Loop mediated isothermal amplification (LAMP) for the detection and subtyping of human papillomaviruses (HPV) in oropharyngeal squamous cell carcinoma (OPSCC).

BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is a growing problem that presents a significant challenge to Otolaryngologist-Head and Neck Surgeons. Knowledge of HPV status yields critical prognostic information, with potential for treatment selection based on tumour HPV status. The current gold standard of diagnosis, PCR, is expensive, demanding and time consuming. Alternatives such as p16 immunohistochemistry are subjective and potentially inaccurate. Loop-mediated isothermal amplification (LAMP) is a rapid, robust and inexpensive molecular diagnostic technique. OBJECTIVES: Our aim was to verify LAMP as a potential bedside diagnostic assay for subtyping of HPV in OPSCC. STUDY DESIGN: DNA from 72 formalin-fixed paraffin embedded (FFPE) OPSCC patient samples was tested. PCR and LAMP were then performed to specifically identify HPV 16, 18, 31, 33 and 35. RESULTS AND CONCLUSIONS: For these high-risk subtypes, LAMP had an overall sensitivity of 99.4% and specificity of 93.2% relative to PCR. LAMP is a promising technology that can accurately diagnose high-risk HPV infection.

Lymphocytic vasculitis in X-linked lymphoproliferative disease.

Systemic vasculitis is an uncommon manifestation of X-linked lymphoproliferative disease (XLP), a disorder in which there is a selective immune deficiency to Epstein-Barr virus (EBV). The molecular basis for XLP has recently been ascribed to mutations within SLAM-associated protein (SAP), an SH2 domain-containing protein expressed primarily in T cells. The authors describe a patient who died as a result of chronic systemic vasculitis and fulfilled clinical criteria for the diagnosis of XLP. Sequencing of this patient's SAP gene uncovered a novel point mutation affecting the SH2 domain. The patient presented with virus-associated hemophagocytic syndrome (VAHS) and later had chorioretinitis, bronchiectasis, and hypogammaglobulinemia develop. He further developed mononeuritis and fatal respiratory failure. Evidence of widespread small and medium vessel vasculitis was noted at autopsy with involvement of retinal, cerebral, and coronary arteries as well as the segmental vessels of the kidneys, testes, and pancreas. Immunohistochemical analysis using antibodies to CD20, CD45RO, and CD8 revealed that the vessel wall infiltrates consisted primarily of CD8(+) T cells, implying a cytotoxic T-lymphocyte response to antigen. EBV DNA was detected by polymerase chain reaction (PCR) in arterial wall tissue microdissected from infiltrated vessels further suggesting that the CD8(+) T cells were targeting EBV antigens within the endothelium. The authors propose that functional inactivation of the SAP protein can impair the immunologic response to EBV, resulting in systemic vasculitis.

Decreased expression of the INK4 family of cyclin-dependent kinase inhibitors in Wilms tumor.

Cyclin-dependent kinase (CDK) inhibitors represented by the INK4 family (including p16(INK4a, CDKN2A), p15(INK4b, CDKN2B), p18(INK4c, CDKN2C), and p19(INK4d, CDKN2D)) are regulators of the cell cycle shown to be aberrant in many types of human cancer. We tested the hypothesis that these CDK inhibitors are a target for altered gene expression in Wilms tumor. Using RT-PCR, gene expression of the INK4 family was found to be decreased in 9 of 38 Wilms tumor samples obtained from the National Wilms Tumor Study Group (NWTSG) tissue bank. All the affected tumor samples were of favorable histology. Methylation-specific PCR revealed that methylation in the p16 promoter region may be responsible for altered expression. The incidence of loss of p16 expression may increase with increasing tumor stage, i.e., 1/10 (10%) with stage I/II FH Wilms tumor, 2/10 (20%) with stage III FH Wilms tumor, and 4/10 (40%) with stage IV FH Wilms tumor. Thus, determining the expression status of the INK4 family may have potential prognostic value in the management of Wilms tumor.

Laser capture microdissection-guided fluorescence in situ hybridization and flow cytometric cell cycle analysis of purified nuclei from paraffin sections.

Laser capture microdissection (LCM) has recently been identified as a quick, simple, and effective method by which microdissection of complex tissue specimens for molecular analysis can be routinely performed. Assessment of gene copy number by fluorescence in situ hybridization (FISH) is useful for the analysis of molecular genetic alterations in cancer. Unfortunately, the application of FISH to paraffin sections of tumor specimens is fraught with technical difficulty and potential artifacts. Our results demonstrate that LCM-microdissected nuclei are suitable for FISH gene copy analysis. Amplification of genes in cancer specimens can be detected as easily in LCM-prepared nuclei as in fresh nuclei from cancer tissue specimens. Furthermore, contamination of tumor specimens by normal cells can make interpretation of flow cytometric cell cycle analysis difficult. Our results show that LCM-microdissected nuclei can also be used for flow cytometric cell cycle and ploidy analysis. LCM/FISH offers the advantages of multicolor FISH in a morphologically defined cell population, without the technical problems of FISH performed on paraffin sections. This technique should further simplify the methodology required to perform copy number analysis of tumor suppressor or protooncogenes in archived cancer specimens. The use of LCM specimens will also improve the specificity and simplify the interpretation of flow cytometric cell cycle and ploidy analysis of breast cancer specimens.

Chromosomal localization and genomic organization of the human retinal rod Na-Ca+K exchanger.

The retinal rod Na-Ca+K exchanger is a unique calcium extrusion protein found only in the outer segments of retinal rod photoreceptors. Rod Na-Ca+K exchanger cDNA (NCKX1) has been cloned from bovine and human retinas. Here, we have used fluorescent in situ hybridization and radiation hybrid mapping to localize the human NCKX1 gene to chromosome 15q22. We have determined the genomic organization of human rod NCKX1 and found one intron in the 5' untranslated region and eight introns within the coding region.

A novel testis-specific gene, SPAG4, whose product interacts specifically with outer dense fiber protein ODF27, maps to human chromosome 20q11.2.

The product of the spermatid-specific rat SPAG4 gene interacts specifically with the major 27-kDa outer dense fiber protein ODF27 in elongating spermatids. In this study we isolated the human homolog of the spermatid-specific rat SPAG4 gene and mapped it by fluorescence in situ hybridization to chromosome 20q11.2.

Human outer dense fiber gene, ODF2, localizes to chromosome 9q34.

We have isolated the human homolog of the rat Odf2 gene. In rat, Odf2, the 84-kDa major outer dense fiber protein, interacts strongly and specifically with Odf1, the 27-kDa major outer dense fiber protein. The interaction is mediated by leucine zippers during ODF assembly along the sperm axoneme. We compared homology and genomic structure to rat and mouse Odf2 genes. Using fluorescence in situ hybridization, we mapped the human Odf2 gene (ODF2) to chromosome 9q34.

The human thrombin receptor gene and the 5q-syndrome.

The human thrombin receptor gene has been localized to band q13 of chromosome 5, a site that is at or contiguous to the common proximal breakpoint found in the majority of patients with interstitial deletions involving 5q (5q- syndrome; refractory anemia with dysmegakaryocytopoiesis). Recent evidence suggests that the thrombin receptor may represent the prototype of an emerging family of proteolytically activated receptors that may be clustered within this region of the human genome. The phenotypic heterogeneity evident in patients with the 5q- syndrome may be explained by two (or more) distinct molecular defects-one associated with megakaryocytic dyspoiesis and the other dysregulated myeloid growth potentially related to development of leukemogenesis. Because the thrombin receptor is known to mediate proliferative effects on diverse cells including vascular smooth muscle cells, endothelial cells and megakaryocytes, we have studied the role of this receptor in the pathogenesis of this syndrome using fluorescent in situ hybridization (FISH) analysis. Dual-label FISH using a q12-specific genomic fragment and the TR gene was completed using interphase and metaphase analysis from seven patients with a del(5)(q13q33). These data unequivocally demonstrate that the thrombin receptor gene is located centromeric to the common proximal breakpoint, and is grossly present in the seven patients containing this specific interstitial deletion. Additionally, one patient demonstrated a small proximal rearrangement, most likely representing a paracentric inversion, which has not previously been described within the intact region centromeric to the breakpoint. The biological properties of proteolytically activated receptors are reviewed in more detail, with a focus on the molecular genetics of the thrombin receptor and its potential role in megakaryoctyopoiesis.

Mutation analysis of the transforming growth factor beta type II receptor in sporadic human cancers of the pancreas, liver, and breast.

The transforming growth factor beta (TGF beta) binds the type II TGF beta growth factor receptor (TGF beta RII) to inhibit growth of most epithelial tissues. Most human colon cancers with microsatellite instability have frameshift mutations in two microsatellites within the TGF beta RII coding region; such mutations truncate the receptor to produce resistance to TGF beta. To investigate this pathway in other tissues, we surveyed sporadic human cancers of the pancreas, liver and breast to determine the frequency of microsatellite mutations in the TGF beta RII. We amplified genomic DNA segments containing two microsatellites plus 72% of domain XI of the serine-threonine kinase region. SSCP analysis showed no evidence of mutation in 32 sporadic cancers (12 pancreas, 10 liver, and 10 breast). We conclude that microsatellite mutations in TGF beta RII are uncommon in sporadic tumors of the pancreas, liver and breast.

Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfected p16INK4.

The function of p16INK4 as a putative tumor suppressor gene was examined by investigating its ability to inhibit the growth of cancer cell lines in vitro and tumor formation in vivo. A p16INK4 cDNA expression vector was transfected into five human cancer cell lines that varied in their p16INK4 and retinoblastoma (Rb) status. Suppression of colony-forming efficiency was seen in four cell lines. Of two cell lines wild type for p16INK4 but null for Rb protein expression, one (Hep 3B) showed inhibition of colony formation, whereas the other (Saos-2) did not. This observation may demonstrate involvement of p16INK4 independent of Rb. The transfected p16INK4 gene was frequently selected against and lost during continued growth in vitro. When compared to the colon carcinoma cell line (DLD-1),p16INK4-transfected DLD-1 clone 1 cells were less tumorigenic in athymic nude mice. Tumors arising from p16INK4-transfected DLD-1 clones, which were growth suppressed in vitro, either lost the integrated exogenous p16INK4 or expressed reduced amounts of p16INK4 protein. Therefore, p16INK4 was also selected against during tumor formation in vivo. These data are consistent with the hypothesis that p16INK4 is a tumor suppressor gene.

Intragenic mutations of the p16(INK4), p15(INK4B) and p18 genes in primary non-small-cell lung cancers.

The p15(INK4B), p16(INK4) and p18 genes are members of the gene family coding for inhibitors of cyclin-dependent kinases 4 and 6. p15(INK4B) and p16(INK4) are located at 9p21, a chromosomal region frequently deleted in many human neoplasms. To examine the role of these 3 genes in lung carcinogenesis, somatic mutations within the genes were analyzed by single-strand conformation polymorphism and DNA sequencing in 71 non-small-cell lung cancer (NSCLC) samples. Six somatic mutations in the p16(INK4) gene and 3 cases with a polymorphic allele were observed. Loss of heterozygosity in the p18 gene was found in 1 sample. We did not find any intragenic mutations in the p15(INK4B) or p18 genes. We conclude that p16(INK4) mutations play a role in the formation of some NSCLCs, whereas the involvement of p15(INK4B) and p18 is uncommon.

The thrombin receptor gene is centromeric to the common proximal breakpoint in patients with the 5q- syndrome: identification of a previously unrecognized chromosome 5 inversion.

The human thrombin receptor (TR) gene has previously been localized to band q13 of chromosome 5, a site that is at or contiguous with the common proximal breakpoint site identified in the majority of patients with the 5q- syndrome (dysmegakaryocytopoiesis and refractory anaemia). Since thrombin has putative effects on the growth and differentiation of megakaryocytes, we hypothesized that the phenotypic abnormalities in megakaryocytopoiesis observed in the 5q- syndrome may be partially explained by involvement of the TR gene in the interstitial deletion. We have utilized molecular and fluorescence in situ hybridization (FISH) analysis to study potential cytogenetic abnormalities of the thrombin receptor gene in patients demonstrating this abnormality. Dual-label FISH with a q12-specific genomic fragment and the TR gene was completed using interphase and metaphase cells from seven patients with a del(5)(q13q33). Our data demonstrate that the TR gene is located on the centromeric side of the common proximal breakpoint, and is grossly present in all patients with the affected 5q- chromosome. In addition, one of the seven patients demonstrated a small proximal rearrangement, most likely representing a paracentric inversion, which was not apparent by conventional cytogenetic techniques. The significance of these alterations is discussed.

The use of archival frozen tumor tissue imprint specimens for fluorescence in situ hybridization.

Fluorescence in situ hybridization for the detection of gene amplification or deletion has great promise as a method of providing diagnostic or prognostic information from tumor specimens. Fine needle aspiration samples or tumor tissue imprints are much easier to use and provide better results than paraffin sections, however, these materials are rarely archived and it may take many months to accumulate enough specimens for a study. Archival breast carcinoma tumor tissue samples, some stored frozen for several years, were used to prepare tumor tissue touch imprints on glass microscope slides. The imprints were subjected to fluorescence in situ hybridization analyses utilizing a biotin-dUTP-labeled genomic DNA probe for the cyclin D1 gene (CCND1/PRAD1) and a digoxygenin-labeled chromosome 11 alpha-satellite probe to control for chromosomal copy number. Amplification of CCND1 was easily detectable in frozen tissue imprints. The results indicate that both cytologic morphology and hybridization capacity are well preserved in archived frozen tissue and easily permit its use for in situ hybridization experiments. The ability to use stored frozen tumor tissue for molecular morphologic analysis should allow more rapid assessment of fluorescence in situ hybridization as a potential adjunct for tumor analysis by the surgical pathologist.

Chromosomal mapping of the human genes CKS1 to 8q21 and CKS2 to 9q22.

The human cdk2/cyclin A kinase complex is a key regulator of the events of S phase. This complex contains several proteins involved in regulating its catalytic activity, including one or more of the CKS proteins, which have recently been shown to inhibit the activation of the cdk2 kinase. To investigate whether the CKS genes may be altered in human neoplasia, we mapped the chromosome locations of CKS1 and CKS2 by fluorescence in situ hybridization (FISH). CKS1 was localized to 8q21, a locus that is seldom grossly altered in cancer. The localization of CKS2 to 9q22 places it very near to a putative tumour suppressor locus suggested to be responsible for susceptibility to the Basal Cell Nervus Syndrome (BCNS or Gorlin's syndrome) familial cancer disorder. Six fibroblast cell lines isolated from patients with BCNS were demonstrated by FISH to have both copies of CKS2 present. Partial sequencing of a genomic clone of CKS2 revealed that the open reading frame lies over three exons. Examination of the six cell lines by SSCP and PCR-based sequencing of the parts of the three exons coding for the full length protein demonstrated no consistent divergence from the reported cDNA sequence in any exon. It is unlikely that CKS2 is the BCNS tumour suppressor gene.

Chromosomal mapping of the genes for the human CDK2/cyclin A-associated proteins p19 (SKP1A and SKP1B) and p45 (SKP2).

Many gene products associated with the cyclin-dependant kinases (CDKs) have been shown to regulate the active kinase complex during the transition points of the cell cycle. Some of these proteins have been implicated in human neoplasia, acting as either oncoproteins or tumour suppressors. The CDK2/cyclin A kinase complex can complex with several proteins, including p21, and PCNA or p45, p19, and p9. It was previously shown that at least two of these proteins, p19 and p45, are abnormally regulated in transformed cell lines. We describe here the mapping by fluorescence in situ hybridization of the gene for the CDK2/cyclin A-associated protein p45 (SKP2) to 5p13 and the p19-related genes p19A (SKP1A) and p19B (SKP1B) to 7q11.2 and 12p12, respectively. All three of these loci are associated with karyotypic alterations, known amplifications, or suspected tumor suppressor genes.

Mutations in the p16INK4/MTS1/CDKN2, p15INK4B/MTS2, and p18 genes in primary and metastatic lung cancer.

We examined the genomic status of cyclin-dependent kinase-4 and -6 inhibitors, p16INK4,p15INK4B, and p18, in 40 primary lung cancers and 31 metastatic lung cancers. Alterations of the p16INK4 gene were detected in 6 (2 insertions and 4 homozygous deletions) of 22 metastatic non-small cell lung cancers (NSCLCs; 27%), but none were detected in 25 primary NSCLCs, 15 primary small cell lung cancers (SCLCs), or 9 metastatic SCLCs, indicating that mutation in the p16INK4 gene is a late event in NSCLC carcinogenesis. Although three intragenic mutations of the p15INK4B gene were detected in 25 primary NSCLCs (12%) and five homozygous deletions of the p15INK4B gene were detected in 22 NSCLCs (23%), no genetic alterations of the p15INK4B gene were found in primary and metastatic SCLCs. The p18 gene was wild type in these 71 lung cancers, except 1 metastatic NSCLC which showed loss of heterozygosity. We also examined alterations of these three genes and expression of p16INK4 in 21 human lung cancer cell lines. Alterations of the p16INK4 and p15INK4B genes were detected in 71% of the NSCLC cell lines (n = 14) and 50% of the NSCLC cell lines (n = 14), respectively, but there were none in the 7 SCLC cell lines studied. No p18 mutations were detected in these 21 cell lines. These results indicate that both p16INK4 and p15INK4B gene mutations are associated with tumor progression of a subset of NSCLC, but not of SCLC, and that p15INK4B mutations might also be an early event in the molecular pathogenesis of a subset of NSCLC.

Chromosomal mapping of the genes for the human cell cycle proteins cyclin C (CCNC), cyclin E (CCNE), p21 (CDKN1) and KAP (CDKN3).

Many gene products associated with the cdk cell cycle kinases are thought to regulate the active kinase complex and thus regulate the transition points of the cell cycle. Genes encoding these proteins may potentially function as oncogenes or tumor suppressor genes. We describe the chromosomal mapping by FISH of the genes for several cdk-associated proteins including human CCNC (cyclin C) to 6q21, CCNE (cyclin E) to 19q12-->q13, CDKN1 (p21) to 6p21.2 and CDKN3 (KAP to 14q22). Some of these sites are near chromosomal translocations or LOH sites common to a variety of human tumors. The potential role for each of these genes in neoplasia is discussed.

Mutations and altered expression of p16INK4 in human cancer.

Cell cycle arrest at the G1 checkpoint allows completion of critical macromolecular events prior to S phase. Regulators of the G1 checkpoint include an inhibitor of cyclin-dependent kinase, p16INK4; two tumor-suppressor proteins, p53 and RB (the product of the retinoblastoma-susceptibility gene); and cyclin D1. Neither p16INK4 nor the RB protein was detected in 28 of 29 tumor cell lines from human lung, esophagus, liver, colon, and pancreas. The presence of p16INK4 protein is inversely correlated with detectable RB or cyclin D1 proteins and is not correlated with p53 mutations. Homozygous deletions of p16INK4 were detected in several cell lines, but intragenic mutations of this gene were unusual in either cell lines or primary tumors. Transfection of the p16INK4 cDNA expression vector into carcinoma cells inhibits their colony-forming efficiency and the p16INK4 expressing cells are selected against with continued passage in vitro. These results are consistent with the hypothesis that p16INK4 is a tumor-suppressor protein and that genetic and epigenetic abnormalities in genes controlling the G1 checkpoint can lead to both escape from senescence and cancer formation.