Association between rb-1 gene alterations and factors of favourable prognosis in human breast cancer,without effect on survival

The retinoblastoma (RB) tumour suppressor gene has been associated not only with retinoblastoma but also with several other tumours like osteosarcoma, small cell lung carcinoma and prostate and breast cancer. We have studied the incidence of RB gene alterations in 96 primary breast tumours using Southern blotting techniques. The outcome has been related with patient and tumour characteristics, oncogene amplifications, p53 mutations and prognosis. RB gene alterations were found to occur more frequently in estrogen receptor (ER)-positive than in ER-negative tumours and less frequently in tumours with oncogene amplification than in tumours without oncogene amplification of HHR2/neu, c-myc or 11q13. RB gene alteration was observed in tumours both with and without a p53 gene mutation. Data on 87 patients (mean age, 59.6 years; median follow-up, 108 months) and RB gene alterations revealed a significant association between the frequency of RB gene alterations and node-negative patients (p < 0.01) or smaller (<2 cm) tumours (p < 0.01), but no relation with age, differentiation grade or (relapse-free) survival. Patients with and without RB gene alterations showed the same relapse-free and overall survival. © 1995 Wiley-Liss, Inc.     

Failure of RB1 to reverse the malignant phenotype of human tumor cell lines.

In addition to retinoblastoma and osteosarcoma, mutation of both alleles of the RB1 gene occurs frequently in several other types of tumors. In order to evaluate the role of RB1 in cancer, the wild type RB1 gene was introduced into the RB1-deleted breast cancer cell line MDA-468-S4 and retinoblastoma cell lines WERI-Rb1 and Y-79. The RB1 complementary DNA was under control of the inducible murine metallothionein promoter in MDA-468-S4 and the thymidine kinase promoter in the retinoblastoma lines. The protein, p110RB1, produced from the exogenously introduced gene appeared normal by immunoprecipitation, Western blot analysis, and nuclear localization and also showed normal cell cycle-dependent phosphorylation and an ability to bind to E1a protein. No changes in growth rate or morphology were observed in either of the reconstituted cell types. Expression of p110RB1 in MDA-468-S4 did not affect anchorage-independent growth when measured by colony formation in soft agar. Although the ability of WERI-Rb1 cells expressing p110RB1 to form colonies in methylcellulose was reduced, the reconstituted retinoblastoma cell lines formed intraocular tumors in immunodeficient mice with the same efficiency as the RB1-negative parent cell lines and the tumors produced by the RB1-reconstituted cells continued to express p110RB1. These experimental results suggest that the malignant phenotype is little affected by the replacement of p110RB1 and that RB1 is a relatively weak tumor suppressor gene.     

Regulation of the G1/S phase of the cell cycle and alterations in the RB pathway in human lung cancer

The retinoblastoma (RB)–Cyclin (CCN)D1–p16 cell cycle pathway has a crucial role in lung tumorigenesis. Impairment of the RB pathway has been shown to occur in almost all lung tumors. A deregulation at any level of this core RB pathway seems to make cells insensitive to the mitogenic signaling that is required for cell cycle progression. To date, almost all participants in this pathway have been shown to be altered to a various degree in lung tumors. Some of the alterations are mutually exclusive, including RB and p16^INK4A. In small cell lung cancer, the RB tumor suppressor gene is inactivated in almost 90% of the tumors, whereas in non-small cell lung cancer, the cyclin-dependent kinase (CDK)4 inhibitor p16^INK4A is inactivated in 40–60% of the tumors. Many mechanisms may be responsible for activating the RB–Cyclin D1 pathway, including activating (CDK4) and inactivating mutations (p16^INK4A), deletions (RB and p16^INK4A), amplifications (CCND1 and CDK4), silencing methylation (p16^INK4A and RB), and hyper-phosphorylation (RB). As some of these alterations, such as p16^INK4A methylation, can also be detected in bronchial lavage and serum, they could potentially serve as useful markers for the early detection of lung cancer. This review summarizes recent experiments describing the variable roles of key-player molecules of the RB pathway and different mechanisms by which the RB pathway can be altered in lung cancer.     

Aberrant RB gene expression in routinely processed,archival tumor tissues determined by three different anti-RB antibodies

The retinoblastoma (RB) susceptibility gene encodes a nuclear phosphoprotein which is likely involved in cell cycle control and cell differentiation. The RB protein is mutated or absent in a variety of human malignancies. Its role as a molecular marker for clinical tumor behavior is under extensive investigation. However, studies on the status of the RB protein in primary or metastatic tumors and their precursor lesions have been slowed by the lack of availability of a sensitive, reliable assay which allows examination of RB expression in selected cell populations within archival tissues. Thus far, meaningful immunohistochemical analysis of RB protein in formalin-fixed, paraffin-embedded specimens has been achieved only with the polyclonal antibody RB-WL-1. We now describe a method which produces excellent staining results in formalin-fixed, routinely processed tissues, using commercially available monoclonal antibodies (MAbs) in conjunction with an antigen-retrieval step. The resulting stains were superior to those on frozen sections and comparable to those obtained with RB-WL-1. Twelve of 51 random invasive bladder cancers (24%) had abnormal expression of the RB gene, as determined by immunohistochemistry. Smaller cohorts of breast, prostate and lung carcinomas had incidences of aberrant RB gene expression ranging from 9% to 24%. Since the staining method was widely applicable to essentially all formalin-fixed, archival tissues, it may expedite studies on the biological and clinical significance of altered RB expression in human neoplasia.     

Intraocular tumor formation of RB reconstituted retinoblastoma cells

It has been reported that replacement of a functional retinoblastoma (RB) gene in RB defective WERI-27 retinoblastoma cells results in complete loss of their tumorigenic potential in nude mice following s.c. injection. We have repeated the identical studies and found that although tumors did not develop s.c., the RB reconstituted cells, either soon after RB virus infection or after long term cultivation, consistently produced tumors when injected intraocularly. These tumor cells, when reestablished in culture, were found to retain a normal RB protein as determined by direct Western blotting and immunocytochemical staining. The tumors, however, occurred with a longer average latency period and with less frequency compared to those produced by the parental RB defective cells. Our results suggest that reintroduction of the RB gene into WERI-27 cells reduces but does not completely suppress their tumorigenic potential. Since retinoblastoma is an eye tumor it also provides further documentation that the use of an orthotopic injection site can be critical when determining the tumorigenicity of a given cell type.     

Cyclin D1 and retinoblastoma susceptibility gene alterations in non-small cell lung cancer

Among the major regulators of the G₁ restriction point are cyclin D1 and the retinoblastoma gene product (RB). In non-small cell lung cancer (NSCLC), the cyclin D1 gene is amplified/over-expressed in almost 50% of cases, and RB is inactivated in 6–32% of cases. It is of interest to evaluate concurrently the alterations of both genes on the same series of NSCLCs, to investigate whether cyclin D1 and RB alterations are alternative pathways leading to inactivation of the G₁ restriction point or if they can occur in the same tumor, possibly exerting an additive effect on cancer progression. We investigated a series of 57 NSCLCs, analyzing cyclin D1 and RB at the gene and protein levels by Southern blot, Northern blot and immunohistochemistry. The cyclin D1 gene was amplified in 18 cases. cyclin D1 immunoreactivity was seen in 25 tumors. Amplification and expression were significantly associated. RB immunohistochemical expression was absent in 9 of 42 informative cases. RB mRNA expression was low to absent in 9 of 45 informative cases. cyclin D1 amplification was associated with normal RB mRNA, and cyclin D1 over-expression was associated with normal RB immunoreactivity, supporting the hypothesis that alterations of cyclin D1 and RB are alternative mechanisms by which tumor cells may escape the G₁ restriction point. A concurrent alteration of RB and cyclin D1 was seen in a small subset of NSCLCs. Abnormalities of cyclin D1 and/or RB at the gene and/or expression level were present in more than 90% of cases, stressing that cyclin D1 and/or RB alterations represent an important step in lung tumorigenesis. Int. J. Cancer 75:187–192, 1998. © 1998 Wiley-Liss, Inc.     

Retinoblastoma gene‐independent G1 phase arrest by flavone,phosphatidylinositol 3‐kinase inhibitor,and histone deacetylase inhibitor

In most human malignant tumors, retinoblastoma tumor‐suppressor gene (RB) product is inactivated by phosphorylation. Therefore, cancer preventive agents or molecular‐targeting agents can inhibit the tumor growth at G₁ phase through RB reactivation. However, little is known about the effectiveness of RB reactivating agents against malignancies with mutated RB. We report here that chemopreventive agent flavone, phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) also induce G₁ phase arrest in malignant tumor cells with mutated RB. In human prostate cancer DU145 cells with mutated RB, flavone increased cyclin‐dependent kinase (CDK) inhibitors p21 and p27, and reduced cdk4 and cdk6, resulting in decrement of phosphorylated RB family proteins p130 and p107. LY294002 also dephosphorylated p107 and p130 proteins, whereas TSA dephosphorylated p130, but not p107. Furthermore, flavone induced G₁ phase arrest in both mouse embryo fibroblast (MEF) wild‐type and MEF RB ^{? / ?} cells, but did not do so in RB, p107, and p130 triple‐knockout MEF cells. These results suggested that p130 and p107 contributed to G₁ phase arrest by flavone in RB‐mutated cells. However, flavone induced tumor suppressor microRNA miR‐34a with reduction of E2F1 and E2F3, known to be downregulated by miR‐34a, raising the possibility that miR‐34a might partially contribute to G₁ arrest by flavone. These results raise the possibility that RB reactivating chemopreventive agents or molecular targeting agents might also be effective against a variety of malignant tumor cells with mutant RB.     

Changes in growth and tumorigenicity following reconstitution of retinoblastoma gene function in various human cancer cell types by microcell transfer of chromosome 13.

Functional loss of the retinoblastoma (RB) gene has been implicated in the initiation or progression of several human tumor types including cancer of the eye, bone, bladder, and prostate. To examine the consequence of adding one RB allele containing its normal regulatory elements back into representative examples of each of these cancer types, as well as to compare the results to those previously reported using various RB complementary DNA constructs, a neomycin resistant marked 13 chromosome was transferred by microcell fusion. Several attempts to obtain RB positive osteosarcoma cells failed. In addition, only one RB positive retinoblastoma clone was isolated. This clone contained many large cells, could not be maintained in long-term culture, and produced only RB negative tumors. Three RB positive bladder cancer cell clones were obtained, all of which grew slower in culture than their RB negative parental counterpart and did not form colonies in soft agar. Tumorigenicity was markedly suppressed in these clones. One clone yielded no tumors, and the other 2 clones produced only one small tumor each, both of which were RB negative. In contrast, the 2 RB positive prostate cancer cell clones isolated had no differences in their cell culture growth properties, including growth in soft agar compared to the parental cells. One of the clones was nontumorigenic, while the other clone produced 4 small tumors, all of which were RB positive. These results indicate that the transfer of one RB allele by microcell transfer produces different levels of growth inhibition as well as tumor suppression, depending on the cell type examined. In the case of prostate cancer, the function of the RB gene in tumor suppression appears to be independent from its growth regulatory function, since no growth inhibition in cell culture was noted in these cells, although tumor suppression was significant.     

Oncogene-dependent engraftment of human myeloid leukemia cells in immunosuppressed mice.

We have developed an in vivo model of differentiated human acute myeloid leukemia (AML) by retroviral infection of the cytokine-dependent AML cell line TF-1 with the v-Src oncogene. When injected either intravenously or intraperitoneally into 300 cGy irradiated SCID mice, animals formed multiple granulocytic sarcomas involving the adrenals, kidneys, lymph nodes and other organs. The mean survival time was 34+/-10 days (n = 40) after intravenous injection and 24+/-3 days (n = 5) after intraperitoneal injection of 20 million cells. The cells recovered from leukemic animals continued to express interleukin-3 receptors and remained sensitive to the diphtheria fusion protein DT388IL3. Further, these granulocytic sarcoma-derived cells grew again in irradiated SCID mice (n = 10). The cytogenetic abnormalities observed prior to inoculation in mice were stably present after in vivo passage. Similar to the results with v-Src transfected TF-1 cells, in vivo leukemic growth was observed with TF-1 cells transfected with the human granulocyte-macrophage colony-stimulating factor gene (n = 5) and with TF-1 cells recovered from subcutaneous tumors in nude mice (n = 5). In contrast, TF-1 cells expressing v-Ha-Ras (n = 5), BCR-ABL (n = 5), or activated Raf-1 (n = 44) did not grow in irradiated SCID mice. This is a unique, reproducible model for in vivo growth of a differentiated human acute myeloid leukemia and may be useful in the assessment of anti-leukemic therapeutics which have human-specific molecular targets such as the interleukin-3 receptor.     

In vivo activation and expansion of T cells by a bi-specific antibody abolishes metastasis formation of human melanoma cells in SCID mice

Bi-specific antibody fragments (bAB) are used in tumour therapy as a means to redirect and to strengthen effector cell function. It would be of great therapeutic advantage if, in addition, recruitment, expansion and the state of activity of effector cells are influenced by targeting through a bAB. This question was explored in the melanoma-bearing SCID mouse. The chemically coupled Fab′ fragments of an anti-CD3 and an anti-p97 monoclonal antibody (MAB) were characterized in vitro for dual binding specificity and support of lymphokine-activated-killer-cell (LAKC) cytotoxicity towards a highly aggressive human melanoma line, which was significantly increased and exceeded levels of antibody-dependent cellular cytotoxicity observed in the presence of the anti-p97 MAB. The in vivo efficacy was tested in the SCID mouse: 5, 10 and 15 days after i.p. application of tumour cells, mice received LAKC (2 × 10⁷) together with bAB (150–100 μg). The application of bAB was repeated at days 20 and 25. Application of LAKC to melanoma-bearing SCID mice prolonged the mean survival time from 22 days of the untreated control group to 41 days. Anti-p97 did not exert any additive effect. In the presence of bAB, melanoma cells did not grow in 3 out of 8 mice. The mean survival time of the 5 mice developing tumours was 45 days. Importantly, none of the mice receiving bAB developed metastases, which were seen in 100% of animals receiving tumour cells or tumour cells plus LAKC or tumour cells plus LAKC plus anti-p97. As revealed by LAKC recovered from the SCID mice, the efficacy of the bAB was based on prolonged persistence of CD8-positive cells as well as on expansion and activation of CD4-positive cells, which was observed only in bAB-treated tumour-bearing mice. The efficiency in recruiting cytotoxic and, in particular, helper T cells suggests bAB as a valuable additive in immunotherapeutic treatment of melanoma patients. Int. J. Cancer 75:908–918, 1998. © 1998 Wiley-Liss, Inc.     

In vivo andin vitro approaches to study metastasis in human prostatic cancer

Summary Prostate cancer is the most common malignancy in American males and is second only to lung cancer as a cause of death in the United States. Clinically, radical prostatectomy offers a patient with locally contained disease an excellent chance for cure. For patients with metastatic disease, the current therapies are merely palliative. Understanding the biology of prostate cancer metastasis should facilitate the development of novel and effective therapeutic modalities. Crucial to this objective is the availability of human tumor systems in which the biology of metastasis can be studied. The present chapter will briefly assess variousin vivo andin vitro approaches to study metastasis in human prostate cancer. Utilization of athymic nude mice has played an important role in maintaining human prostatic cancer cells as xenografts and has provided an opportunity to establish site-specific subpopulations of the parental cell lines for further characterization and investigation. At present, a few established cell lines have been useful for this purpose. Fresh tumor specimens, unfortunately, have shown limited ability to grow in nude mice. The recent development of novel approaches to permit the maintenance of freshly harvested prostate cancers has been encouraging. The use of reconstituted basement membrane (Matrigel) for co-injection with cancer cells into the subcutaneous tissues has supported growth of biologically indolent tumors. Another approach is to administer tumor cells orthotopically into the prostate of recipient nude mice. Bone marrow metastases in nude mice have been rare in the past. Recently, three approaches have been shown to be successful in accomplishing bony metastasis with PC-3 cells. They include intrasplenic injection, tail vein injection with vena cava occlusion, and tail vein injection into SCID (severe combined immunodeficient) mice. Other routes of tumor cell injection are also useful for the establishment of various organ-related metastatic subpopulations derived from unselected parental cell lines. The use of a modified Boyden chamber assay system has offered a rapidin vitro assessment of the invasive potential of specific cancer cell lines. The variousin vivo andin vitro approaches described in the present chapter make it possible to enrich site-specific variants of different prostatic cancer cell lines for further characterization and elucidation of the molecular mechanisms underlying metastasis in prostate cancer. These approaches also provide an opportunity for testing and developing novel therapeutic modalities.     

Immortalization of fibroblasts from two patients with hereditary retinoblastoma.

Two RB fibroblastic strains from patients with hereditary retinoblastoma (RB), namely, RB80F/250R and RB110F, were transfected with plasmid DNAs encoding SV-40 large T-antigen at passage 31 and 10, respectively, These transfected fibroblasts developed into two immortalized cell lines. RB80F/250R/ori- and RB110F/gpt. RB110F/gpt had a similar doubling time as the parental cells, whereas RB80F/250R/ori- grew more rapidly with a doubling time of 19 hours compared to the parental cells which had a doubling time of 50 hours. The RB80F/250R/ori- cell line was particularly interesting cytogenetically since no normal chromosome 13 was present, although a marker chromosome 13 was found. In contrast, two apparently normal chromosome 13s were present in RB110F/gpt cells, but these cells had a marker chromosome which involved chromosome 14 (14 p+). Both the cell lines also had an abnormal chromosome 1 (1q-). RFLP analysis using the chromosome 13 specific VNTR probe, pTH162, assigned to 13q14.1 showed that the DNA from the RB80F/250R/ori- cells contained only a 6.1kb allelic fragment whereas the DNAs from parental RB80F/250R and RB80F cultures demonstrated both the polymorphic 8.0kb and 6.1kb allelic fragments. However, the RB gene per se was normal at the DNA level. Both cell lines expressed SV-40 large T-antigen together with elevated levels of p53 protein. In addition, levels of RB protein were the same in exponentially growing nontransformed parental cell strains and their immortalized cell lines. However, at confluency the levels of RB protein were greatly reduced in nontransformed cells but not in immortalized cell lines under similar conditions. In future studies using these immortalized cell lines, we shall make an attempt to discern the role of the RB gene and other tumor suppressor genes in the regulation of normal and malignant growth.     

Loss of expression of the p16INK4/CDKN2 gene in cutaneous malignant melanoma correlates with tumor cell proliferation and invasive stage

The G₁/S checkpoint of the cell cycle is regulated by p16, p53 and RB tumor suppressor genes. Loss of expression of the p16^INK4 tumor suppressor protein, the product of the CDKN2 gene, has been associated with a wide variety of human malignancies. Mutations, loss of heterozygosity and deletions of the CDKN2 locus have been reported in sporadic and familial cutaneous malignant melanomas (CMM). To investigate the role of the alterations of p16 expression in melanoma, we evaluated by immunohistochemistry the p16 expression and cell proliferation in 79 primary CMM and 10 benign melanocytic nevi (BMN). Forty-six melanomas (58%) and all BMN were found to be p16 positive; 33 melanomas (42%) were considered p16 negative. The extent of invasion according to Clark was significantly higher in p16-negative tumors than in p16-positive tumors. Cell proliferation as expressed by the proportion of positive cells in Ki-67 immunostaining was found to be significantly higher in p16-negative tumors than in p16-positive tumors, although there was no significant difference in the mitotic index between p16-positive and p16-negative tumors. In p16-positive tumors, the number of Ki-67-positive cells correlated with the mitotic index; in p16-negative tumors, there was no correlation between these parameters. Our data suggest that loss of p16 expression is more common in advanced melanomas, and that G₁/S checkpoint regulation is disrupted in p16-negative melanomas. Our results show that loss of p16 expression is a common event in primary melanomas, which further substantiates the role of p16 as a major tumor suppressor. Int. J. Cancer 74:255-259, 1997. © 1997 Wiley-Liss, Inc.     

Identification of tumorigenic retinal stem-like cells in human solid retinoblastomas

Retinoblastoma (RB) is the most common malignant tumor of the retina in human children. Although it has been hypothesized for a long time that RB derives from multipotent retinal stem cells (RSCs) or retinoblasts, the direct evidence that the presence of tumorigenic RSCs in RB tumors is still lacking. Some studies indicate that malignant tumors contain tumor stem cells similar to their normal tissue stem cell counterparts. With in vitro culture and differentiation method we demonstrate that tumorigenic retinal stem-like cells (RSLCs) indeed exist in RB lesions and that RB tumor-derived cultures encompass undifferentiated cells capable of extensive proliferation as clonal nonadherent neurospheres and can differentiate into different retinal cells in vitro. Interestingly, cultured cells expressed retinal development related genes including nestin, CD133, pax6, chx10 and Rx, and overexpressed Bmi-1, a gene required for self-renewal and proliferation of stem cells. Significantly, when these cultured cells were intraocularly transplanted into SCID mice, they gave rise to new tumors with histomorphological features and immunophenotypes similar to their parental primary RBs. The results show that RBs contain tumorigenic RSLCs that contribute to tumorigenesis. This study provides a new insight to investigate the histogenesis of RBs and establishes a model for other RB research.     

Susceptibility of skin fibroblasts from individuals genetically predisposed to cancer,to transformation by the tumour promoter 12-o-tetradecanoylphorbol-13-acetate

Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited “initiation” event which is present in every cell. Thus, it might be predicted that skin fibroblasts from these patients would exhibit increased susceptibility to in vitro transformation by tumour promoters alone. In the case of skin fibroblasts from RB patients, transformation as assessed by the ability of the cells to grow in semisolid medium and their migration in collagen gels did not occur. However, experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone, although the pattern of migration of the parent cell population in collagen gels was unchanged and they were non-tumorigenic in nude mice. The clones which grew in semi-solid medium, although stable with regard to anchorage-independent growth, were also unaltered in terms of their migration pattern in collagen gels and their tumorigenicity in nude mice, and were considered not to be completely transformed. Parallel cytogenetic analysis showed that, during the course of these transformation studies, TPA significantly increased not only tetraploidy but also the chromosome aberration frequency. Several quadriradial figures were noted.     

Disruption of the RB pathway and cell-proliferative activity in non-small-cell lung cancers

The pathway consisting of retinoblastoma protein (pRB), cyclin D1 and p16 (RB pathway) which is involved in the phosphorylation of pRB plays an important role in G₁/S progression. The disruption of this RB pathway has been reported in several types of human neoplasm. An immunohistochemical study of 101 non-small-cell lung cancers (NSCLCs) showed loss of p16 is in 47 tumors (46.5%) and loss of pRB in 42 tumors (41.6%). In 79 of 101 NSCLCs (78.2%), the expression of p16 and pRB was complementary (p < 0.0001). Methylation of the cdkn2 gene was detected in 50% of p16-negative tumors and in 11% of p16-positive tumors. Aberrant expression of cyclin D1 was found in 45 tumors (44.5%). The cyclin-D1-positive tumors had significantly higher Ki-67 indices than the cyclin-D1-negative tumors irrespective of the tumor p16 or pRB expression. Thus, 91 (90%) of 101 NSCLCs showed disturbed expression of at least 1 of the 3 components of the RB pathway. Our results suggest that the disruption of the RB pathway plays an important role in tumorigenesis in NSCLCs and that increased cyclin-D1 expression leads to strong proliferative activity which may over-ride the suppressive effect of p16 and pRB. Int. J. Cancer (Pred. Oncol.) 79:111–115, 1998.© 1998 Wiley-Liss, Inc.     

Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth

Human melanoma is composed of distinct cell types reminiscent of neural crest derivatives and contains multipotent cells that express the neural crest stem cell markers CD271(p75(NTR)) and Sox10. When isolated from solid tumors by using a method that leaves intact cell surface epitopes, CD271-positive, but not CD271-negative, cells formed tumors on transplantation into nude or nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. These tumors fully mirrored the heterogeneity of the parental melanoma and could be passaged more than 5 times. In contrast, in more immunocompromised NOD/SCID/IL2rγ(null) mice, or in natural killer cell-depleted nude or NOD/SCID mice, both CD271-positive and CD271-negative tumor cell fractions established tumors. However, tumors resulting from either fraction did not phenocopy the parental tumors, and tumors derived from the CD271-negative cell fraction could not be passaged multiple times. Together, our findings identify CD271-positive cells as melanoma stem cells. Our observation that a relatively high frequency of CD271/Sox10-positive cells correlates with higher metastatic potential and worse prognosis further supports that CD271-positive cells within human melanoma represent genuine cancer stem cells.