Reduced Level of CD44 and Hyaluronan Associated with Unfavorable Prognosis in Clinical Stage I Cutaneous Melanoma
2000; Elsevier BV; Volume: 157; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64608-1
ISSN1525-2191
AutoresJari Karjalainen, Raija Tammi, Markku Tammi, Matti Eskelinen, Ulla M. Ågren, Jyrki Parkkinen, Esko Alhava, Veli-Matti Kosma,
Tópico(s)Immunotherapy and Immune Responses
ResumoThe cell surface glycoprotein CD44 and its ligand, hyaluronan (HA), enhance growth and metastatic capacity of melanoma cells in vitro, but their clinical significance in primary cutaneous melanoma is still unclear. Therefore, we studied whether the levels of CD44 and HA associate with disease progression and survival of cutaneous melanoma. A series of 292 clinical stage I cutaneous melanomas was analyzed by immunohistochemistry using an anti-CD44H antibody (clone 2C5). HA was demonstrated histochemically using a biotinylated HA-specific affinity probe (bHABC). The reduced staining levels of CD44 and HA were associated with each other and indicators of progressive disease. Reduced CD44 and HA level, high tumor thickness, high pT category, high Clark's level, bleeding, and male gender predicted short univariate recurrence free survival (RFS) and overall survival (OS). In Cox's multivariate analysis (N = 251), the decreased level of CD44, high tumor thickness, and bleeding predicted independently short RFS. High tumor thickness and bleeding were associated with short OS. We conclude that the reduced cell surface CD44 and HA levels associate with poor prognosis in clinical stage I cutaneous melanoma. The notion that the decreased level of CD44 independently predicts short RFS suggests that reduced cell surface CD44 enhances the spreading potential in localized cutaneous melanoma and that quantification of CD44 offers a prognostic tool for its clinical evaluation. The cell surface glycoprotein CD44 and its ligand, hyaluronan (HA), enhance growth and metastatic capacity of melanoma cells in vitro, but their clinical significance in primary cutaneous melanoma is still unclear. Therefore, we studied whether the levels of CD44 and HA associate with disease progression and survival of cutaneous melanoma. A series of 292 clinical stage I cutaneous melanomas was analyzed by immunohistochemistry using an anti-CD44H antibody (clone 2C5). HA was demonstrated histochemically using a biotinylated HA-specific affinity probe (bHABC). The reduced staining levels of CD44 and HA were associated with each other and indicators of progressive disease. Reduced CD44 and HA level, high tumor thickness, high pT category, high Clark's level, bleeding, and male gender predicted short univariate recurrence free survival (RFS) and overall survival (OS). In Cox's multivariate analysis (N = 251), the decreased level of CD44, high tumor thickness, and bleeding predicted independently short RFS. High tumor thickness and bleeding were associated with short OS. We conclude that the reduced cell surface CD44 and HA levels associate with poor prognosis in clinical stage I cutaneous melanoma. The notion that the decreased level of CD44 independently predicts short RFS suggests that reduced cell surface CD44 enhances the spreading potential in localized cutaneous melanoma and that quantification of CD44 offers a prognostic tool for its clinical evaluation. The incidence and mortality of cutaneous malignant melanoma has increased worldwide during past decades among white populations.1Berwick M Halpern A Melanoma epidemiology.Curr Opin Oncol. 1997; 9: 178-182Crossref PubMed Scopus (71) Google Scholar Local disease is in most cases curable by surgical excision.2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar However, the probability of development of disseminated disease is progressively higher as the depth of the primary melanoma increases. 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This retrospective study consists of primary melanomas derived from a consecutive series 369 clinical stage I cutaneous melanoma patients with sufficient clinicopathological and long-term follow-up data.36Karjalainen JM Kellokoski JK Eskelinen MJ Alhava EM Kosma V-M Downregulation of transcription factor AP-2 predicts poor survival in stage I cutaneous malignant melanoma.J Clin Oncol. 1998; 16: 3584-3591Crossref PubMed Scopus (92) Google Scholar The patients were diagnosed and treated in the district of Kuopio University Hospital between 1974 and 1989. The histological diagnosis, Breslow thickness and Clark level were re-examined from 1 to 4 original sections of the primary tumor by the same pathologist (VMK), unaware of the clinical data. Of the original 369 cases, 292 had enough archival tumor material available for the present study (Table 1). The most representative block was cut into new 5-μm-thick consecutive sections for CD44 and HA stainings. Depending on the availability of representative tumor material as compared with the original sections, 282 stainings with an antibody recognizing all forms of CD44 and 277 stainings for HA were eventually evaluable.Table 1Patient and Tumor Characteristics of 292 Patients with Stage I Cutaneous Malignant MelanomaCharacteristicn%Total no. of patients292100Age (years) Mean (SD)55.9 (15.2) Range19.0–89.7Anatomic site Head and neck4716 Trunk and perineum14550 Upper limbs4415 Lower limbs5619Bleeding*Either as an anamnestic information or as a clinical sign observed by the treating physician. of the primary tumor Yes7626 No11941 Unknown9733Sex Male14349 Female14951Disease recurrence Yes8429 No20871Cause of death Malignant melanoma5218 Other4014 Alive20068Clark's level I124 II5418 III8228 IV11941 V259Tumor thickness ≤0.75 mm6422 0.76–1.50 mm6923 1.51–4.0 mm9934 >4.0 mm4114 Not possible to analyze196TNM category pT1-T2, N0, M013145 pT3, N0, M011539 pT4, N0, M04616* Either as an anamnestic information or as a clinical sign observed by the treating physician. Open table in a new tab CD44 was demonstrated by using a mouse IgG anti-CD44H antibody (clone 2C5) (R&D Systems, Abingdon, UK) which recognizes all forms of CD44.37Fox S Fawcett J Jackson D Collins I Gatter KC Harris AL Gearing A Simmons DL Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms.Cancer Res. 1994; 54: 4539-4546PubMed Google Scholar Adjacent 5-μm sections from tumors were deparaffinized and rehydrated using xylene and graded alcohols. The sections were microwaved in a 0.01 mol/L citrate buffer (pH 6.0) for 3 × 5 minutes, incubated in a citrate buffer for 18 minutes, and washed twice for 5 minutes with phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked by 5% hydrogen peroxide for 5 minutes, followed by a wash twice for 5 minutes with PBS. The sections were incubated with 1% bovine serum albumin (BSA) and PBS for 30 minutes at 37°C. The primary antibody was diluted with 1% BSA to 1:2000 and incubated on the slides overnight at 4°C. After another washing step, the bound antibody was localized using a biotinylated secondary antibody and an avidin-biotin-peroxidase detection kit (Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, CA), and the slides were developed with diaminobenzidine tetrahydrochloride (DAB) (Sigma, St. Louis, MO), counterstained with Mayer's hematoxylin, dehydrated, cleared and mounted with DePex (BDH Poole, UK). In each batch, a melanoma specimen processed without primary antibodies served as a negative control, and one CD44-positive melanoma block served as a source for positive control sections. In addition, the adjacent normal epidermis within the tumor served as positive internal control. The biotinylated complex of hyaluronan binding region and link protein (bHABC) was prepared from bovine articular cartilage as described previously.38Tammi R Ågren U Tuhkanen AL Tammi M Hyaluronan metabolism in skin.Prog Histochem Cytochem. 1994; 29: 1-77Crossref PubMed Scopus (136) Google Scholar, 39Tammi R Ripellino JA Margolis RU Tammi M Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe.J Invest Dermatol. 1988; 90: 412-414Abstract Full Text PDF PubMed Google Scholar Briefly, the proteoglycans were extracted from the cartilage with 4 mol/L guanidinium chloride. The extract was dialyzed against distilled water in the presence of high molecular weight hyaluronan. The C-terminus of the proteoglycan molecule was cleaved off with trypsin, and the resultant complex of hyaluronan binding region and link protein (HABC) with HA was purified using hydroxylapatite chromatography and gel filtration. The complex was biotinylated, and the bHABC was separated from HA using gel filtration under dissociative conditions. The purity of the preparation was tested by polyacrylamide gel electrophoresis and Western blotting. The sections were deparaffinized in xylene, rehydrated with graded alcohols and washed with PB. Endogenous peroxidase was blocked with 1% hydrogen peroxide for 5 minutes and nonspecific binding was blocked with 1% BSA in PB for 30 minutes. The sections were incubated in bHABC (2.5 μg/ml, diluted in 1% BSA) overnight at 4°C. The slides were washed with PB and treated with avidin-biotin-peroxidase kit (1:200 dilution) for 1 h at room temperature. Following wash with PB the color was developed with 0.05% DAB and 0.03% hydrogen peroxide in PB at room temperature. The slides were counterstained with Mayer's hematoxylin for 2 minutes, washed, dehydrated, and mounted in Depex. The specificity of the staining was controlled by digesting some sections with 100 TRU/ml of Streptomyces hyaluronidase (Seikagaku Kogyo Co., Tokyo, Japan) in the presence of protease inhibitors before the staining, or preincubating the bHABC-probe with hyaluronan oligosaccharides to block the specific binding site (Figure 1A).39Tammi R Ripellino JA Margolis RU Tammi M Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe.J Invest Dermatol. 1988; 90: 412-414Abstract Full Text PDF PubMed Google Scholar Throughout the evaluations, the observers were unaware of the clinical data. In all stainings, scoring was performed with a dual head microscope (field diameter 490 μm) by two observers (JMK and VMK) in the whole tumor area on the slide. In both CD44 and HA stainings, the positivities were assessed qualitatively, ie, the tumor cells were considered as positive when there was a homogeneous and clearly visible signal present, and negative if the signal was absent. According to this principle, the fraction of positively stained cancer cells in the entire slide was evaluated within each specimen. The frequency distribution and percentiles for the HA- and CD44-positive cancer cell fractions within each specimen were analyzed within the whole series (Figure 2). For both stainings, the tumors were eventually categorized as high (91 to 100% of positively stained cancer cells for CD44 and 71 to 100% of positively stained cancer cells for HA) or reduced (0 to 90% of positively stained cancer cells for CD44 and 0 to 70% of positively stained cancer cells for HA) expressors according to the median percentage of positively stained cancer cells.40Simon R Altman DG Statistical aspects of prognostic factor studies in oncology.Br J Cancer. 1994; 69: 979-985Crossref PubMed Scopus (497) Google Scholar The staining intensity of hyaluronan in the intratumoral stroma was compared qualitatively to the adjacent normal HA-positive epidermis and categorized as follows: +, weaker than epidermis; ++, as strong as epidermis; and +++, stronger than epidermis. The SPSS-Win 7.5 program package was used in a PC computer for basic statistical calculations. The χ2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar test was used to compare the frequency distributions of clinicopathological features between the original database (n = 369) and the population available for the current study. A Spearman correlation coefficient was used to test the relationships between continuous variables. Nonparametric tests (Kruskal-Wallis) were applied for the comparisons of staining levels between different clinicopathological categories. Frequency tables were analyzed using a 2-sided Fisher's exact test. In univariate survival analyses the EGRET statistical software package was used for calculation of Kaplan-Meier estimates of survival rates and the log rank analysis41Kaplan EL Meier P Nonparametric estimation from incomplete observations.J Am Stat Assoc. 1958; 53: 457-481Crossref Scopus (47473) Google Scholar to test the differences between the survival curves. Cox's multivariate survival analysis was done using the Log likelihood ratio significance test in a forward stepwise manner.42Cox DR Regression models and life tables with discussion.J Stat Soc B. 1972; 34: 187-192Google Scholar The adequacy of the proportional hazards assumption was tested by logminlog plots. Overall survival (OS) analysis included as an event only the deaths due to malignant melanoma. Deaths due to postoperative complications within 30 days were excluded. Recurrence-free survival (RFS) was defined as the time elapsed between the primary treatment and the recurrent melanoma. For all statistical tests, probability values less than 0.05 were regarded as significant. The frequency distributions of the clinicopathological characteristics, as well as the follow-up and survival times between the original database (N = 369)36Karjalainen JM Kellokoski JK Eskelinen MJ Alhava EM Kosma V-M Downregulation of transcription factor AP-2 predicts poor survival in stage I cutaneous malignant melanoma.J Clin Oncol. 1998; 16: 3584-3591Crossref PubMed Scopus (92) Google Scholar and the patients with valid material available for CD44 and HA stainings were almost identical (Table 1). The mean follow-up time of all 292 patients was 6.3 ± 3.3 (SD) years (median, 5.4 years; range, 0.5–18 years). The CD44 positivity (median value 90%) was confined to tumor cell membranes (Figure 1B). Most tumors showed intense membranous and cytoplasmic HA-positivity (median value 70%), as did the consistently positive epidermis (Figure 1C). The dermal stroma within and outside the tumor was always HA positive (Figure 1A). The histograms of the cellular CD44 and HA levels are shown in Figure 2 and the cellular CD44 and HA levels and stromal HA intensity in different categories in Table 2.Table 2Expression of CD44 and Hyaluronan in Clinical Stage I Cutaneous Malignant MelanomaStainingn%CD44 0–90%15354 91–100%12946 Total282100Cellular HA 0–70%14051 71–100%13749 Total277100Stromal HA intensity +4315 ++21879 +++166 Total277100 Open table in a new tab CD44 positivity was strongly associated with cellular HA according to Spearman's statistics (r = 0.309;P < 0.00005; N = 267) and a 2-sided Fisher's exact test (P = 0.005). Decreasing levels of cancer cell-associated CD44 (Figure 1D) and HA (Figure 1E)were both related to increasing Breslow thickness (χ2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar = 20.5, P < 0.00005 for CD44 and χ2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar = 16.4, P = 0.001 for HA), increasing Clark level (χ2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar = 35.1, P < 0.00005 for CD44 and χ2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar = 26.5, P < 0.00005 for HA), and increasing pT category (χ2=34.8, P < 0.00005 for CD44 and χ2=19.1, P < 0.00005 for HA), and this trend was evenly distributed within Breslow's thickness, Clark's level and pT categories. Decreasing CD44 and HA levels also associated with bleeding (χ2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar = 7.4, P = 0.024 for CD44 and χ2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar = 9.0, P = 0.011 for HA) and with recurrent disease (χ2Kirkwood JM Resnick GD Cole BF Efficacy, safety and risk-benefit analysis of adjuvant interferon α-2b in melanoma.Semin Oncol. 1997; 24: S16-23PubMed Google Scholar = 18.2, P < 0.00005 for CD44 and χ2=6.7, P = 0.01 for HA). Stromal HA intensity did not show statistically significant correlation with CD44 level or with any of the clinicopathological variables. During the follow-up, 84/292 patients (29%) had a recurrence, 52 patients (18%) died of melanoma, and 40 patients (14%) died of other causes. The 5-year rates for crude, overall, and recurrence-free survivals were 78%, 86%, and 75%, respectively. Reduced levels of CD44 (0 to 90% positively stained cancer cells) and cancer cell-associated HA (0 to 70% positively stained cancer cells) predicted short OS (P = 0.0077 and P = 0.0146, Figure 3) and short RFS (P = 0.0001 and P = 0.0141, Figure 4) (Table 3). Reduced CD44 level predicted poor RFS also within the low-risk (≤1.5 mm) subgroup, (P= 0.0147, N = 127, other data not shown). Stromal HA intensity was not significantly related to OS or RFS. The conventional parameters predicting poor RFS and OS were high tumor thickness and high pT category (P < 0.00005 for both RFS and OS, respectively), high Clark's level of invasion (P < 0.00005 for RFS and P = 0.0001 for OS), bleeding (P = 0.0001 for both RFS and OS) and male gender (P = 0.0292 for RFS and P = 0.0339 for OS) (Table 3). In addition, the reduction of CD44 and HA staining associated fairly well with unfavorable prognosis also by using the 33rd and 66th percentiles of frequency distribution as cut off points (RFS: P = 0.0027 for HA and P = 0.001 for CD44; OS:P = 0.0632 for HA and P = 0.0112 for CD44, other data not shown).Figure 4A: RFS according to cell surface CD44 expression. N = 282. B: RFS according to tumor cell associated HA expression. N = 277.View Large Image Figure ViewerDownload (PPT)Table 3Clinical, Histological, Immunohistochemical, and Histochemical Factors Related to Survival in 292 Clinical Stage I Cutaneous Malignant MelanomaCategory (variable)No. of patients5-year RFS (95% CI)P*Log rank analysis.5-year OS (95% CI)P*Log rank analysis.Sex0.02920.0339 Male1430.69 (0.60–0.76)0.81 (0.73–0.87) Female1490.80 (0.73–0.86)0.90 (0.83–0.94)Bleeding0.00010.0001 Yes760.65 (0.52–0.75)0.80 (0.68–0.88) No1190.86 (0.78–0.91)0.93 (0.86–0.97) Unknown970.69 (0.59–0.78)0.81 (0.71–0.88)Clark's level<0.000050.0001 I120.88 (0.39–0.98)0.88 (0.39–0.98) II540.98 (0.88–1.0)0.98 (0.87–1.0) III820.79 (0.68–0.87)0.93 (0.84–0.97) IV1190.63 (0.54–0.71)0.79 (0.70–0.86) V250.61 (0.38–0.78)0.63 (0.39–0.80)Tumor thickness (mm)<0.00005 4.0410.48 (0.31–0.63)0.60 (0.40–0.75)TNM category<0.00005 90%1290.83 (0
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