the 5 cases of adenocarcinomas, 3 (50%) showed HPV-18, and 3 (50%) showed HPV-16. HPV DNAs were detected in histologically negative paraaortic lymph nodes (31%; 14/45): the HPV-positive PCR products from paraaortic nodes were only hybridized to HPV-16. The nature of the relationship between the presence of HPV DNA and node tumor involvement is still not known. HPV-16 DNA in paraaortic nodes may suggest subclinical early metastases or tumor cells destroyed by immune cells and may provide important information in decisions regarding postoperative adjuvant treatment. The prognostic significance of HPV DNA in histologically negative paraaortic lymph nodes remains to be established after several years of follow-up.

Search for herpes simplex virus type 2 (HSV-2) and human papillomavirus (HPV) in the normal and abnormal cervical samples

The specimens of 111 cervical carcinomas, 68 chronic cervicitis and 43 normal cervical exfoliated epithelial cells were examined for the presence of HSV-2 DNA sequences with DNA hybridization using HSV-2 BgL I N fragment probe labelled by 32P-dCTP. The result showed that the infection rates of HSV-2 in the samples of cervical cancer, chronic cervicitis and normal epithelial cells were 14.41% (16/111), 27.94% (19/68) and 25.58% (11/43), respectively. It was implied that early stages carcinogenesis of cervical epithelial cells might be correlated with the HSV-2 infection. Sixteen HSV- 2 positive samples of cervical carcinomas were also examined for the presence of the sequences homologous to human papillomavirus (HPV) type 6B/11, 16 and 18 DNA using dot blot hybridization (Tm-17C). The result indicated that 13 out of 16 were HPV 16 DNA hybridization positive accounting for 81.2% of all HSV-2 positive samples and none of them were positive for HPV type 6B/11 and 18. The result indicated that double infection of HSV-2 and HPV16 in the same cervical carcinoma tissues may provide a strong evidence of the viral synergistic interaction in the induction of female cervical carcinomas.

Functions of human papillomavirus proteins.

Cervical cancer is the second leading cause of deaths from cancer among women worldwide with approximately 500 000 deaths annually. Epidemiologic studies have implicated a sexually transmitted agent as a cause of cervical cancer, and molecular virology studies over the past 10 years have established a strong association between specific human papillomavirus (HPV) types and certain anogenital carcinomas, including cervical cancer (reviewed in zur Hausen and Schneider 1987). Over 65 different HPV types have now been described, and each is associated with a specific clinical entity (DeVilliers 1989). Approximately 20 or 25 HPVs have been associated with anogenital lesions; these HPVs have been further classified as either “low-risk” or “high-risk” types based on the preneoplastic character of the clinical lesions with which they are associated. Low-risk HPVs such as HPV-6 and HPV-11 are generally associated with venereal warts or condyloma acuminata which only rarely progress to malignancy. The high-risk HPVs include HPV-16 and HPV-18 and these are associated with squamous intraepithelial neoplasias which are potentially precancerous. In the cervix, they are associated with cervical intraepithelial neoplasia, or CIN. These CIN lesions are considered preneoplastic in that a small percentage of high-grade CIN lesions will progress to cervical cancer. Approximately 70% of human cervical cancers contain either HPV-16 or HPV-18 DNA (zur Hausen and Schneider 1987). Indeed, HPV-16 and HPV-18 DNA were originally isolated from human cervical carcinoma tissues (Durst et al. 1983; Boshart et al. 1984). Other high-risk HPVs, including types 31, 33, 35, 39, 45, 51, and 52, have subsequently been identified and have also been associated with CIN lesions and with invasive cervical carcinomas. All together, approximately 85% of cervical cancers can be shown to contain DNA of one of the high-risk HPV types (Riou et al. 1990).

Human papillomavirus, Epstein-Barr virus, and cervical carcinoma in Greenland.

The incidence of cervical cancer in Greenland is one of the highest in the world. This is in accordance with known epidemiologic risk factors, in particular of sexual lifestyle and tobacco smoking. Yet a recent study of cervical smears from randomly selected Greenlandic women failed to demonstrate any elevated prevalence of human papillomavirus (HPV), of which particularly HPV types 16 and 18 are assumed to play a role in the development of cervical dysplasia and carcinoma. Another oncogenic virus, Epstein-Barr virus (EBV) is known to be widespread in the Greenlandic population and the virus has been reported to infect the female genital tract. We therefore used the polymerase chain reaction to examine paraffin-embedded tissues of cervical carcinomas from 11 indigenous Greenlandic women and, for comparison, also cervical carcinoma tissues from 11 Danish patients, for EBV, HPV 16 and HPV 18 DNA sequences. None of the 22 cervical tissues contained EBV DNA. Six Greenlandic and five Danish carcinomas harbored HPV 16 DNA, and one carcinoma of each group was HPV 18-positive. In conclusion, cervical carcinoma is not associated with EBV infection. Further, the oncogenic HPVs are probably encountered at the same rate in carcinomas from Greenland and from Denmark.

Ectopic production of prolactin in uterine cervical carcinoma

Sera from 743 patients with uterine cervical carcinoma were assayed for levels of circulating prolactin (PRL). Markedly abnormal serum prolactin levels were observed in 30.8% (229/743) of patients. During the early stages of cancer, the degree of PRL elevation in serum and incidence of abnormal PRL levels are much greater than those found for carcinoembryonic antigen, which is generally considered to be a circulating cervical tumor marker. Surgical removal of cervical carcinoma resulted in normalization of serum PRL concentrations. Immunohistochemical staining techniques and primary tissue culture of cervical carcinoma were used to verify the presence of PRL in the uterine cervical carcinoma cells. Twenty-two of forty-nine cervical carcinoma tissue sections showed cytoplasmic staining for prolactin, while normal cervix tissues tested were negative. Production of prolactin was directly demonstrated in the early tissue cultures of cervical carcinoma. These studies show ectopic production of PRL by uterine cervical carcinoma. Prolactin production may be a potential marker for detecting early occult tumors or for gauging the effectiveness of therapy for human cervical carcinoma.

Localization and therapy of human cervical tumor xenografts with radiolabeled monoclonal antibody 1H10

Murine IgG 3 monoclonal antibody (Mab) 1H10, which recognizes a tumor-associated antigen expressed on the surface of more than 40% of human cervical carcinoma tissues, was used for in vivo localization and therapy of cervical tumor xenografts. A human cervical carcinoma cell line, CaSki, was used as our experimental tumor system. Mab 1H10 antigen expression on the surface of CaSki cells was found to be cell-cycle independent. The ability of Mab 1H10 F(ab′) 2 to bind to CaSki tumor xenografts was verified by direct immunohistochemical staining of thin tumor sections with a Mab 1H1O-peroxidase conjugate. Radioimmunoscintigraphy of nude mice bearing CaSki tumors after iv administration of [ 131I]1H10 F(ab′) 2 showed clear tumor images 48 hr after Mab injection. Radiolabeled Mab 1H10 F(ab′) 2 was found to specifically localize in solid CaSki tumors 96 hr after antibody injection. Radioactivity in tumor tissue was 4 times higher than that in kidney tissue and over 6 times higher than that in liver tissue. Mab 1H10 F(ab′) 2 binding to xenografted CaSki tumors was 17 times greater than a control IgG 3 F(ab′) 2 after 96 hr. Therapy of athymic mice bearing established CaSki tumors with three iv injections of 100 μCi [ 131I]1H10 F(ab′) 2 resulted in extensive tumor necrosis and significant suppression ( p < 0.05) of tumor growth compared to that in control mice. These results indicate that Mab 1H10 F(ab′) 2 may be clinically useful for detection or treatment of cervical cancer.

Doxorubicin: monoclonal antibody conjugate for therapy of human cervical carcinoma.

Doxorubicin (Dox) was conjugated via a dextran linker to the F(ab')2 fragment of monoclonal antibody (MAb) 1H10 which recognizes an antigen expressed on the surface of human cervical carcinoma cells and tissues. Drug-antibody conjugates (1H10-Dox) with a molar ratio of Dox to MAb ranging from 40:1 to 60:1 retained antigen-binding and pharmacological activities. Anti-tumor activity of the conjugate in vitro was evaluated by measuring inhibition of [5-3H]-uridine incorporation into cellular RNA. 1H10-Dox was found to be 30 times more toxic to cervical tumor cells than a control MAb-Dox conjugate and 150 times more potent than Dox coupled to dextran. In addition, 1H10-Dox was less toxic to antigen-negative cells in vitro, suggesting that 1H10-Dox killing of cervical carcinoma cells was antibody-mediated. 125I-labeled 1H10-Dox preferentially localized in solid human cervical carcinoma xenografts in athymic mice with tumor-to-blood ratios of 1H10-Dox reaching 17.9 after 24 hr and 32.8 after 48 hr. Treatment of athymic mice bearing human cervical tumors with 1H10-Dox resulted in a dose-dependent inhibition of tumor growth. Multiple administrations of 1H10-Dox at a dose corresponding to 20 micrograms doxorubicin significantly suppressed the growth of human cervical tumors in nude mice without significant side effects (weight loss), and this suppression was antibody specific. Both i.p. and i.v. administration of 1H10-Dox were found to be equally effective. Our results suggest that 1H10-Dox may be useful for the treatment of human cervical carcinoma.

Histochemical study of estrogen and progesterone receptors in cervical carcinoma

Estrogen and progesterone receptors were determined in 56 cases of primary cervical carcinoma tissue by histochemical methods. 42.86% of the 56 cases were ER positive and 46.43% PR positive. No correlation was found between ER and PR status and age, menopausal status of the patient and clinical stage. Adenocarcinoma had significantly higher ER and PR positive rate than squamous carcinoma. Highly differentiated carcinoma had higher ER and PR positive rate than middle- and dedifferentiated carcinoma. A positive correlation between ER and PR and cellular infiltration of stroma and peritumoral reaction of fibrin was found. The patients with ER and PR positive had a significantly higher 5-year survival. ER and PR may be used as prognostic factors in cervical carcinoma.

Ectopic production of prolactin in uterine cervical carcinoma

Sera from 743 patients with uterine cervical carcinoma were assayed for levels of circulating prolactin (PRL). Markedly abnormal serum prolactin levels were observed in 30.8% (229/743) of patients. During the early stages of cancer, the degree of PRL elevation in serum and incidence of abnormal PRL levels are much greater than those found for carcinoembryonic antigen, which is generally considered to be a circulating cervical tumor marker. Surgical removal of cervical carcinoma resulted in normalization of serum PRL concentrations. Immunohistochemical staining techniques and primary tissue culture of cervical carcinoma were used to verify the presence of PRL in the uterine cervical carcinoma cells. Twenty-two of forty-nine cervical carcinoma tissue sections showed cytoplasmic staining for prolactin, while normal cervix tissues tested were negative. Production of prolactin was directly demonstrated in the early tissue cultures of cervical carcinoma. These studies show ectopic production of PRL by uterine cervical carcinoma. Prolactin production may be a potential marker for detecting early occult tumors or for gauging the effectiveness of therapy for human cervical carcinoma.

Human papillomaviruses and small cell carcinoma of the uterine cervix

The in vitro DNA amplification technique of polymerase chain reaction was used to evaluate the possible presence of human papillomavirus (HPV) in small cell carcinoma of the uterine cervix. None of the 12 cases examined contain detectable amounts of either HPV type 16, 18, 31, or 33 DNA. On the other hand, HPV types 16 and 18 DNA were found in 14 (93.3%) and 9 (60.0%) of 25 invasive cervical squamous carcinoma tissues. The results seem to suggest that these types of HPV are not present or are present in extremely small quantities in cervical small cell carcinoma. Such an absence of HPV DNA makes it unlikely that these types of HPV play any etiological role in the pathogenesis of cervical small cell carcinoma.

Stage related detection rate of human papillomavirus DNA in invasive cervical cancer

SummaryHistologically proved cervical carcinoma tissue samples from 50 patients were analysed for the presence of human papillomavirus types 6, II, 16 and 18 deoxyribonucleic acid sequences. P'2 labelled papillomavirus test-DNAs were analysed by Southern blot hybridisation. Viral types 16 and 18 have high prevalences in cervical carcinoma tissues and they may play a role in cervical carcinogenesis. Increasing detection rates of oncogenic viral types were found in more advanced stages. Results suggest that human papillomaviruses are involved in the progression of the disease as well as in its initiation.

Analysis of the unoccupied site of an integrated human papillomavirus 16 sequence in a cervical carcinoma

We have previously cloned and analyzed the structure of a type 16 human papillomavirus (HPV16) integration in a primary cervical carcinoma tissue, M50 (Choo et al., J. Virol. 62, 1659–1666, 1988). We found that specific nucleotide sequences within the HPV16 genome influenced the genomic organization of the integrated viral genome. Using the viral-cellular junctions of the M50 DNA as probes, we have now cloned the unoccupied site from a human genomic library. Mapping analysis showed that a deletion of about 1.1 kilobase pairs (kb) had occurred at the integration site of M50. Sequencing of the integration junctions of the unoccupied site and comparison with the viral sequence has revealed short regions of sequence homology between the viral and the cellular genomes at both junctions. Our results are consistent with a mechanism of integration of the HPV16 sequences in the M50 carcinoma involving illegitimate recombination events using short patches of homologous sequences between the two heterologous genomes for anchorage and as guides for crossover. Preferred topoisomerase I cleavage sites and alternating purine and pyrimidine bases, which favor the formation of Z-DNA, could also be identified at the integration regions, supporting a proposed role for the topoisomerase I enzyme in the illegitimate recombination in the viral integration process.

Cervical Cancer Cell Lines Containing Herpesvirus Markers

Five epithelioid cell lines were established from invasive cervical carcinoma tissue and were examined for herpes simplex virus (HSV) antigens or virus-induced changes. Immunofluorescence studies with antiserum to an HSV type 2 nonstructural polypeptide detected perinuclear antigens. In these cell lines, concentrations of a membrane fucopeptide that is regularly present in normal cells were reduced to levels similar to those previously observed in virus-transformed cells. One of the cell lines inoculated into athymic mice produced tumors.

Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma

A heterologous antiserum for human cervical squamous cell carcinoma was prepared and specificity determined by Ouchterlony immunodiffusion and immunofluorescence studies. With this antiserum, a tumor antigen was purified from human cervical squamous cell carcinoma tissue. The specificities of the antigen and the antiserum were then re-examined by a radioimmunoassay method using 125 I-labeled purified antigen. Although normal cervical tissue extract showed a moderate cross-reactivity in the radioimmunoassay, the circulating antigen activity could not be detected in normal women or in several patients with carcinomas, whereas 27 of 35 patients with cervical squamous cell carcinoma showed detectable serum antigen activity. All aptients with advanced stages of cervical squamous cell carcinoma showed detectable antigen levels. These results indicate that there is a quantitative abnormality, at least, of this tumor antigen in patients with cervical squamous cell carcinoma and that the radioimmunoassay for the antigen is a potentially useful tool in clinical care.