Biomedical subjects

Dirk J Ruiter

Publications and source records attributed to Dirk J Ruiter.

At least 19 recordsLinked to original sources

PubMed2005

Post-surgery adjuvant therapy with intermediate doses of interferon alfa 2b versus observation in patients with stage IIb/III melanoma (EORTC 18952): randomised controlled trial.

BACKGROUND: Individuals affected by melanoma with thick primary tumours or regional node involvement have a poor outlook, with only 30-50% alive at 5 years. High-dose and low-dose interferon alfa have been assessed for the treatment of these patients, with the former having considerable toxicity and a consistent effect on disease free survival, but not on overall survival, and the latter no consistent effect on either. Our aim was, therefore, to assess the effect of two regimens of interferon of intermediate dose versus observation alone on distant metastasis-free interval (DMFI) and overall survival in such patients. METHODS: We did a randomised controlled trial in 1388 patients who had had a thick primary tumour (thickness > or = 4 mm) resected (stage IIb) or regional lymph node metastases dissected (stage III) and had been assigned to 13-months (n=553) or 25 months (n=556) of treatment with subcutaneous interferon alfa 2b, or observation (n=279). Treatment comprised 4 weeks of 10 million units (MU) of interferon alfa (5 days per week) followed by either 10 MU three times a week for 1 year or 5 MU three times a week for 2 years, to a total dose of 1760 MU. Our primary endpoint was DMFI. Analyses were by intent to treat. FINDINGS: After a median follow-up of 4.65 years, we had recorded 760 distant metastases and 681 deaths. At 4.5 years, the 25-month interferon group showed a 7.2% increase in rate of DMFI (hazard ratio 0.83, 97.5% CI 0.66-1.03) and a 5.4% improvement in overall survival. The 13-month interferon group showed a 3.2% increase in rate of DMFI at 4.5 years (0.93, 0.75-1.16) and no extension of overall survival. Toxicity was acceptable, with 18% (195 of 1076) of patients going off study because of toxicity or as a result of refusal of treatment because of side-effects. INTERPRETATION: Interferon alfa as used in the regimens studied does not improve outcome for patients with stage IIb/III melanomas, and cannot be recommended. With respect to efficacy of the drug, duration of treatment seemed more important than dose, and should be assessed in future trials.

Adult↗

PubMed2005

Stromal responses in human primary melanoma of the skin.

Tumour development and progression has long been considered as the consequence of an imbalance between apoptosis and proliferation of transformed cells. However, whereas genetic aberrations leading to the activation of oncogenes and/or loss of tumour suppressor genes are crucial for the transformation towards aberrant cell growth, progression towards a full blown malignancy requires a dynamic interaction between tumour cells and the environment in which they thrive. Over the recent years, it has become evident that the (early) inflammatory and angiogenic response, and remodelling of the extracellular proteins are key factors in creating a microenvironment that sustains tumour growth and metastasis. The host response towards cutaneous melanoma has received relatively little attention, most likely because the majority of these tumours develop without evoking a strong stromal response as can be observed in, e.g., carcinomas. This review discusses potential critical modulators of melanoma growth: turn-over of the most abundant extracellular matrix protein in skin (i.e. type I collagen), the early inflammatory response and angiogenesis.

Collagen Type I↗

PubMed2005

Immunomonitoring tumor-specific T cells in delayed-type hypersensitivity skin biopsies after dendritic cell vaccination correlates with clinical outcome.

PURPOSE: Tumor-specific immunomonitoring is essential to evaluate the efficacy of vaccination against cancer. In this study, we investigated the predictive value of the presence or absence of antigen-specific T cells in biopsies from delayed-type hypersensitivity (DTH) sites. PATIENTS AND METHODS: In our ongoing clinical trials, HLA-A2.1+ melanoma patients were vaccinated with mature dendritic cells (DC) pulsed with melanoma-associated peptides (gp100 and tyrosinase) and keyhole limpet hemocyanin. RESULTS: After intradermal administration of a DTH challenge with gp100- and tyrosinase peptide-loaded DC, essentially all patients showed a positive induration. In clinically responding patients, T cells specific for the antigen preferentially accumulated in the DTH site, as visualized by in situ tetramer staining. Furthermore, significant numbers of functional gp100 and tyrosinase tetramer-positive T cells could be isolated from these DTH biopsies, in accordance with the applied antigen in the DTH challenge. We observed a direct correlation between the presence of DC vaccine-related T cells in the DTH biopsies of stage IV melanoma patients and a positive clinical outcome (P = .0012). CONCLUSION: These findings demonstrate the potency of this novel approach in the monitoring of vaccination studies in cancer patients.

Adjuvants, Immunologic↗

PubMed2005

The MMA1 gene family of cancer-testis antigens has multiple alternative splice variants: characterization of their expression profile, the genomic organization, and transcript properties.

Previously, we reported the identification of MMA1A by screening for differential gene expression in two human melanoma cell lines displaying diverse metastatic behavior after subcutaneous inoculation into nude mice. Splice variant MMA1B, which also was identified through database homology searches, showed a high degree of similarity with the MMA1A for exons 1, 2, and 4, but was missing exon 3. Through extensive expression profiling among normal and tumor samples, both MMA1A and -1B were found to belong to the family of cancer-testis antigens. In this study, we identified four additional alternatively spliced MMA1 variants, named MMA1C, MMA1D, MMA1E, and MMA1F. Generally, these novel MMA1 transcripts differ from MMA1A in that exon 2 or exon 3 is enlarged because of the use of alternative splice sites in intron 2 of the MMA1 gene. Moreover, MMA1E also lacks exon 3, as was previously seen in MMA1B. In screening for expression of the novel MMA1 transcripts in normal and tumor tissues, we demonstrated that MMA1C, -1D, and -1E also are members of the cancer-testis antigen family. MMA1F was found in only one melanoma metastasis sample and therefore is believed to have been expressed incidentally. Furthermore, we comprehensively elucidated the genomic structure of the MMA1 gene and the characteristic features of the alternatively spliced MMA1 transcripts.

Alternative Splicing↗

PubMed2005

Decrease in circulating anti-angiogenic factors (angiostatin and endostatin) after surgical removal of primary colorectal carcinoma coincides with increased metabolic activity of liver metastases.

Removal of a primary colorectal tumor resulted in an increase in metabolic activity in its liver metastasis. Concomitantly, levels of angiostatin and endostatin in urine and plasma, respectively, dropped. This finding indicates that the primary tumor suppressed angiogenesis in its distant metastasis, and that removal of the primary lesion caused a flare-up in vessel neoformation and, thus, enhanced metabolic activity in its liver metastasis.

Aged↗

PubMed2005

INK4-ARF and p53 mutations in metastatic cutaneous squamous cell carcinoma: case report and archival study on the use of Ink4a-ARF and p53 mutation analysis in identification of the corresponding primary tumor.

So far, histopathologic, immunohistochemical and molecular properties of metastatic cutaneous squamous cell carcinomas (CSCCs) are relatively unexplored. In patients with multiple CSCCs, as for instance renal transplant recipients (RTRs), it might prove difficult to identify the primary tumor responsible for metastasis. We report a case of an RTR with multiple CSCCs, one of which metastasized. By using p53 and INK4a-ARF mutation analysis, we identified the responsible primary tumor due to an identical mutation in exon 2 of the INK4a-ARF locus. Archival study yielded 14 cases of metastatic CSCC (present case included). In only 8 of 14 metastases, DNA quality was sufficient to perform PCR reactions. In 7 of 8 metastases, either an INK4a-ARF (6 of 8 cases) and/or p53 (3 of 8 cases) mutation was present. In 6 of 7 cases, the corresponding primary could be identified by an identical mutation in p53 and/or INK4a-ARF. In conclusion, molecular analysis using a combination of p53 and INK4a-ARF mutation analysis can identify the corresponding primary skin tumor in case of CSCC metastases in the majority of cases. This is facilitated by the high frequency of these mutations in metastatic CSCC when compared with frequency spectra reported in the literature in primary CSCCs. The major limitation was formed by insufficient DNA quality in archival tissue.

ADP-Ribosylation Factor 6↗

PubMed2005

Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.

Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.

Aneuploidy↗

PubMed2005

Analysis of mutations in B-RAF, N-RAS, and H-RAS genes in the differential diagnosis of Spitz nevus and spitzoid melanoma.

A definite diagnosis cannot be established based on histologic features alone in a large number of Spitz nevi and spitzoid melanomas. In a vast majority of common benign and malignant melanocytic lesions, B-RAF and N-RAS mutations were described, but these were not detected in Spitz nevi. In contrast, H-RAS mutations were frequently encountered in Spitz nevi, but only rarely in melanomas. To date, B-RAF mutation analysis has not been reported in atypical Spitz nevi, and there are only a few reports of it in spitzoid melanomas. We analyzed 96 formalin-fixed, paraffin-embedded spitzoid melanocytic lesions for hotspot mutations in B-RAF, N-RAS, and H-RAS genes to test the assumption whether mutation analysis would assist a more accurate diagnosis of spitzoid melanocytic lesions, which are notoriously difficult to classify. B-RAF or N-RAS mutations were observed in 31 of 36 (86%) spitzoid melanomas, and in 6 of 7 (86%) spitzoid melanoma metastases. In contrast, none of the 14 Spitz nevi and none of the 16 atypical Spitz nevi had mutations in any of the three genes. A B-RAF or N-RAS mutation was found in 8 of 23 (35%) spitzoid lesions suspected for melanoma. H-RAS mutations were detected in 4 of 14 (29%) Spitz nevi, in 3 of 22 (14%) atypical Spitz nevi, in 1 of 15 (7%) spitzoid tumors suspected for melanoma, but in none of the spitzoid melanomas. These results strongly indicate that Spitz nevi and spitzoid melanomas are genetically unrelated entities. Furthermore, we can conclude that mutation analysis may be useful as an additional diagnostic tool to distinguish between benign and malignant spitzoid lesions.

Adolescent↗

PubMed2005

Functional and morphologic analysis of the fluid-conducting meshwork in xenografted cutaneous and primary uveal melanoma.

PURPOSE: In primary uveal and cutaneous melanoma lesions, extracellular matrix (ECM) is often deposited in arcs, loops, and network patterns. Based on prognostic relevance, these patterns appear to play a significant role in facilitating metastasis. It has been demonstrated that these patterns were capable of transmitting fluid. The current study was undertaken to elucidate further the functional role of these patterns in tumor perfusion and to examine the composition of the patterns by immunohistochemistry. METHODS: To study the role of these patterns in perfusion, fluorochrome-labeled bovine serum albumin, bovine insulin, and dextrans of different molecular sizes were injected intravenously into nude mice bearing subcutaneous human cutaneous melanoma xenografts. Distribution of the human melanoma cells and murine host cells was analyzed by DNA in situ hybridization. To elucidate the composition of these patterns, human uveal melanoma tissues were analyzed for expression of ECM components by immunohistochemistry. RESULTS: Small molecules (Stokes' radius <4.4 nm) crossed the vessel wall and spread along the ECM patterns within 2 to 10 minutes, whereas larger molecules (Stokes' radius approximately 5.8 nm) required 30 to 45 minutes to enter. Murine host cells were found exclusively in the ECM pattern compartment. In primary uveal melanoma, different types of collagen, ECM-associated heparan sulfate proteoglycans, and different types of cells were present in the patterns. CONCLUSIONS: The data suggest that the ECM deposited as arcs, loops, and network patterns, accommodate the transport of plasma-derived molecules, (e.g., nutrients), to the tumor lesion, thus enhancing tumor growth and progression, and facilitating infiltration of tumor tissue by host-derived cells.

Animals↗

PubMed2004

Vascular density in colorectal liver metastases increases after removal of the primary tumor in human cancer patients.

In animal models, explosive growth of metastases after removal of the primary tumor has been attributed to abolishment of angiogenesis inhibition. We investigated the influence of (removal of) the primary tumor on vascularization of liver metastases in human colorectal cancer patients. We analyzed vascular density in synchronous liver metastases from patients with the primary tumor in situ, in synchronous metastases from patients with the primary tumor resected and in metachronous metastases. In a limited number of cases, biopsies from metastases from the same patient before and within 3 months after resection were analyzed. In addition, vascular density in metastases was compared to the vascular density in the corresponding primary tumor. Peritumoral and intratumoral vascular density were determined by staining for endothelial antigens CD31 and CD34, respectively. Both peritumoral and intratumoral vascular density were elevated in synchronous metastases from patients with the primary tumor removed compared to synchronous metastases from patients with the primary tumor in situ. Comparable results were observed in patients with metachronous metastases. An increase in vascular density after resection of the colorectal malignancy was also observed in biopsies taken from the same patient before and after tumor resection. Remarkably, vascular density in the liver metastases was always lower than that in the corresponding primary tumor. Our data show for the first time in humans that the presence of a primary tumor is correlated with decreased vascularization of its distant metastases. Resection of the primary tumor results in an increased vascularization of metastatic lesions.

Adult↗

PubMed2004

Anti-tumor activity of a combination of plasminogen activator and captopril in a human melanoma xenograft model.

Angiostatin, a proteolytic fragment of plasminogen consisting of the first 3 or 4 kringle domains, reduces tumor growth by specifically inhibiting tumor angiogenesis. Angiostatin is generated in vitro in a 2-step process. First, plasminogen is converted to plasmin by plasminogen activators. Next, plasmin excises the angiostatin fragment from plasminogen, a process requiring molecules that are able to donate a free sulfhydryl group. In this study, we investigated whether stimulation of in vivo angiostatin generation by administration of plasminogen activator and a free sulfhydryl group donor (FSD) has anti-tumor activity. First, we determined the optimal conditions for in vitro angiostatin generation by incubating murine plasma with different concentrations of plasminogen activator and/or the FSD captopril. Angiostatin generation was monitored by western blot analysis. Our results were extrapolated to the in vivo situation by administering the optimal dose of tissue-type plasminogen activator (tPA, i.v. injection 3 times/week) and captopril (in drinking water) to mice and analyzing the presence of angiostatin in the circulation. Angiostatin was readily detectable in mice receiving both tPA and captopril, but not in mice receiving either one of the agents. Finally, the anti-tumor activity of the tPA/captopril treatment was tested in a human melanoma xenograft model. Administration of tPA alone had only a marginal effect on tumor growth. Captopril alone reduced tumor growth by about 60%, whereas treatment with both captopril and tPA resulted in 83% inhibition of tumor growth.

Angiogenesis Inhibitors↗

PubMed2004

Presence and localization of T-cell subsets in relation to melanocyte differentiation antigen expression and tumour regression as assessed by immunohistochemistry and molecular analysis of microdissected T cells.

Melanoma-associated antigens may be the driving force behind the lymphocytic infiltrates in melanomas and the occurrence of melanoma regression. To investigate the clinical relevance of melanoma differentiation antigens (MDAs) as T-cell targets, the relationship between the presence and localization of T-cell subsets and the expression of MDAs was studied by immunohistochemistry and the diversity of CD8+ T cells in regressive melanomas was assessed using laser-assisted microdissection. While MDA expression as well as T-cell subset distribution, as assessed by immunohistochemical analysis, was heterogeneous within and between lesions, they were histologically independent phenomena. In four lesions studied in detail by PCR analysis of microdissected T cells, a limited T-cell diversity and evidence for clonally expanded tumour infiltrating lymphocytes were found. However, no major differences in T-cell diversity, as assessed by PCR analysis, between peri-and intra-tumoural areas became apparent, this despite the known clinical significance of the specific localization of a T-cell infiltrate. T cells of clonal origin did not show preferential localization to regressive tumour areas. Moreover, clonally related cells were found in two lesions with a non-brisk infiltrate, while in two lesions with a brisk infiltrate (clinically, a good prognostic factor) no evidence for clonally expanded tumour infiltrating lymphocytes was found. These data support the notion that specific immune reactivity and homing of specific cells to the tumour can occur in melanoma patients. However, they also show that the presence of clonally expanded T cells in the tumour is not necessarily associated with an effective anti-tumour immune response and may, for instance, represent regulatory cells. It appears that the clinical impact of an anti-tumour immune response is largely decided at the tumour site, where micro-environmental conditions dictate the functional state of the T cells. Full understanding of these processes can only be achieved by performing more dynamic analyses of the local host-tumour interactions.

Antigens, Neoplasm↗

PubMed2004

Type I collagen synthesis parallels the conversion of keratinocytic intraepidermal neoplasia to cutaneous squamous cell carcinoma.

Neoplastic progression of solid tumours is often characterized by a simultaneous increase in matrix protein (eg collagen) synthesis and degradation, and results in the formation of a tumour stroma. At the tumour periphery, this process is believed to facilitate angiogenesis and invasive growth of tumour cells. In various types of carcinoma, differentiation of fibroblasts towards myofibroblasts is thought to play an important role in extracellular matrix remodelling as their emergence coincides with architectural changes in the tumour stroma. Here, distinct architectural changes in collagen fibres are reported in cutaneous squamous cell carcinomas (cSCC) with respect to normal skin and precursor lesions, ie keratinocytic intraepidermal neoplasia (KIN). Simultaneously, type I collagen mRNA was observed in fibroblasts in close proximity to cSCC lesions (19/19) but only in 2 of 10 KIN lesions tested. Interestingly, whereas emerging of myofibroblasts correlated with reduced differentiation of cSCCs, it was not a prerequisite for type I collagen synthesis. These data indicate that type I collagen synthesis by fibroblasts parallels the malignant transformation of human KIN to cSCC.

Carcinoma in Situ↗

PubMed2003

Histopathology training in Europe: a lesson for other specialties?

Critical shortages of trained histopathologists limit the capacity of cancer and other clinical services. A survey of histopathology training in 18 European countries conducted by the European Union of Medical Specialists Section of Pathology/European Board of Pathology revealed a considerable shortage of both trained and trainee histopathologists in several of the responding countries. Demographic data indicate a high preponderance of trained histopathologists over 55 years of age and a notable proportion of part-time personnel. Although the training capacity in some countries has been increased, the immediate future of histopathology and the clinical services dependent on the specialty is worrisome. The histopathology workload is growing, and, in some countries, recruitment of trainees is insufficient for future needs. To avert the serious consequences for dependent clinical specialties and their patients, there should be a concerted European action to promote and expand histopathology training.

Curriculum↗

PubMed2003

Maturation of dendritic cells is a prerequisite for inducing immune responses in advanced melanoma patients.

PURPOSE: We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo. EXPERIMENTAL DESIGN: Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients). RESULTS: All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. In a second patient, DTH-derived T cells showed specific lysis of tumor cells expressing the antigens used for DC pulsing. Only patients vaccinated with mature DCs showed objective clinical responses. Interestingly, both patients with long-term progression-free survival (22 and >40 months) were both vaccinated with mature DCs and demonstrated antigen-specific T-cell reactivity of DTH-derived T cells. CONCLUSIONS: We conclude that mature DC are superior to immature DC in the induction of immunological responses in melanoma patients, which may translate into improved clinical results.

Adjuvants, Immunologic↗

PubMed2003

Heparan sulphate epitope-expression is associated with the inflammatory response in metastatic malignant melanoma.

Heparan sulphate (HS) represents a heterogeneous class of molecules on cell membranes and extracellular matrices. These molecules are involved in a variety of biological processes, including immune responses, through their binding and functional modulation of proteins. Recently a panel of HS-epitope-specific, human single chain antibodies have been generated by phage display, facilitating analysis of the structural heterogeneity of HS in relation to pathological conditions. In a pilot study a heterogeneous staining pattern in melanoma metastases was observed with one of the clones (EW4G1). Using a double-staining technique, the expression of this epitope was studied in 12 metastatic melanoma lesions in relation to the presence of a CD3(+) cell infiltrate. Different staining patterns with EW4G1 were observed in the different lesions. The different staining patterns were associated with the presence and pattern of inflammation with CD3(+) cells. A pronounced staining pattern of blood vessels with EW4G1 was associated with a more or less brisk presence of CD3(+) cells, while a pronounced staining of tumour cells or tumour cell matrix or absence of staining with EW4G1 was associated with absence of CD3(+) cells. These results suggest a dualistic role for HS in the recruitment and intratumoural migration of CD3(+) cells, depending on the location of expression of its epitope recognized by EW4G1. Further characterization of the structural diversity of HS and its function in T-cell recruitment and migration is therefore warranted, since detailed understanding of this relation may provide new targets for therapeutic intervention, such that better homing and migration of T cells (in)to tumours might be achieved in immunologically based treatment strategies.

Antibody Affinity↗

PubMed2003

Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma.

We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood supply (B. Kusters et al., Cancer Res., 62: 341-345, 2002). Here, we compare the activities of the 121, 165, and 189 VEGF-A isoforms in this model by transfecting Mel57 cells with the respective cDNAs and by injecting the resulting stably transfected cell lines in the internal carotid arteries of nude mice (n = 10 for each isoform). Although the three isoforms had similar potency to induce endothelial cell proliferation, VEGF(121) expression did not result in sprouting angiogenesis but rather led to extensive vasodilation and increased permeability of pre-existing, predominantly peritumoral vessels. Sometimes, proliferating endothelial cells accumulated in vessel lumina, giving these a microvascular, glomeruloid, proliferation-like appearance. Expression of VEGF(165) or VEGF(189) was associated with induction of an intratumoral neovascular bed. In VEGF(165)-expressing tumors, daughter endothelial cells were distributed among newly formed vessels that were extensively dilated. This also occurred in VEGF(189) tumors, but there, vasodilation was less pronounced. Using contrast-enhanced magnetic resonance imaging, the different vascular phenotypes were visualized on characteristic radiological images. VEGF(165) expression was the most unfavorable of the three. Mice carrying VEGF(165) tumors became moribund earlier than those carrying VEGF(121)-expressing tumors (16 +/- 4 days versus 22 +/- 3 days). Our data demonstrate that VEGF-A isoforms differ in angiogenic properties that can be visualized by contrast-enhanced magnetic resonance imaging.

Animals↗

PubMed2003

The human SPANX multigene family: genomic organization, alignment and expression in male germ cells and tumor cell lines.

Multigenicity is one of the features of cancer/testis-associated genes. In the present study we analyzed the number and expression of genes of the SPANX(CTp11) family of cancer/testis-associated genes. Genomic database analysis, next to the four previously described SPANX genes, revealed the presence of a novel gene: SPANXE. Moreover, we detected an allelic variant of SPANXB resulting in one amino acid substitution in the encoded protein: SPANXB'. Most SPANX genes are present on contig NT_011574 located at Xq26.3-Xq27.1. Based on expressed sequence tag databases and RT-PCR analysis three additional novel SPANX sequences were identified, though not represented so far in the human genome sequence. Sequence alignments justify a subdivision of this gene family based on the absence (SPANXA-likes) or presence (SPANXB) of an 18 base pair sequence stretch in the open reading frame. The alignments also reveal an unusually high level (99%) of intron homology. Furthermore, the nucleotide variations in the open reading frame almost all lead to amino acid substitutions. Southern blot and database analyses indicate that SPANX sequences are exclusively present in primates. With RT-PCR analysis on human sperm cell precursors and tumor cell lines most family members could be detected. SPANXB was only found in sperm cell precursors and could not be detected in the tumor cell lines tested. Overall SPANXA was the most frequently expressed SPANX variant in melanoma and glioblastoma cell lines.

Amino Acid Sequence↗