Kaposi sarcoma (KS) is a low-grade vascular neoplasm first described by Kaposi in 1872 1. It has a complex pathogenesis involving a disordered cytokine network, immune alterations, and active infection with human herpes virus-8 (HHV-8) 2,3. Four epidemiologic forms of KS have been described: classic form (C-KS); African-endemic KS; immunosuppressive drug-related KS; and acquired immunodeficiency syndrome-related KS. All forms of KS have the same histology 3. KS poses problems in a histologic diagnosis because of its broad morphologic spectrum and similarity to many benign vasoproliferative lesions (e.g. pyogenic granuloma, bacillary angiomatosis, and microvenular hemangioma) and tumors with a prominent spindle cell component (e.g. spindle cell hemangioma, spindle cell angiosarcoma, and dermatofibrosarcoma protuberans) 4,5.
Identification of the early KS lesions in the skin can be particularly problematic. In 1994, Chang et al.6 discovered HHV-8, or the KS-associated herpes virus, as the causative agent of acquired immunodeficiency syndrome-associated KS and it has subsequently been found in all epidemiologic forms. The latent nuclear antigen-1 of HHV-8 (HHV-8 LNA-1) is a nuclear antigen expressed in all cells latently infected by the virus 7. HHV-8 LNA-1 is a protein encoded for by open reading frame-73 of the viral genome. The protein is expressed predominantly during viral latency and appears to play a role in viral integration into the host genome 8. It also interferes in apoptosis through interactions with p53 9. Biological factors such as tumor angiogenesis and cellular proliferation characteristics are fundamental determinants of the behavior of many tumor types. CD31 has been largely used for immunohistochemical analysis of tumor angiogenesis 10. CD31 is a glycoprotein located on endothelial cells, platelets, monocytes, and granulocytes 11. It has been shown to be a sensitive and specific indicator of endothelial differentiation 10. The CD34 protein is a member of family of single-pass transmembrane sialomucin proteins that is also expressed on vascular endothelia in the adults and on hematopoietic cells 12.
Cyclins are important protein molecules involved in the induction and control of the cell cycle. Factors modulating exit from G0 and progression through the G1 phase of the cell cycle are critical in determining the overall growth rates of cells and, more importantly, cellular sensitivity to cytotoxic agents and radiotherapy. Cyclin D1 regulates G1-phase progression by activating the protein kinase activity of p34cdc2 and phosphorylation of the retinoblastoma protein 13. Progression through the cell cycle is governed by a series of cyclin-dependent kinases (CDKs), which are activated by binding to cyclin proteins, regulated by phosphorylation, and inhibited by CDK inhibitors 14. Two families of CDK inhibitors that modulate the activity of cyclin–CDK complexes have been described: the Cip/Kip and Ink 4 families 14. The Cip/Kip family includes p21Cip1, p27Kip1, and p57Kip2 proteins 15. CDK inhibitor p27Kip1 seems to provide a central signal that coordinates varied inputs from the extracellular environment and serves as a threshold for progression to the S phase or exiting the cell cycle 16. One of the easiest and most reliable methods for the assessment of tumor cell proliferation is Ki-67 antigen expression 17. Ki-67 is a nonhistone protein that is detected in the nucleus in the G1, S, G2, and M phases of the cell cycle, but not in the G0 phase. The number of cells with Ki-67 is used to mark cell proliferation 17. p53 is a tumor suppressor gene, which is known as the guardian of the genome. This is because of its ability to regulate the cell cycle under stressful conditions and to maintain genome integrity 18. p53 protein is critical in negatively controlling the cell cycle and the loss of p53 function plays an important role in the development of diverse cancers 19. Tumor cells can evade the normal control of cell growth and survival by several mechanisms, including autocrine stimulation and overexpression of proto-oncogenes that function as antagonists of apoptosis. One of the most important negative regulators of apoptosis is Bcl-2 and its related proteins (Bcl-2 family members), whose expression is regulated by cytokines and other death survival signals at different levels 20.
The aim of this study was to evaluate the role of HHV-8 LNA-1, CD31, and CD34 as diagnostic markers in classical cutaneous KS and its mimickers. Cyclin D1, p27Kip1, Ki-67, p53, and Bcl-2 were also evaluated as prognostic markers in classical cutaneous KS.
Patients and methods
The present study included a total of 42 patients: 15 cases of classical cutaneous KS and 27 cases of KS mimickers [spindle cell hemangioma (seven), dermatofibrosarcoma protuberans (five), angiosarcoma (five), and pyogenic granuloma (10)] diagnosed between 2006 and 2010. These patients were recruited from Dermatology and Plastic Surgery Departments, Faculty of Medicine, Tanta University Hospitals, in addition to the Oncology Institute in Tanta [25 cases (15 KS and 10 pyogenic granuloma)] and from their files [paraffin blocks (17 cases)]. KS patients were classified into two groups: group I included seven patients (patch/plaque stage) and group II included eight patients (nodular stage). The inclusion criteria included C-KS patients (with cutaneous lesions only and HIV negative confirmed by the blood enzyme-linked immunosorbent assay test). After informed consent was obtained, an excision biopsy was taken. It was formalin fixed and paraffin embedded. For all specimens, hematoxylin and eosin-stained preparations were examined to confirm the clinical diagnosis.
Four-micrometer-thick sections were cut from original formalin fixed-embedded tissues from the primary tissue blocks and placed on silanized slides and examined for immunohistochemical staining using a standard streptavidin–biotin–peroxidase complex with antibodies 21 as shown in Table 1. Each specimen was blindly evaluated by the pathologist. The positive staining with the markers was evaluated using a four-point scoring system, with 0 for no staining, +1 for mild staining (up to 50% positive cells), +2 for moderate staining (50–75% positive cells), and +3 for marked staining (75–100% positive cells).
All data obtained were transferred to the statistical package for the social sciences version 15 (IBM Co., New York, New York, USA) for analysis. Data were summarized as mean±SD using Student’s t-test. Comparisons between groups were carried out using the χ2-test and Fisher’s exact test for quantitative variables. Statistical significance was determined at a level of P value equal to or less than 0.05.
The study population included a total of 15 cutaneous (HIV negative) C-KS patients (Fig. 1); all were men. The mean age of the studied groups was 64.16±11.8 years in group I and 56.85±12.33 years in group II. The difference between the studied groups revealed no significant changes. The mean duration of KS was 18.42±9.64 and 10.14±3.33 months in group I and group II, respectively. Comparison of the duration of the disease in the studied groups revealed significant differences between group I and group II (P=0.050). Comparison of the anatomic distribution of the lesions in the studied groups revealed no significant differences between group I and group II (Table 2).
The study population also included 27 cases of KS mimickers. The histologic features of KS included spindle-shaped tumor cells surrounding hyperemic vascular slits, often accompanied by extravasated erythrocytes, hemosiderin, and fibrosis (Fig. 2a and b). Similar to KS, cases of spindle cell hemangioma were composed of bland spindle cell proliferations between vascular lumens. However, unlike KS, vacuolated cells were sometimes found to have lining lumens as well as epithelioid endothelial cells. Infiltrative lesions with bland spindle cells in a tight, storiform pattern were found for cases of dermatofibrosarcoma protuberans. Cutaneous angiosarcomas were studied in five patients. Infiltrating, anastomosing vascular channels lined by numerous plump spindled-to-epithelioid cells with large hyperchromatic nuclei characterized these cases. All pyogenic granulomas were nodular proliferations of small capillaries with epidermal ulceration, resembling granulation tissue.
CD31 and CD34 positivities were demonstrated as microvessel density in all cases of KS (Figs 3 and 4) and also in other mimickers, but with variable intensity (Table 3). All 15 KS cases showed positive nuclear staining for HHV-8 (100%) with variable intensity (Fig. 5); group II generally had more positive spindle cells compared with group I. Normal vascular endothelial cells were negative in all cases of KS. KS mimickers were all negative for this antigen (Table 3). The level of cyclin D1 expression (nuclear) detected in group II was higher than that of group I, with a significant difference between both groups (P=0.007) (Fig. 6, Table 4). All cases of KS were p27Kip1 positive and showed staining of variable intensity with a nuclear pattern (Fig. 7). Markedly higher intensity was detected in group I than in group II, with a significant difference between both the groups (P=0.002) (Table 4). Ki-67 positivity was nuclear in most cases of KS, but varied in intensity (Fig. 8); there was no significant difference between the two groups (P=0.489) (Table 4). A negative (Fig. 9) and weak nuclear staining pattern for p53 was found for the majority of cases in group I. The positive cases were higher in group II (Fig. 10), with a significant difference between the two groups (P=0.006) (Table 4). A diffuse cytoplasmic staining pattern was found for Bcl-2 in KS-positive patients throughout the entire lesion, but varied in intensity. A positive intensity was detected in group II (Fig. 11) compared with group I and the difference was statistically significant (P=0.005) (Table 4).
The pathogenesis of KS, a tumor of probable vascular origin, remains unclear. It is still unclear whether KS is a true malignancy or whether it represents a reactive polyclonal process 22. Differentiation of KS from other benign or malignant vascular tumors as well as other nonvascular spindle cell soft-tissue neoplasm may, on occasion, be difficult. The histologic features of these lesions overlap, leading to difficulties in diagnosis. With the discovery of HHV-8 in all forms of KS, it became possible to consider virus detection as a potential diagnostic test. Molecular methods have traditionally been used to identify HHV-8 in human tissues. These include PCR amplification, 23 direct in-situ hybridization 24, in-situ PCR 25, and reverse transcriptase in-situ PCR 26. These methods are labor intensive, time consuming, and require highly skilled laboratory personnel. In addition, highly sensitive PCR methodologies have led to a recent controversy. The commercial availability of a monoclonal antibody to HHV-8 LNA-1 has made cost-effective, tissue-localized identification of HHV-8 in fixed human specimens possible. The reliable detection of HHV-8 in fixed tissues by immunohistochemistry (IHC) could enable one to differentiate KS from other histologically similar entities 5,27. For this reason, the current work studied the sensitivity and specificity of this antibody in the detection of HHV-8 LNA-1 in KS. All KS cases demonstrated positive, nuclear staining for HHV-8 (100% sensitivity), whereas all cases of KS mimickers were negative for this antigen (0%). The uniform expression of HHV-8 in all of the present study cases of C-KS confirms the observations of previous investigators using IHC 7,28–33. The current results were similar to those of Dupin et al.7, as they observed a similar pattern of staining, with fewer immunoreactive spindle cells noted in patch/plaque lesions compared with nodular lesions. These authors also found LNA-1 expression in cells of other HHV-8-positive lesions such as multicentric Castleman’s disease and primary effusion lymphoma, but no staining was found in samples of multiple myeloma, prostate cancer, or angiosarcoma. Unlike Dupin et al.7, the current study also studied examples of spindle cell lesions that might enter the differential diagnosis of KS and found them to be uniformly negative for HHV-8 LNA-1. In the study of Cheuk et al.34, HHV-8 LNA-1 was applied to five cases of pyogenic granuloma; the results were positive in four cases and negative in one case. Considering the demonstrated high sensitivity and specificity of the immunostaining, they reported that the four positive cases probably represent an early phase or a morphologic variant of KS, whereas the negative case represents a pyogenic granuloma-like lesion. The current study did not find HHV-8 LNA-1 in any of the cases of angiosarcomas, in contrast to some studies using PCR-based methods 35,36. The current study indicates that immunohistochemical analysis may be more reliable because there is immunolocalization of the viral product to the lesional cells. False-positive results in previous PCR-based studies might be attributable to contamination by HHV-8-positive passenger lymphocytes, nonspecific amplification, or misdiagnosis of KS as angiosarcoma. Thus, it seems that the association of HHV-8 with angiosarcoma is probably rare, if it occurs at all 36.
Cell cycle progression requires the coordinated performance of a series of regulating molecules that orchestrate cycle transitions through either mitogenic or antiproliferative signals 37. One of the many cell-cycle-regulating molecules is cyclin D1 37. Amplification of the cyclin D1 gene with protein overexpression can allow cancer cells to progress from the resting G0 phase to G1 and/or G1 to S phases of the cell cycle, resulting in enhanced DNA replication. Increased cyclin D1 expression is associated with features of malignancy, disease recurrence, and poorer prognosis in a number of malignancies such as ovarian tumors 38, and esophageal 39 and pancreatic cancers 40, detected by IHC and in-situ hybridization, and may be an important prognostic factor. Furthermore, inhibition of cyclin D1 expression in colonic cancer cells with antisense can reverse the tumorigenicity of these cells 41. The results of these studies suggest that cyclin D1 may be a potentially useful target in the treatment of malignancies that overexpressed the protein. The present study found that cyclin D1 overexpression was prevalent in classic nodular lesions. This is in agreement with Hong et al.42, who used IHC, in a study of 41 KS cases, and found a strong link between cyclin D1 overexpression, HIV negativity, and nodular subtypes. It seems that for the progression of KS to a more advanced, nodular stage lesion, the effect of overexpressed cyclin D1 protein and its resultant effect on the cell cycle is important in HIV-negative patients 43.
The p27Kip1 regulates cell proliferation by binding and inhibiting G1 cyclin–CDK complexes and therefore negatively regulating progression through the G1 and S phases of the cell cycle 15. In addition, p27Kip1 associates with cyclin E–CDK2, cyclin A–CDK2, and cyclin D–CDK4 complexes, abrogating their activity. Moreover, high levels of p27Kip1 are found in quiescent cells, suggesting that it can play a role in maintaining cells in the G0 phase 15. It has been suggested that the loss of p27Kip1 protein expression may result in tumor development and/or progression 14,44–46. The results of the present study indicated that there is a relationship between decreased p27Kip1 expression and progression of C-KS lesions from patch/plaque to nodular. In nodular lesions, the great majority of the cells are KS cells, but in earlier lesions of KS, it is difficult to differentiate between a KS abnormal cell and a background normal vascular cell or fibroblast. Thus, higher p27Kip1-positive cell counts may be expected in early lesions. The association between the loss of p27Kip1 protein and uncontrolled proliferation of cancer cells is congruent with the function of p27Kip1 as a negative regulator of cyclins E and A, which, in complex with their catalytic partner CDK2, drive cells into the S phase. In the normal cell cycle, the G0/G1 phase is characterized by high p27Kip1 levels. A reduction in p27Kip1 levels appears, therefore, to be a common but late event in C-KS. These results are in agreement with the results of Fernández-Figueras et al.47 and Penin et al.48.
In the present study, we also investigated the proliferative activity of the KS lesions using the Ki-67 protein, a well-established marker of cell proliferation 49. No significant difference was found in the Ki-67 proliferation index in different histologic stages of KS lesions; this result is in agreement with Haupt et al.17. Other studies were in disagreement with the present study in that Ki-67 was found to be positively correlated with the histopathologic stage of the disease 50,51.
The p53 protein is critical in negatively controlling the cell cycle and the loss of p53 function plays an important role in the development of diverse cancers 19 Abnormalities of the p53 protein can be investigated using IHC; a small amount of wild-type p53 protein is present in the nucleus of all normal mammalian cells at a concentration usually below the detection level of IHC. The accumulation of p53 protein to a detectable level is believed to be caused by stabilization of the p53 protein. The most frequent mechanism in malignant neoplasms is a mis-sense mutation of the gene, which leads to a synthesis of a mutated inactive form of protein that is more stable than the wild-type protein. Alternate mechanisms of p53 stabilization and inactivation include interaction of the wild type with either viral or cellular proteins that prevents its degradation and causes loss of its function 52. In the present immunohistochemical study, it was found that p53 immunoreactivity in KS correlates significantly with the histopathologic stage of the disease. There was a statistically significant difference in p35 expression between the KS groups. Because the histopathologic subtypes of KS are considered to correspond to the developmental stages of the tumor, these findings suggest that accumulation and mutation of p53 protein may play a role in the tumor progression of C-KS. This is in agreement with many studies that suggest that p53 mutations arise relatively late in the neoplastic progression 53 and with the observation of Hodak et al.51; they found that spindle cells, the proportion of which increases with the histopathologic progression of KS, were more often positive, displaying more extensive staining than the endothelial cell population of the tumor, and tended to show greater staining intensity. The present results confirmed the results of previous studies 54,55 in which p53 overexpression was found to be a late event in KS observed mainly in the spindle cells. Dada et al.56 studied the p53 protein expression, using different monoclonal antibodies, in 20 cases of KS of various clinical types, finding p53 immunoreactivity in all of the cases. Data on the histopathologic stage of these cases were not provided, although comparison of studies using different p53 antibodies has its limitations.
The Bcl-2 family is a group of at least 15 related cytoplasmic proteins that control an evolutionarily conserved mechanism for the regulation of apoptosis. Some of the members of this family, including Bcl-2, Bcl-xL, and Mcl-1, are blockers of programmed cell death, whereas others, such as Bax, Bak, and Bcl-xS, are promoters of apoptosis. Mutations of the genes encoding for Bcl-2 family proteins can lead to neoplasia 21. The angiogenic activity attributed to the vascular endothelial growth factor may be due in part to its ability to enhance endothelial cell survival by inducing the expression of Bcl-2, as has been shown in human dermal microvascular endothelial cells in vitro57. In the current study, we found statistically significant differences in the average intensity of immunohistochemical staining according to the type of lesions (progression stages). The cellular composition of lesions could explain some of the differences; patch-stage lesions of KS contain more neoplastic vessels and nodules contain more spindle cell fascicles. Thus, the contribution of Bcl-2 overexpression toward the progression of KS lesions by an antiapoptotic mechanism seems to be crucial, even though other antiapoptotic molecules and mechanisms may also be involved. The results of the current study confirmed those obtained in previous studies 21,58,59.
Vascular tumors strongly expressed CD31 and CD34. It seems essential to search for HHV-8 LNA-1 within endothelial cells of any vascular lesion mimicking KS or any other vascular proliferation that is difficult to classify. Ki-67 showed no significant difference in different histologic stages of KS. Downregulation of p27Kip1 expression and upregulation of cyclin D1, p53, and Bcl-2 may be implicated in KS progression through its various cutaneous histopathological stages (prognostic markers).
Conflicts of interest
There are no conflicts of interest.
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