PCR clonality detection in Hodgkin lymphoma.

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein-Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.

Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies.

The BIOMED-2 Concerted Action BMH4-CT98-3936 on 'Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders' developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB). As no comparable TCRB PCR method pre-existed and only a limited number of samples was tested within the BIOMED-2 study, we initiated this study for further validation of the newly developed TCRB PCR approach by comparing PCR data with previously generated Southern blot (SB) data in a series of 66 immature (ALL) and 36 mature T-cell malignancies. In 91% of cases, concordant PCR and SB results were found. Discrepancies consisted of either failure to detect SB-detected TCRB rearrangements by PCR (6.5%) or detection of an additional non-SB defined rearrangement (2.5%). In 99% of cases (99/100), at least one clonal TCRB rearrangement was detected by PCR in the SB-positive cases. A predominance of complete Vbeta-Jbeta rearrangements was seen in TCRalphabeta(+) T-cell malignancies and CD3-negative T-ALL (100 and 90%, respectively), whereas in TCRgammadelta(+) T-ALL, more incomplete Dbeta-Jbeta TCRB rearrangements were detected (73%). Our results underline the reliability of this new TCRB PCR method and its strategic applicability in clonality diagnostics of lymphoproliferative disorders and MRD studies.

Transition of horizontal to vertical growth phase melanoma is accompanied by induction of vascular endothelial growth factor expression and angiogenesis.

Melanoma progression in general is characterized by an increase in both metastatic frequency and the vascular density of the tumour tissue. Although a direct correlation between these two parameters in individual cases seems to be lacking, it is clear that metastasis is invariably preceded by angiogenesis. One of the angiogenic factors that is produced by human melanoma cells is vascular endothelial growth factor (VEGF). To investigate the role of this factor in the angiogenic process in primary cutaneous melanoma we determined the mean vascular density and the presence of VEGF protein in biopsies of human lesions. The results were compared with those found in normal skin or uninvolved skin from melanoma patients. In addition, we studied morphological and antigenic features of the proliferating neovasculature. We show that (1) the mean vascular density gradually rises along with melanoma progression, (2) transition of horizontal to vertical growth phase melanoma is accompanied by induction of VEGF protein expression and accumulation of this factor in the stroma, (3) vertical growth phase melanoma is often organized in nodules separated by septa containing blood vessels, but without lymphatics, and (4) blood vessel lumina in vertical growth phase melanoma are separated from tumour nodules by two basal lamina containing collagen type IV and the endothelium shows activated morphology and focal expression of the adhesion molecule E-selectin. Our findings indicate that VEGF is a prominent angiogenic factor in melanoma angiogenesis. Although its expression is induced during progression, the effect of VEGF on the incidence of metastasis is probably indirect.

Lack of lymphangiogenesis in human primary cutaneous melanoma. Consequences for the mechanism of lymphatic dissemination.

Cutaneous melanoma has an initial preference for lymphatic spread. Remarkably, melanoma progression toward this metastasizing phenotype is accompanied by intense blood vessel angiogenesis (hemangiogenesis), but lymphangiogenesis, the formation of new lymph vessels in the tumor, has never been reported. To investigate how primary melanoma cells interact with the existing lymphatic microvasculature, and whether lymphangiogenesis occurs, an immunostaining was developed that differentially decorates blood and lymph vessels in frozen tissue sections. The density and distribution of both these vessel types in and around thin (< or = 1.5 mm) and thick (> or = 1.5 mm) primary melanoma lesions and in normal and uninvolved skin were determined. Although especially in thick melanoma lesions a significant increase in blood vessel density was observed, lymphatic density remained unaltered, showing that lymphangiogenesis did not occur. Morphological analysis indicated, however, that melanoma progression is accompanied by a sequence of events that involves hemangiogenesis supporting tumor expansion, especially in the vertical growth phase. Often, stromal sepia are formed around the blood capillaries in the tumor neovasculature protecting them from invasion. Lymph vessels inside the tumor were infrequently observed. However, subepidermal lymph vessels often seemed to be entrapped and penetrated by the expanding tumor mass. In this way, hemangiogenesis, as the driving force behind tumor expansion, might indirectly increase the chance of lymphatic invasion in the absence of lymphangiogenesis. This model explains the paradox that, although melanoma metastasis seems to require angiogenesis, a consistent relation of prognosis with blood capillary density in primary cutaneous melanoma is lacking.

Analysis of the tumor vasculature and metastatic behavior of xenografts of human melanoma cell lines transfected with vascular permeability factor.

Vascular permeability factor (VPF) is an important mediator of vascular development in tumors. Some human melanoma cell lines have a low VPF expression level in culture, but this level is upregulated when growing as a tumor in nude mice. Other melanoma lines have a constitutively high VPF expression. To compare the biological behavior of tumors with these two expression patterns, a human melanoma cell line with an inducible VPF expression was transfected with VPF expression constructs. In this way, several lines were obtained that constitutively produce either the soluble VPF121 or the matrix-associated VPF189 variant at levels of 4 to 30 times the VPF level in mature tumors derived from the parental line. The recombinant VPF RNA, which lacks most of the 5' noncoding sequences present in the endogenous VPF mRNA, was much more efficiently translated than the endogenous messenger. Upon injection in nude mice, all VPF-transfected lines developed tumors with aberrations in vascularization and in distribution of matrix components. In these tumors the blood vessels were hyperpermeable for an i.v. injected protein tracer. Transfection did not influence the in vitro growth rate of the cell lines, but the tumors from the VPF-transfected lines had higher growth rates in vivo than tumors from the parental line or the vector-transfected line. Although the incidence of lung metastasis was similar in all lines, the number of metastases per affected lung was significantly increased in mice carrying VPF-transfectant tumors. We conclude that the pattern and the level of VPF expression in a tumor are important determinants of the architecture and functionality of the vascular bed, but that overexpression of VPF does not necessarily lead to an increase of microvascular density or metastatic spread. The role of VPF in melanoma progression is obviously complex and may be difficult to derive in its generality from a single experimental model.

Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice.

We studied the expression of the angiogenic factor vascular permeability factor) (VPF, also called vascular endothelial growth factor), in human melanoma cells in vitro and in vivo. Melanoma lines that develop tumors with a low metastatic potential in nude mice were found to have low expression levels of VPF in vitro, and the VPF expression levels in melanoma lines that yield highly metastatic xenografts were high. However, in vivo the correlation between VPF mRNA levels and the frequency of metastasis was lost; in all xenografts equally high levels of VPF mRNA were found, independent of the parental cell line. Hence, in vivo VPF gene expression was upregulated in the low expressing lines. The external factor responsible for this induction may be hypoxia, given that we found that low oxygen tension caused a (reversible) increase in the VPF mRNA levels in otherwise low expressing melanoma lines in vitro. A melanoma line with an inducible VPF expression was engineered into a line with a constitutive VPF expression. In the xenografts from this line a change in the vascular architecture was seen, indicating that the pattern or the level of VPF expression is important for tumor angiogenesis in melanoma xenografts.

Covalent dimerization of vascular permeability factor/vascular endothelial growth factor is essential for its biological activity. Evidence from Cys to Ser mutations.

Vascular permeability factor, or vascular endothelial growth factor (VPF/VEGF) is an important factor in the regulation of vascular growth and vascular permeability. VPF is a secreted, dimeric protein and has 8 cysteine residues conserved with platelet-derived growth factor (PDGF). To study the role of some of these cysteine residues in maintaining the structure and function of VPF, we replaced the codons for the second, third, fourth, and fifth cysteine by serine codons, and expressed the mutant proteins in a mammalian expression system. Cysteine residues 2 and 4 in VPF were found to be directly involved in anti-parallel interchain disulfide bonds, as in PDGF. VPF mutants lacking one of these cysteins were severely impaired in their S-linked dimerization, while upon coexpression of both mutants the ability to form dimers was restored. The VPF mutants lacking cysteine residue 2 or 4 also competed poorly for receptor binding of labeled VPF and had low biological activity, but these defects were also complemented by coexpressing the two mutants, indicating that for efficient receptor binding and activation VPF needs to be a covalent dimer, unlike PDGF-BB. Furthermore, cysteine residue 5 was found to be essential for VPF dimerization and activity, while the mutant lacking cysteine residue 3 was only mildly affected in its ability to dimerize and had partial biological activity.