Dataset from a proteomics analysis of tumor antigens shared between an allogenic tumor cell lysate vaccine and pancreatic tumor tissue.

The data described was acquired as part of a clinical study with the aim to investigate the potential of tumor-reactive T-cell response as response to vaccination of pancreatic cancer patients with an allogenic tumor cell lysate vaccine (Lau et al., 2022). Proteomics analysis was carried out to identify tumor antigens that are shared between the allogeneic tumor cell lysate used for the vaccine and pancreatic ductal adenocarcinoma (PDAC) tissue samples. To this objective, cell lysates of the vaccine and of nine tissue samples were enzymatically digested and isotopically labeled with tandem mass tags (TMT) in a so-called six-plex manner (Thermo Fisher Scientific). Three pools were prepared by mixing the samples according to their TMT-labels. Subsequently, the three sample pools were fractionated into 24 fractions with high-pH reversed phase chromatography. These fractions were first analyzed on a nano-liquid chromatography (LC) system online coupled to a high-resolution Eclipse Orbitrap mass spectrometer (MS) equipped with a high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) source using a data-dependent MS2 shotgun method. Overall, 126,618 unique peptide sequences, on basis of 768,638 peptide spectra matches and corresponding to 7,597 protein groups, were identified in the total sample set including 61 tumor antigens (Supplement Table S2 in Lau et al. 2022) that were prioritized by Cheever and co-workers as vaccine target antigens on basis of a series of objective criteria (Cheever et al., 2009). In the second phase of the experiment, this set of tumor antigens was targeted using a serial precursor selection (SPS) MS3 method. From this data, ion trap MS2 and Orbitrap MS3 fragment spectra were extracted for peptide identification (protein sequence database-dependent search) and relative quantification using the TMT labels, respectively. The dataset ultimately allowed the identification and quantification of 51 proteins and 163 related peptide precursors with the TMT labels (see Fig. 2B and Supplemental Fig. 8, Lau et al. 2022).

Autologous dendritic cells pulsed with allogeneic tumour cell lysate induce tumour-reactive T-cell responses in patients with pancreatic cancer: A phase I study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor prognosis even after curative resection. Responses to immunotherapy are rare and related to inadequate T-cell priming. We previously demonstrated the potency of allogeneic lysate-dendritic cell (DC) vaccination in a preclinical model. Here we translate this concept to patients. METHODS: In this phase I study, patients with resected PDAC were included when they demonstrated no radiologic signs of recurrence after standard-of-care treatment. Allogeneic tumour lysate-loaded autologous monocyte-derived DCs were injected at weeks 0, 2, 4 and at months 3 and 6. Objectives are feasibility, safety and immunogenicity of allogeneic tumour-DCs. The presence of tumour antigens shared between the vaccine and patient tumours was investigated. Immunological analyses were performed on peripheral blood, skin and tumour. RESULTS: Ten patients were included. DC production and administration were successful. All patients experienced a grade 1 injection-site and infusion-related reaction. Two patients experienced a grade 2 fever and 1 patient experienced a grade 3 dyspnoea. No vaccine-related serious adverse events were observed. Shared tumour antigens were found between the vaccine and patient tumours. All evaluated patients displayed a vaccine-induced response indicated by increased frequencies of Ki67+ and activated PD-1+ circulating T-cells. In addition, treatment-induced T-cell reactivity to autologous tumour of study patients was detected. Seven out of ten patients have not experienced disease recurrence or progression at a median follow-up of 25 months (15-32 months). CONCLUSION: Allogeneic tumour lysate-DC treatment is feasible, safe and induces immune reactivity to PDAC expressed antigens.

Comparative proteomic analysis of human mesenchymal stromal cell behavior on calcium phosphate ceramics with different osteoinductive potential.

In recent years, synthetic calcium phosphate (CaP) ceramics have emerged as an alternative to bone grafts in the treatment of large critical-sized bone defects. To successfully substitute for bone grafts, materials must be osteoinductive, that is, they must induce osteogenic differentiation and subsequent bone formation in vivo. Although a set of osteoinductive CaP ceramics has been developed, the precise biological mechanism by which a material directs cells toward osteogenesis and the role of individual chemical and physical properties in this mechanism remain incompletely understood. Here, we used proteomics to compare serum protein adsorption to two CaP ceramics with different osteoinductive potential, namely an osteoinductive ß-tricalcium phosphate (TCP) and a non-osteoinductive hydroxyapatite (HA). Moreover, we analyzed the protein profiles of human mesenchymal stromal cells (hMSCs) cultured on these two ceramics. The serum protein adsorption experiments in the absence of cells highlighted the proteins that are highly abundant in the serum and/or have a high affinity to CaP. The extent of adsorption was suggested to be affected by the available surface area for binding and by the ion exchange dynamics on the surface. Several proteins were uniquely expressed by hMSCs on TCP and HA surfaces. Proteins identified as enriched on TCP were involved in processes related to wound healing, cell proliferation, and the production of extracellular matrix. On the other hand, proteins that were enriched on HA were involved in processes related to protein production, translation, localization, and secretion. In addition, we performed a separate proteomics analysis on TCP, HA, and two biphasic calcium phosphates with known osteoinductive potential and performed a clustering analysis on a combination of a set of proteins found to be enriched on osteoinductive materials with a set of proteins already known to be involved in osteogenesis. This yielded two protein networks potentially involved in the process of osteoinduction - one consisting of collagen fragments and collagen-related enzymes and a second consisting of endopeptidase inhibitors and regulatory proteins. The results of this study show that protein profiling can be a useful tool to help understand the effect of biomaterial properties on the interactions between a biomaterial and a biological system. Such understanding will contribute to the design and development of improved biomaterials for (bone) regenerative therapies.

Mass Spectrometry for Identification, Monitoring, and Minimal Residual Disease Detection of M-Proteins.

BACKGROUND: Monoclonal gammopathies (MGs) are plasma cell disorders defined by the clonal expansion of plasma cells, resulting in the characteristic excretion of a monoclonal immunoglobulin (M-protein). M-protein detection and quantification are integral parts of the diagnosis and monitoring of MGs. Novel treatment modalities impose new challenges on the traditional electrophoretic and immunochemical methods that are routinely used for M-protein diagnostics, such as interferences from therapeutic monoclonal antibodies and the need for increased analytical sensitivity to measure minimal residual disease. CONTENT: Mass spectrometry (MS) is ideally suited to accurate mass measurements or targeted measurement of unique clonotypic peptide fragments. Based on these features, MS-based methods allow for the analytically sensitive measurement of the patient-specific M-protein. SUMMARY: This review provides a comprehensive overview of the MS methods that have been developed recently to detect, characterize, and quantify M-proteins. The advantages and disadvantages of using these techniques in clinical practice and the impact they will have on the management of patients with MGs are discussed.

Trophoblast calcyclin is elevated in placental tissue from patients with early pre-eclampsia.

The aetiology of pre-eclampsia is thought to originate from aberrant spiral artery remodelling and invasion evoking cellular oxidative stress. Previously, we discovered differentially expressed proteins in trophoblast cells of pre-eclamptic pregnancies. One of these proteins is calcyclin (S100A6); a Ca(2+)-binding protein associated with cellular stress response. By immunohistochemistry on formalin-fixed paraffin-embedded placental tissue, calcyclin expression was compared between women with early pre-eclampsia (n=72) and non-hypertensive control patients (n=66) (χ(2), p=0.006) blindly by two observers. Significantly more intense staining was seen in trophoblast cells of pre-eclamptic pregnancies compared to control placentas suggesting that trophoblast calcyclin is elevated in early pregnancy.

Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method.

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5-100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1-1,000 nM for all MTXPGn (R(2) > 0.99). The coefficient of variation ranged from 1-4% for intraday precision and 6-15% for interday precision. Samples were stable for at least 1 month at -80 °C. Recovery ranged from 98-100%, and the relative matrix-effect varied from 95-99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.

Serum proteomic patterns for ovarian cancer monitoring.

We set out to discover ovarian cancer biomarkers useful for monitoring progression during and after chemotherapy and possibly for diagnosis. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to create serum protein profiles of ovarian cancer patients before chemotherapy or at progression (n = 51) (trial initiated by the Gynecological Cancer Cooperative Group of the European Organization for Research and Treatment of Cancer trial) that were compared with those of healthy individuals (n = 31). In addition, sera profiles from ovarian cancer patients after chemotherapy (n = 12) were compared with those of ovarian cancer patients at progression (n = 24). One of the discovered biomarkers was identified and subsequently confirmed and validated using enzyme-linked immunosorbent assay (ELISA). Eight primary (sens = 94%, spec = 97%, P < 0.0001) and seven progression tumor biomarkers (sens = 91%, spec = 97%, P < 0.0001) were discovered. In addition, we discovered eight potential progression monitoring biomarkers (sens = 75%, spec = 83%, P = 0.0008) of which one, a biomarker of 11.7 kd, was further identified as serum amyloid A1. Independent validation (ELISA) showed an elevated expression of this protein at relapse in four of the seven ovarian cancer patients tested. Combining the eight newly discovered progression monitoring biomarkers with CA125 resulted in a clear increase of the sensitivity (91-100%). These biomarkers, in combination with for instance CA125, should be validated in large ovarian cancer and control groups. The resulting multimarker assay could be suitable for disease monitoring during and after therapy and might also be useful for ovarian cancer screening.

Hepatocyte-derived fibrinogen-related protein-1 is associated with the fibrin matrix of a plasma clot.

In order to study the multiple functions of fibrinogen and fibrin, we are investigating which proteins bind to the fibrin matrix of a plasma clot by using a proteomic approach. Extracts from washed plasma clots were analysed by 2-D gel electrophoresis. A relatively abundant spot was identified as hepatocyte-derived fibrinogen-related protein-1 (HFREP-1) by MALDI-TOF analysis, molecular mass (34 kDa), iso-electric point (pI 5.5) as well as by Western blot analysis. HFREP-1 in plasma almost completely bound to the fibrin matrix during clot formation. Several purified fibrinogen preparations proved to be contaminated with HFREP-1. It is concluded that HFREP-1 (also named hepassocin), a protein with liver cell growth regulatory properties, occurs in plasma and strongly associates with fibrin and possibly fibrinogen.

Innovations in serum and urine markers in prostate cancer current European research in the P-Mark project.

An overview is given of serum and urine prostate cancer markers that are currently under investigation and subsequently the P-Mark project is introduced. There are many markers showing promise to overcome the limitations of prostate specific antigen (PSA). Eventually, these markers should be able to increase the specificity in diagnosis, differentiate between harmless and aggressive disease and identify progression towards androgen independence at an early stage. In the P-Mark project, several recently developed, promising markers will be evaluated using clinically well-defined biorepositories. Following successful evaluation, these markers will be validated on a sample set derived from two large, European, prostate cancer studies and used for the identification of special risk groups in the general population. In addition, novel markers will be identified in the same biorepositories by different mass spectrometry techniques.

Spatial variability of genomic aberrations in a large glioblastoma resection specimen.

In the present study, the distribution of genetic aberrations in a glioblastoma resection specimen of unusually large size (9x8x2 cm) was investigated using comparative genomic hybridization (CGH). CGH was performed on 20 samples taken from the specimen, and the genetic aberrations found were compared with the regional histology. The samples were histopathologically graded according to WHO criteria, and a division in high- and low-grade areas and infiltration rims was made. In high-grade areas, low-grade areas as well as infiltration rims, gains on 10p11.2-pter (14/20), 11q12-q22 (6/20) and losses on 4q13-qter (9/20), 10q22-qter (8/20), 11p14-pter (5/20), 13q12-qter (7/20) were revealed. Gains on 1q21-32 (2/4) and losses on 7p21-pter (3/4) were exclusively found in the high-grade areas. In the low-grade tumor samples and in the infiltration rim, gains on 16p11.2-pter (6/16), 17p11.2-pter (6/16), 17q11.2-qter (5/16), 20q11.2-q13 (3/16) and deletions on 5q31-qter (4/16) were detected. Gains on 7q21-qter (8/11) and 8q11.2-qter (6/11), and loss of chromosome 9 (4/11) and the Y-chromosome (4/11) were found in the high-grade and low-grade samples, not in the infiltration rims. The finding of a set of identical chromosomal aberrations throughout the resection specimen, most of which have been previously reported in gliomas, confirms a mechanism of clonal tumor proliferation operative in gliomas. The previously unreported genetic alterations which were predominantly traced in the tumor rims, might reflect either selection for properties related to infiltrating behavior, or genomic instability of subclones. The findings illustrate the importance of searching for high-grade genetic aberrations in low-grade tumor samples taken from cases in which sampling error is suspected.

Glial fibrillary acidic protein and its fragments discriminate astrocytoma from oligodendroglioma.

In the last few years it has been shown that anaplastic oligodendrogliomas, in contrast to anaplastic astrocytomas, are responsive to a three drug regimen chemotherapy. The histologic criteria for the discrimination between oligodendrogliomas and astrocytomas are subject to substantial interobserver variability, particularly in anaplastic and mixed gliomas. In the present study a two-dimensional electrophoresis technique (2-DE) has been applied to glioma samples in an attempt to discriminate the glioma subtypes. It was found that the presence of glial fibrillary acidic protein (GFAP) fragments distinguishes oligodendroglioma from astrocytoma. One-dimensional (1-DE) immunoblots were compared with immunohistologically stained tissue sections in which various GFAP-positive cell types were seen. It is concluded that 2-DE and 1-DE GFAP immunoblotting provide accurate information for the reliable discrimination of anaplastic astrocytomas and oligodendrogliomas.

Expression of MAGE, gp100 and tyrosinase genes in uveal melanoma cell lines.

In order to determine the possible use of uveal melanoma cell lines as stimulators in immunotherapy, we evaluated the expression of the human genes for MAGE-1, -2 and -3, gp100 and tyrosinase in uveal melanoma cell lines. mRNA expression of the MAGE-1, -2 and -3, gp100 and tyrosinase genes and the HLA class I specificity were determined in five primary and three metastatic uveal melanoma cell lines. Expression of the examined genes was heterogeneous in the primary and metastatic cell lines. The cell lines OCM-1 and OMM-1 expressed MAGE-1, -2 and -3, whereas EOM-3, MEL202, 92-1 and OMM-3 were negative for these antigens. gp100 was expressed in all cell lines, and tyrosinase in all but three (EOM-29, OMM-2 and OMM-3). Except for EOM-3, the HLA-A type of all the cell lines could be determined by complement-dependent microlymphocytotoxicity assay. Since at least two melanoma-associated antigens can be found in uveal melanoma cell lines, as well as the HLA class I molecules, these cell lines may be applicable as immunogens for specific immunotherapy against metastatic uveal melanoma.

The Han:SPRD rat is not a genetic model of human autosomal dominant polycystic kidney disease type 1.

Human autosomal dominant polycystic kidney disease (ADPKD) is a high incidence disorder leading to renal failure in many patients. The majority of cases results from a mutation in the PKD1 gene. The only well documented animal model of ADPKD is the Han:SPRD-Pkd strain. Its genetic basis is unknown as yet. In the current study we determined whether the disease in these rats is genetically linked to the rat homologue of the PKD1 gene. We used the protamine gene as a polymorphic marker (Prm1) of the PKD1 region. Matings of Han:SPRD-Pkd with BB rats and backcross of the offspring with BB yielded animals informative for linkage analysis. This analysis revealed random segregation of the defect and the Prm1 marker, indicating that the model is not caused by a mutation in the PKD1 gene. We conclude that the Han:SPRD-Pkd rat strain is not a genetic model of PKD1.

HNK-1 antigens on uveal and cutaneous melanoma cell lines.

The HNK-1 epitope has been associated with the metastatic behaviour of uveal melanomas. We characterized HNK-1 antigens on four human uveal (primary and metastatic) and two primary cutaneous melanoma cell lines by immunocytochemistry and Western blot analysis. We also determined the involvement of the HNK-1 epitope in cell-cell interactions on a matrigel layer. Three uveal melanoma cell lines (one primary and two metastatic) and one cutaneous melanoma cell line showed HNK-1 expression by immunocytochemistry. On matrigel, only the HNK-1-positive cutaneous melanoma cell line Bowes grew in a honeycomb-like structure which disappeared after adding HNK-1 antibodies to the culture medium. Immunoblot analysis of the primary uveal melanoma cell line EOM-3 revealed five HNK-1-positive protein bands with apparent molecular weights of 200, 160, 115, 95 and 75 kDa. The cutaneous melanoma cell line Bowes showed three HNK-1-positive protein bands with apparent molecular weights of 150, 135 and 90 kDa. This study shows that two uveal (primary and metastatic) and one primary cutaneous melanoma cell lines express HNK-1 antigens on immunoblot. Only in the HNK-1-positive cutaneous melanoma cell line Bowes did the HNK-1 epitope have a function in intercellular adhesion. Although the primary uveal melanoma cell line EOM-3 showed a similar HNK-1 immunoreactivity, we could not demonstrate HNK-1-mediated cell adhesion. On immunoblot, the two cell lines displayed different HNK-1 antigens, which may explain the difference in cell adhesion.

Metastatic uveal melanoma. A morphologic and immunohistochemical analysis.

BACKGROUND: Uveal melanoma often metastasizes late and preferentially to the liver, in contrast to cutaneous melanoma. The objective of the current study was to evaluate the histopathologic and immunohistochemical changes in primary uveal melanomas and their corresponding metastases. METHODS: The morphology and immunohistochemical reactivity for the melanoma-associated antibodies HMB-45, S-100 protein, and NKI-C3 were assessed for 29 primary uveal melanomas and their corresponding metastatses. RESULTS: A significant difference in cell type of the primary and the metastatic uveal melanoma was found (P = 0.0001). The metastases derived from the 29 patient's revealed 82.5% epithelioid or nonclassifiable cells. Positive staining of the primary uvea melanomas and their metastases was found to be 93% and 91%, respectively, for HMB-45, 80% and 66%, respectively, for S-100, and 56% and 71%, respectively, for NKI-C3. CONCLUSIONS: Metastases of uveal melanomas are comprised of a higher grade of malignant cell types. Nonclassifiable cells can be observed in 40% of metastatic lesions. In the current study, HMB-45 proved to be the most sensitive immunohistochemical marker in the analysis of metastatic uveal melanoma and should be used as part of a panel of monoclonal antibodies in the analysis of any metastatic tumor of unknown origin.

Association between NM23-H1 gene expression and metastasis of human uveal melanoma in an animal model.

PURPOSE: To evaluate the role of nm23 gene expression in the development of metastases of human uveal melanomas in an animal model. METHODS: Seven human uveal melanoma cell lines and two murine skin melanoma cell lines were subjected to Northern blot analysis for the detection of nm23-H1 mRNA and to immuno-histochemistry to detect nm23 antigen. Each tumor cell line was transplanted intracamerally into nude mice, and the metastatic behavior was evaluated by histopathologic analysis of the livers and by determining host survival times. RESULTS: There was a strong inverse correlation between the levels of nm23 mRNA expression and nm23 antigen expression and the development of metastases of all seven human uveal melanomas and both murine skin melanomas transplanted intracamerally. Host survival time also was correlated with the degree of nm23 gene expression. CONCLUSIONS: The expression of nm23 mRNA and nm23 antigen in human uveal melanomas is correlated closely with reduced metastatic behavior in experimental animals and may serve as a sensitive prognostic indicator of malignancy and survival in patients with uveal melanomas.

Identification of glycosaminoglycans in age-related macular deposits.

We investigated the presence and localization of glycosaminoglycans in basal laminar deposit and drusen in age-related maculopathy. Conventional histological staining techniques and monoclonal antibodies specific for several glycosaminoglycans were used on paraffin-embedded human maculae. Furthermore, macular homogenates were analyzed with two-dimensional electrophoresis. Quantitative analysis of glycosaminoglycans was done spectrophotometrically using dimethylmethylene blue. Immunohistochemically, all basal laminar deposit stained positive for chondroitin 4-sulfate and focally positive for heparan sulfate proteoglycan. Drusen were not stained with any of the monoclonal antibodies. With two-dimensional electrophoresis, it was demonstrated that macular extracts with and without age-related maculopathy contained chondroitin sulfate. Heparan sulfate was only expressed in maculae with age-related maculopathy. The total amount of glycosaminoglycans was significantly higher in maculae with basal laminar deposit than in maculae without basal laminar deposit (P = .001). There were significant differences in the amount and composition of glycosaminoglycans between maculae with and without age-related maculopathy.

Establishment and characterization of primary and metastatic uveal melanoma cell lines.

We report on the establishment and characterization of 2 primary (EOM-3, EOM-29) and 3 metastatic uveal melanoma cell lines (OMM-1, OMM-2, OMM-3) and further cytogenetic characterization of a previously described primary uveal melanoma cell line (OCM-1). Only a few long-term growing primary uveal melanoma cell lines have as yet been established, while of metastatic uveal melanoma cell lines we have found no descriptions. The morphology of the in vitro cultured cells varied from spindle to epithelioid. The cell lines were characterized by immunocytochemistry, electron microscopy and cytogenetical analysis. The relative growth rate was determined by bromodeoxyuridine (BUdR) incorporation. The melanocytic origin of the cell lines was determined by positive staining with antibodies identifying melanoma-associated antigens. Melanosomes and pre-melanosomes were indeed observed by electron microscopy in all cell lines. The stem-cell karyotype was found to be normal in 3 cell lines (EOM-29, OMM-2, OMM-3) and abnormal in 3 others (EOM-3, OCM-1, OMM-1) showing a net loss of chromosome 6. The OCM-1 and the OMM-1 cell lines even demonstrated a large amount of structural chromosomal aberrations, the former being near-tetraploid and the latter triploid. The EOM-29 cell line, cultured from a ciliary body melanoma, did not show the previously described chromosome 3 and 8 abnormalities.

Chorionic villus sampling and materno-fetal transfusions: an immunological pathogenesis of vascular disruptive syndromes?

Experimental materno-embryonic transfusions with serum that is immunologically active against blood group antigens cause congenital malformations in the rat embryo. In view of the possible increased incidence of vascular disruptive syndromes after chorionic villus sampling (CVS), we investigated the occurrence of materno-fetal transfusions (MFTs) in this procedure. In 18 pregnant women experiencing two needle introductions at CVS, we looked immunohistochemically at the presence of haemoglobin A1-containing maternal erythrocytes in the fetal circulation of the separately collected first and second chorionic villus samples. In 4 of 18 patients (22 per cent), a significant increase of maternal cells was observed in the second sample compared with the first sample, indicating the occurrence of MFT by CVS. On the rare occasion of maternal immunization against fetal antigens, a CVS-associated MFT might provoke immunological damage to the fetus.

Neural cell adhesion molecule distribution in primary and metastatic uveal melanoma.

Tumor cell adhesion, detachment, and aggregation play an important part in tumor invasion and metastasis, and a variety of cell adhesion molecules have been found on tumor cells. Cell adhesion molecules, including those of the immunoglobulin superfamily, are associated with the development of metastatic behavior in cutaneous melanomas. The neural cell adhesion molecule (NCAM) belongs to this family. To investigate its possible role in the development metastatic behavior of uveal melanomas, the authors studied immunohistochemically the expression of NCAM by using an antibody that recognizes all three major isoforms of NCAM and an antibody that recognizes the HNK-1 epitope present on some isoforms of NCAM. The authors studied 32 primary uveal melanomas from 32 patients (among these, 12 were rapidly metastasizing and 16 slowly metastasizing) and 29 metastases from 19 patients. From 13 patients the primary, as well as the metastatic, tumors were available. With one exception, all HNK-1 positive primary and metastatic tumors were also positive for NCAM. NCAM was significantly more expressed in aggressive, rapidly metastasizing primary tumors (P = .02 and .04, respectively) and in metastases. HNK-1 was significantly (P = .04) more expressed in larger tumors. In liver metastases HNK-1 immunoreactivity was significantly (P = .005) less frequently expressed than NCAM. Therefore, NCAM isoforms that lack the HNK-1 epitope might play a role in the organ specific metastatic behavior of uveal melanomas.