Characterization of monoclonal antibodies directed against squamous cell carcinoma antigens: report of the TD-10 Workshop.

Thirteen monoclonal antibodies directed against squamous cell carcinoma antigens (SCCA1 and SCCA2) were obtained from five international collaborating laboratories participating in the ISOBM TD-10 Workshop. Native and recombinant forms of SCCA were used in a wide variety of approaches to determine the reactivity and specificity of these antibodies. Based on reactivity, the antibodies could be divided into three groups: the SCCA1-reactive group containing those that reacted only with recombinant SCCA1 (rSCCA1) and native SCCA1 (nSCCA1) antigens, the SCCA2-reactive group containing those that reacted only with recombinant SCCA2 (rSCCA2), and the pan-reactive group containing those antibodies that reacted with rSCCA1, nSCCA1, and rSCCA2. Binding to radioiodinated rSCCA1 showed that all reactive antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Binding to labelled rSCCA2 demonstrated that five antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Antibody reactivity on Western blots was tested with nonreduced and reduced native and recombinant SCCA1 and SCCA2. In general, these findings showed that reduction had little effect on binding to SCCA1, but often a strong effect on the binding to SCCA2. Binding of antibodies to rSCCA1 and rSCCA2 in complexes with cathepsin L and G, respectively, was used to assist in the localization of epitope regions in enzyme-complexed SCCA. Cross-inhibition experiments showed that SCCA1-reactive antibodies represent two different epitope groups, and this is supported by their ability to make SCCA1-specific assays by combining antibodies from the two epitope groups. The SCCA2-reactive group represents two related antibodies and one unique as seen in cross-inhibition, but they do not form successful assay combinations. Classification of the pan-reactive antibodies is more difficult, as some epitope groups differ when results from rSCCA1 are compared with rSCCA2 as the target. However, two antibodies are outstanding, SCC107 and SCC113, as they are high-affinity antibodies which react equally well with free and protease complexes of SCCA1 and SCCA2. The precise location of epitopes was further studied using sequential overlapping peptides and homology modelling. The findings from this workshop strongly indicate that the recombinant antigens (rSCCA1 and rSCCA2) are very similar in epitope structure to the native counterparts in saliva, and squamous epithelium from normal and cancer tissues. Therefore, it is reasonable to conclude that the specificities found are reliable and have application for antibody measurement of all forms of squamous cell carcinoma in serum except SCCA2 in complex with its protease.

Cytogenetic and molecular genetic analyses of liposarcoma and its soft tissue simulators: recognition of new variants and differential diagnosis.

Liposarcoma is one of the most common sarcomas of adults. Its differential diagnosis and accurate subclassification are often problematic; the latter is also important with regard to appropriate treatment and prognosis. We studied a series of 23 liposarcomas that had unusual or previously undescribed features and 10 liposarcoma simulators and correlated the morphologic, cytogenetic, and molecular genetic findings. We found that use of cytogenetic-molecular genetic techniques aids in the distinction between myxoid-round cell liposarcoma and their simulators, chondroid lipoma, myxoid spindle cell-pleomorphic lipoma, cellular intramuscular myxoma, and myxofibrosarcoma. Poorly differentiated forms of round cell liposarcoma lacking morphologic evidence of lipogenesis can also be diagnosed using these techniques; however, the techniques do not aid in distinguishing low-grade myxoid from high-grade round cell liposarcomas. This study also shows that retroperitoneal liposarcomas with myxoid liposarcoma-like zones are part of the morphologic spectrum of well-differentiated-dedifferentiated liposarcoma rather than true myxoid liposarcomas. Perhaps most importantly, our results provide the first molecular genetic evidence that true mixed liposarcomas (mixed well-differentiated and myxoid liposarcoma) do indeed exist. They also unequivocally demonstrate the existence of small, round cell variants of pleomorphic liposarcoma that closely simulate myxoid-round cell liposarcoma.

Expression and mutation patterns of p53 in benign and malignant salivary gland tumors.

The expression and mutation patterns of p53 were studied in a series of 68 benign pleomorphic adenomas and 237 malignant salivary gland tumors. p53 overexpression (nuclear staining exceeding 10%) was detected in 20% of the malignant salivary gland tumors, with the highest prevalence observed in polymorphous low grade adenocarcinoma, squamous cell carcinoma, and carcinoma ex pleomorphic adenoma and the lowest in adenoid cystic carcinoma and acinic cell carcinoma. In contrast, none of the 68 benign pleomorphic adenomas had nuclear staining exceeding 10%. SSCP and nucleotide sequence analysis of exons 4 to 9 of p53 in 19 malignant tumors revealed 9 mutations in 7 tumors. Our findings indicate that p53 may be a useful marker to help discriminate between benign and malignant salivary gland tumors.

Chromosomal localization of three human genes encoding bone morphogenetic protein receptors.

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily that play a pivotal role in bone formation during embryogenesis and fracture repair. BMP signaling occurs via hetero-oligomeric serine/threonine kinase complexes of BMP type I (BMPR-IA or BMPR-IB) and type II receptors (BMPR-II). BMPR-IA and IB are closely related receptors, with sequence differences conserved between different species, suggesting that they serve distinct functions. Here we report the cDNA cloning of human BMPR1B and the chromosomal localization of all three BMPR genes. Using somatic cell hybrid and FISH analyses, the BMPR1A, BMPR1B, and BMPR2 genes were assigned to 10q23, 4q22-24, and 2q33-34, respectively. A processed BMPR1A pseudogene was mapped to 6q23.

Conserved mechanism of PLAG1 activation in salivary gland tumors with and without chromosome 8q12 abnormalities: identification of SII as a new fusion partner gene.

We have previously shown (K. Kas et al, Nat. Genet., 15: 170-174, 1997) that the developmentally regulated zinc finger gene pleomorphic adenoma gene 1 (PLAG1) is the target gene in 8q12 in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between PLAG1 and the constitutively expressed gene for beta-catenin (CTNNB1), leading to activation of PLAG1 expression and reduced expression of CTNNB1. Here we have studied the expression of PLAG1 by Northern blot analysis in 47 primary benign and malignant human tumors with or without cytogenetic abnormalities of 8q12. Overexpression of PLAG1 was found in 23 tumors (49%). Thirteen of 17 pleomorphic adenomas with a normal karyotype and 5 of 10 with 12q13-15 abnormalities overexpressed PLAG1, which demonstrates that PLAG1 activation is a frequent event in adenomas irrespective of karyotype. In contrast, PLAG1 was overexpressed in only 2 of 11 malignant salivary gland tumors analyzed, which suggests that, at least in salivary gland tumors, PLAG1 activation preferentially occurs in benign tumors. PLAG1 over-expression was also found in three of nine mesenchymal tumors, i.e., in two uterine leiomyomas and one leiomyosarcoma. RNase protection, rapid amplification of 5'-cDNA ends (5'-RACE), and reverse transcription-PCR analyses of five adenomas with a normal karyotype revealed fusion transcripts in three tumors. Nucleotide sequence analysis of these showed that they contained fusions between PLAG1 and CTNNB1 (one case) or PLAG1 and a novel fusion partner gene, i.e., the gene encoding the transcription elongation factor SII (two cases). The fusions occurred in the 5' noncoding region of PLAG1, leading to exchange of regulatory control elements and, as a consequence, activation of PLAG1 gene expression. Because all of the cases had grossly normal karyotypes, the rearrangements must result from cryptic rearrangements. The results suggest that in addition to chromosomal translocations and cryptic rearrangements, PLAG1 may also be activated by mutations or indirect mechanisms. Our findings establish a conserved mechanism of PLAG1 activation in salivary gland tumors with and without 8q12 aberrations, which indicates that such activation is a frequent event in these tumors.

Fluorescence in situ hybridization mapping of breakpoints in pleomorphic adenomas with 8q12-13 abnormalities identifies a subgroup of tumors without PLAG1 involvement.

Recently, we identified the PLAG1 gene as the target gene in pleomorphic adenomas with chromosome abnormalities involving 8q12. The majority of breakpoints were shown to reside within the 5' noncoding region of the gene. We now report three pleomorphic adenomas with breakpoints located distal to PLAG1 in band 8q13. These tumors had the following chromosome 8 abnormalities: ins(8;12)(q12-13;q14q15), t(8;12)(q13;q15), and t(6;8)(p21.3-22;q13). Fluorescence in situ hybridization mapping of the chromosome 8 breakpoints revealed a yeast artificial chromosome clone spanning the breakpoints in two tumors. In none of the cases was PLAG1 activated and/or disrupted. Three candidate genes, N8, HMGIC, and HMGIY, were analyzed for rearrangements and/or abnormal expression by using reverse transcriptase-polymerase chain reaction, rapid amplification of 3' cDNA ends, and Northern blot analyses.

Identification of NFIB as recurrent translocation partner gene of HMGIC in pleomorphic adenomas.

Approximately 12% of all pleomorphic adenomas of the salivary glands are characterized by chromosome aberrations involving the chromosome segment 12q13-15. Several chromosomes have been found as translocation partners of chromosome 12, and some of these recurrently. Recently, the HMGIC gene was identified as the target gene affected by the 12q13-15 aberrations. Here, we report the identification and characterization of a new translocation partner gene of HMGIC in pleomorphic adenomas. 3'-RACE analysis of a primary adenoma with an apparently normal karyotype revealed an HMGIC fusion transcript containing ectopic sequences derived from the human NFIB gene, previously mapped to chromosome band 9p24.1. The HMGIC NFIB fusion transcript was also confirmed by RT-PCR. Since the chromosome segment 9p12-24 is repeatedly involved as translocation partner of chromosome 12q13-15 in pleomorphic adenomas, we tested whether NFIB might be a recurrent partner of HMGIC. RT-PCR analysis of a second adenoma with an ins(9;12)(p23;q12q15) as the sole anomaly, revealed that also in this tumor an HMGIC/NFIB hybrid transcript was present. The reciprocal NFIB/HMGIC fusion transcript, however, could not be detected in any of these tumors. Nucleotide sequence analysis of the fusion transcripts indicated that the genetic aberration in both tumors resulted in the replacement of a carboxy-terminal segment of HMGIC by the last five amino acids of NFIB. In conclusion, our results reveal the recurrent involvement of the NFIB gene as translocation partner gene of HMGIC in pleomorphic adenomas.

A 2-Mb YAC contig and physical map covering the chromosome 8q12 breakpoint cluster region in pleomorphic adenomas of the salivary glands.

Pleomorphic adenomas are benign epithelial tumors originating from the major and minor salivary glands. Extensive cytogenetic studies have demonstrated that they frequently show chromosome abnormalities involving chromosome 8, with consistent breakpoints at 8q12. In previous studies, we have shown that these breakpoints are located in a 9-cM interval between MOS/D8S285 and D8S260. Here, we describe directional chromosome walking studies starting from D8S260 as well as D8S285. Using the CEPH and ICRF YAC libraries, these studies resulted in the construction of two nonoverlapping YAC contigs of about 2 and 5 Mb, respectively. Initial fluorescence in situ hybridization (FISH) analysis suggested that the majority of 8q12 breakpoints clustered within the 2-Mb contig, which was mapped to the centromeric part of chromosome band 8q12. This contig has at least double coverage and consists of 34 overlapping YAC clones. The localization of the YACs was confirmed by FISH analysis. On the basis of mapping data of landmarks with an average spacing of 65 kb as well as restriction enzyme analysis, a long-range physical map was established for the chromosome region spanned by the 2-Mb contig. The relative positions of various known genes and expressed sequence tags within this contig were also determined. Subsequent FISH analyses of pleomorphic adenomas using YACs as well as cosmids revealed that all but two of the 8q12 breakpoints in the primary tumors tested mapped within a 300-kb interval between the MOS proto-oncogene and STS EM156. The target gene affected by the chromosome aberrations mapping within this interval was recently shown to be the PLAG1 gene, which encodes a novel zinc finger protein.

Observations by chromosome banding, FISH and immunohistochemistry in an adenoid cystic carcinoma with del(17)(p13) as the sole deviation.

We report on an adenoid cystic-carcinoma (ACC) with a clonal deletion of 17p as the only karyotypic abnormality. Using different chromosome 17-derived probes we showed by FISH that the deletion encompassed the p53 tumour suppressor gene. Immunohistochemical analysis revealed overexpression of p53 protein in a subpopulation of cells, suggesting a mutation in the remaining p53 allele in these cells. Our findings provide novel information about possible progressional pathways in ACC, and demonstrate the value of combining conventional cytogenetic analysis with-molecular cytogenetic and immunohistochemical methods. This approach is particularly useful in cases with minor cytogenetic abnormalities at the border of visibility.

Amplification of multiple regions of chromosome 12, including 12q13-15, in chronic lymphocytic leukaemia.

Trisomy 12 is a frequent abnormality in chronic lymphocytic leukaemia (CLL). The biological importance of trisomy 12 is still poorly understood but it has been suggested that one or several genes are duplicated leading to malignant transformation. We present a case with amplification of 12q13-22 found in a clinically aggressive relapse of CLL. A smaller region, 12q13-15, was amplified most frequently and a YAC containing the MDM2 gene gave the highest number of signals. Additionally, in a subclone an amplicon containing at least 5 copies of a cosmid from 12q23-24 was detected. The case shows that small duplications of chromosome 12, not revealed by cytogenetic analysis, may occur in CLL. Also, it shows that cytogenetic clonal evolution can occur in CLL without morphological evidence of blast transformation. Our results indicate that the 12q13-15 region carries an important gene for CLL progression.

Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations.

Pleiomorphic adenoma of the salivary glands is a benign epithelial tumour occurring primarily in the major and minor salivary glands. It is by far the most common type of salivary gland tumour. Microscopically, pleiomorphic adenomas show a marked histological diversity with epithelial, myoepithelial and mesenchymal components in a variety of patterns. In addition to a cytogenetic subgroup with normal karyotypes, pleiomorphic adenomas are characterized by recurrent chromosome rearrangements, particularly reciprocal translocations, with breakpoints at 8q12, 3p21, and 12q13-15, in that order of frequency. The most common abnormality is a reciprocal t(3;8)(p21;q12). We here demonstrate that the t(3;8)(p21;q12) results in promoter swapping between PLAG1, a novel, developmentally regulated zinc finger gene at 8q12, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein interface functioning in the WG/WNT signalling pathway and specification of cell fate during embryogenesis. Fusions occur in the 5'-non-coding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. Due to the t(3;8)(p21;q12), PLAG1 is activated and expression levels of CTNNB1 are reduced. Activation of PLAG1 was also observed in an adenoma with a variant translocation t(8;15)(q12;q14). Our results indicate that PLAG1 activation due to promoter swapping is a crucial event in salivary gland tumourigenesis.

Identification of Smad2, a human Mad-related protein in the transforming growth factor beta signaling pathway.

Transforming growth factor-beta (TGF-beta) superfamily members are multifunctional cytokines that exert their effects via heteromeric complexes of two distinct serine and threonine kinase receptors. Drosophila mothers against decapentaplegic and related genes in Caenorhabditis elegans, Xenopus, and mammals were shown to function downstream in the intracellular signaling pathways of TGF-beta superfamily members. Here we report the cloning of a Mad-related protein, termed Sma- and Mad-related protein 2 (Smad2). TGF-beta stimulated the phosphorylation and nuclear translocation of Smad2 in nontransfected Mv1Lu cells. In addition, we demonstrated that TGF-beta and activin mediated phosphorylation of Smad2 after its overexpression with appropriate type I and II receptors in COS cells. Smad2 and Smad1 were found to be broadly expressed in human tissues. Smad2 is closely linked to DPC4 on chromosome 18q21.1, a region often deleted in human cancers. Cells that lack Smad2 may escape from TGF-beta-mediated growth inhibition and promote cancer progression.

Expression of reciprocal hybrid transcripts of HMGIC and FHIT in a pleomorphic adenoma of the parotid gland.

The developmentally regulated HMGIC gene, which encodes an architectural transcription factor, has recently been linked to the pathogenesis of benign solid tumors with chromosome aberrations involving 12q13-15. Among these tumors are pleomorphic adenoma of the salivary glands, lipoma, uterine leiomyoma, hamartomas of the breast and lung, fibroadenoma of the breast, angiomyxoma, and endometrial polyps. For most tumor types, however, the translocation partners are variable. At present, no translocation partner genes of HMGIC are known for pleomorphic adenomas. Here, we report that in a primary pleomorphic adenoma of the parotid gland, the FHIT gene, which spans the chromosome 3p14.2 fragile site FRA3B, and is frequently disrupted in tumors, acts as a fusion partner of HMGIC. In addition to normal HMGIC and FHIT transcripts, an HMGIC/FHIT hybrid transcript as well as its reciprocal counterpart, FHIT/HMGIC, were found to be expressed by reverse transcription-PCR. The results establish the concurrent disruption of two tumor-associated genes in a benign tumor.

Mapping of the 8q12 translocation breakpoint to a 40-kb region in a pleomorphic adenoma with an ins(8;3)(q12;p21.3p14.1).

The translocation t(3;8)(p21;q12) is the most common chromosome abnormality observed in pleomorphic adenomas of the salivary glands. In this paper we describe the physical mapping of the breakpoints in an adenoma with a variant t(3;8), viz., an ins(8;3)(q12;p21.3p14.1). Using sequence-tagged sites (STSs) corresponding to landmarks within a previously identified yeast artificial chromosome (YAC) spanning the breakpoint in adenomas with t(3;8), cosmids isolated from a chromosome 8-specific cosmid library. The 8q12 insertion breakpoint was mapped by FISH to a 300-kb region flanked by MOS and a new STS, CH129. A cosmid within this region was shown to span the breakpoint. To test whether the recently identified FHIT gene, which maps to 3p14.2, was disrupted by the 3p rearrangement, we also isolated an FHIT YAC and mapped this YAC by FISH distal to the most proximal 3p breakpoint. In addition, RT-PCR analysis revealed only a normal-sized FHIT transcript, suggesting that FHIT is not affected by the 3;8-rearrangement.

Identification of a yeast artificial chromosome spanning the 8q12 translocation breakpoint in pleomorphic adenomas with t(3;8)(p21;q12).

A subgroup of pleomorphic adenomas of the salivary glands is characterized by translocations involving chromosome 8, with consistent breakpoints at 8q12. As part of a positional cloning effort to isolate the gene(s) affected by these translocations we now report the mapping of the 8q12 breakpoint in two primary pleomorphic adenomas with the recurrent t(3;8)(p21;q12). Yeast artificial chromosome (YAC) clones corresponding to eight different loci in 8q11-12 were isolated and mapped by fluorescence in situ hybridization (FISH). The t(3;8) breakpoint was mapped within a 1 Mb region flanked by MOS proximally and by the genetic marker D8S166 distally. One YAC within this region was shown to span the t(3;8) breakpoint in two tumors. This YAC will provide an excellent tool for isolating the gene(s) at the breakpoint(s) in adenomas with t(3;8).

Regional assignment of the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) to 12q24-->qter by fluorescence in situ hybridization.

Using a panel of human-rodent somatic cell hybrids, we have previously mapped the gene (HPD, previously called PPD) encoding 4-hydroxyphenylpyruvate dioxygenase to the distal half of the long arm of human chromosome 12, region q14-->qter. To obtain a genomic probe useful for fluorescence in situ hybridization (FISH) analysis we screened a human leukocyte genomic library and isolated a 13.4-kb phage clone, which by restriction fragment and sequence analyses was shown to contain exons 1-10 of HPD and approximately 2-kb upstream sequences. We now report the subregional localization of HPD to 12q24-->qter based on two color FISH analysis employing this clone.

Fish analysis of supernumerary ring chromosome in dermatofibrosarcoma protuberans.

We describe the light- and electron microscopic, immunohistochemical, cell culture characteristics and cytogenetic findings of a case of dermatofibrosarcoma protuberans (DFSP). Cytogenetically, the lesion exhibited trisomy 8 and a supernumerary ring chromosome as the only clonal abnormalities found in about 35% of the cells analyzed. FISH-analysis of metaphase chromosomes revealed that the ring chromosome contained chromosome 17 sequences. Hybridization with a chromosome 17 centromere specific probe gave three signals in about 19% of the interphase nuclei suggesting that the ring also had a centromere derived from chromosome 17. These observations add to the evidence that supernumerary ring chromosomes, preferentially derived from chromosome 17, and trisomy 8 are non-random abnormalities in DFSP. Our findings also demonstrate the usefulness of FISH for identifying the origin of marker ring chromosomes.