Non-isotopic In Situ Hybridisation Research Articles

Demonstration of Kaposi's Sarcoma-Associated Herpes Virus Cyclin D Homolog in Cutaneous Kaposi's Sarcoma by Colorimetric In Situ Hybridization Using a Catalyzed Signal Amplification System

Kaposi's sarcoma-associated herpes virus (KSHV)/human herpes virus 8 (HHV8) DNA sequences have been demonstrated in Kaposi's sarcoma (KS), as well as in some acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (NHL) and in multicentric Castleman's disease. Although KSHV DNA generally is abundant in KSHV-associated lymphomas, few copies of the virus are present in KS, a property that confounds detection by in situ methods. Previous in situ studies, which identified KSHV in lesions of KS, relied on the use of polymerase chain reaction (PCR) to amplify target DNA sequences before in situ hybridization (ISH) for localization or used ISH with radioactively-labeled probes to obtain adequate levels of detection sensitivity. In this study, a novel nonisotopic nucleic acid ISH method using catalyzed signal amplification and colorimetric detection without PCR-dependent target amplification was used to identify KSHV-specific sequences. The level of sensitivity was increased further by using a probe that detects viral cyclin D homolog transcripts, which are expressed at significant levels during latent viral infection. Thirty cutaneous lesions of KS (25 AIDS-related and five classical European type) were evaluated. AIDS-related NHL and cell lines derived from patients with AIDS-related NHL, all of which were known to harbor KSHV by Southern blot analysis, were used as positive controls. NHL and benign cutaneous vascular lesions not associated with AIDS were used as negative controls. For each of the 30 KS lesions studied, hybridization signals were detected in most of the spindle cells surrounding the atypical slit-like vascular channels and also were detected in some endothelial cells in well-formed blood vessels in the perilesional dermis. Plaque and nodular lesions generally contained more labeled cells than did early patch lesions. All AIDS-related NHL and cell lines contained KSHV-specific sequences; however, the non-AIDS–related NHLs and benign vascular lesions were negative. These results confirm the presence of KSHV sequences in cutaneous KS and provide in situ evidence of infection by this virus in early patch-stage lesions. This study also defines the in situ expression of the KSHV cyclin D homolog viral oncogene in cutaneous KS. The use of this sensitive nonisotopic ISH method should allow detection of other KSHV-specific gene products, further defining the pathobiology of this virus.

Human Papillomavirus 16 and 18 Infection is Absent in Urinary Bladder Carcinomas

The role of human papillomavirus (HPV) in the pathogenesis of bladder neoplasia remains controversial. Studies to date have mainly utilized polymerase chain reaction and DNA blot techniques to detect HPV DNA. The detection rates have varied from 0 to 81%. One of the major limitations of these techniques is that they lack topographic information. We have used a highly sensitive in situ hybridization (ISH) technique which detects single copies of viral genome and provides topographic information for investigation of HPV infection in bladder carcinomas.Thirty-one samples of formalin-fixed paraffin-embedded bladder carcinomas (4 adenocarcinomas, 5 squamous cell and 22 transitional cell carcinomas) were examined using non-isotopic ISH with biotin-labelled DNA probes of HPV 16 and 18 subtypes. HPV-positive skin and anal carcinoma samples were used as controls.No positive signals for HPV 16 or 18 were detected in any of the bladder carcinoma samples. Given the sensitivity of the technique, the result indicates that HPV 16 and 18 are not implicated in the development of bladder neoplasia compared to certain other epithelial malignancies. The possibility that other subtypes of HPV contribute to bladder epithelial carcinogenesis remains to be clarified.

Human papillomavirus and mixed epithelial tumors of the endometrium

Strong epidemiological evidence links human papilloma viruses (HPV) with the development of cervical intraepithelial neoplasia (CIN) and invasive cancers of the uterine cervix. The localization of HPV DNA sequences high up in the female genital tract (in benign and malignant lesions) is not that uncommon, but its precise significance is uncertain. In particular, the detection of HPV DNA sequences by polymerase chain reaction (PCR) needs careful interpretation, because the source of the amplicon may emanate from tumor cells, direct contamination from the cervix, or possibly from extratumoral sites in the endometrium. We have previously reported the identification of koilocyte-like changes in the squamous epithelium of some endometrial adenoacanthomas. Adenoacanthomas (adenocarcinoma with squamous metaplasia) are mixed epithelial tumors arising in the endometrium composed of malignant glandular areas admixed with benign metaplastic squamous epithelium. The rarer adenosquamous carcinoma containing both malignant glandular and squamous areas is also described. The origin of benign/malignant squamous epithelial islands in endometrial tumors has been the subject of speculation, with some investigators considering an origin from metaplastic glandular endometrial cells. In this study, we examined 10 normal endometrial samples, 20 adenocarcinomas, 41 adenocarcinomas with squamous metaplasia, and two adenosquamous carcinomas, (including control cervical material where possible) for the presence of HPV DNA sequences using nonisotopic in situ hybridization (NISH), type-specific HPV PCR, general primer PCR (to detect sequenced and unsequenced HPVs), and PCR in situ hybridization (PCR-ISH). We did not identify HPV DNA sequences in normal endometrial tissue. In adenocarcinomas (endometrioid type), HPV was only identified in 2 of 20 cases by PCR, both of which were HPV 11 positive. We were unsuccessful in identifying HPV in endometrial carcinomas by NISH or by PCR-ISH, raising the possibility of contamination from the cervix in the two positive cases. In adenoacanthomas, a low-risk HPV type (HPV 6) was found in 19 of 41 cases. NISH signals were intranuclear in location in squamous regions of adenoacanthomas. Additional positive nuclei were uncovered using PCR-ISH, which increases the sensitivity of standard NISH detection. HPV DNA sequences were located in some malignant endometrial glandular epithelial cells, but this accounted for a minority of samples. HPV DNA sequences were not detected in extraepithelial sites. Mixed infection by two different HPV types was identified in two cases. Most cases showed similar HPV types in cervical and endometrial lesions, although discordant cases were uncovered. In adenosquamous carcinomas, one case showed mixed infection with HPV 6 and 33 by PCR. The apparent segregation of low-risk HPV type (HPV 6) with benign squamous metaplastic epithelium in adenocarcinoma with squamous metaplasia, and high-risk type (HPV 33) with malignant squamous epithelium in adenosquamous carcinoma, raises important questions in relation to the role of HPVs in mixed epithelial tumors of the endometrium and their interplay in the pathogenesis of squamous metaplasia at extracervical sites.

In situ hybridization detection of low copy nucleic acid sequences using catalyzed reporter deposition and its usefulness in clinical human papillomavirus typing.

In situ hybridization (ISH) detection of low copy DNA and RNA sequences using nonisotopic probes has been difficult in the past because of a lack of sensitivity. Several techniques, such as ISH with radioisotopic-labeled probes, in situ polymerase chain reaction, in situ reverse transcription polymerase chain reaction, self-sustained sequence replication, and chemiluminescence, have allowed increased sensitivity but have required specialized and often expensive equipment, lengthy protocols, and in the case of radioactive probes, there has been an associated increased health risk. Catalyzed reporter deposition (CARD) combined with ISH (CARD-ISH) increases the signal-generating potential of labeled hybridized probes and allows the detection of low copy sequences of nucleic acids in formalin-fixed, paraffin-embedded tissue sections. To determine the sensitivity of CARD-ISH to detect nucleic acids in routinely processed specimens, we analyzed the detection of HPV 16 and 18 infection in formalin-fixed, paraffin-embedded sections of cultured cell lines, including CaSki cells with 400-600 copies of HPV 16, HeLa 229 cells with 10-50 copies of HPV 18, and SiHa cells with 1-2 copies of HPV 16 using a conventional ISH method and by CARD-ISH. In addition, 20 cases of clinical specimens previously analyzed for HPV 6, 11, 16, 18, 31, 33, and 51 with the Enzo PathoGene kit (Enzo Diagnostics, Inc., Farmingdale, NY, U.S.A.) were reexamined with the CARD-ISH method. The CARD-ISH system detected one to two copies of HPV 16 in the SiHa cells whereas the conventional ISH method did not. Both methods detected HPV 16 and 18 in CaSki and HeLa 229 cells, respectively. Three clinical cases that were previously negative and two weakly positive cases of HPV infection were all strongly positive with the CARD-ISH system, a 25% increase in the detection of positive cases by CARD-ISH. We also showed for the first time that a cocktail of six biotinylated oligonucleotide probes was capable of detecting one to two copies of HPV 16 in SiHa cells. These results show that the CARD-ISH method increases the sensitivity of nonisotopic ISH to the level of detecting one to two copies of HPV DNA in formalin-fixed, paraffin-embedded tissue sections using biotinylated cDNA or oligonucleotide probes.

Comparison of in situ hybridisation and polymerase chain reaction in the diagnosis of B cell lymphoma.

AIM: To compare the sensitivity of the detection of immunoglobulin light chain messenger RNA (mRNA) restriction by in situ hybridisation (ISH) and clonal immunoglobulin heavy chain gene rearrangements by polymerase...

Cellular human and viral DNA detection by nonisotopic in situ hybridization.

Abstract In situ hybridization may be defined as the detection of nucleic acids in situ in cells, tissues, chromosomes, and isolated cell organelles. The technique was described in 1969 by two separate groups who demonstrated repetitive ribosomal sequences in nuclei of Xenopus oocytes using radiolabeled probes (1,2). Refinements in recombinant DNA technology and the development of nonisotopic probe labeling and detection (3) obviate the need for radiation protection and disposal facilities, and have converted nonisotopic in situ hybridization (NISH) from a purely research technique to one that can be used in routine laboratory testing.KeywordsCaSki CellReporter MoleculeCervical SmearGlycerol JellyFume CupboardThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of natural occurring Pneumocystis carinii pneumonia in pigs by histopathology, electron microscopy, in situ hybridization and PCR amplification.

Macroscopic, histologic, ultrastructural, microbiologic, in situ hybridization (ISH) and PCR detection results in three 8-week-old pigs naturally infected with Pneumocystis carinii (PC) are described. All animals had a nonsuppurative interstitial pneumonia and intra-alveolar Pneumocystis organisms with foamy eosinophilic and PAS positive appearance. Ultrastructurally, PC trophozoites and cysts were observed in pigs No. 2 and No. 3, with the former being much more numerous. PC organisms were located on the alveolar surface or within the alveolar septa. Trophozoites had numerous filopodia and were thick-walled. Cysts had no or few filopodia, were thick-walled and contained intracystic bodies. Using non-isotopic ISH on formalin-fixed, paraffin-embedded lung tissue sections, PC DNA from pigs No. 2 and No. 3 hybridized with a probe specific for PC ribosomal RNA (rRNA). Using primers specific for mitochondrial rRNA gene (pAZ102-E/pAZ102-H), and for the internal transcriber spacers of ribosomal gene of PC, PCR methods amplified a product in the lung of pigs No. 2 and No. 3 using either frozen or formalin-fixed and paraffin-embedded lung tissue. DNA from Pig No. 1 samples did not amplify with any primer. This is the first time that molecular biology techniques (in situ hybridization and PCR) have been applied to the study of porcine pneumocystosis.

Assignment of the Y4Receptor Gene (PPYR1) to Human Chromosome 10q11.2 and Mouse Chromosome 14

The human and mouse genes for the neuropeptide Y4receptor have been isolated, sequenced, and shown to contain no introns within the coding region of the gene. Nonisotopicin situhybridization and interspecific mouse backcross mapping have localized the genes to human chromosome 10q11.2 and mouse chromosome 14. Five nucleotide variants, which do not alter the protein sequence, have been identified within the coding region of the human receptor gene. The human Y4subtype is most closely related to the Y1-receptor subtype (42%), suggesting that it evolved from an ancestral Y1-like receptor via an RNA-mediated transpositional event.

Genotypic mapping of HPV and assessment of EBV prevalence in endocervical lesions.

AIMS: To examine the prevalence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) in low grade glandular intraepithelial lesions of the cervix, adenocarcinoma with high grade glandular intraepithelial lesions combined,...

Adenoid Basal Carcinoma of the Uterine Cervix

This study investigated the role of human papillomavirus (HPV) in adenoid basal carcinoma, a rare neoplasm of the uterine cervix. Nine archival paraffin-embedded tumors were analyzed with non-isotopic in situ hybridization (NISH) for HPV types 6, 11, 16, 18, 31, and 33 using digoxigenin-labelled probes. The polymerase chain reaction (PCR) was performed on each of the cases using E6 consensus primers to HPV. A total of 67% of adenoid basal carcinomas harbored the HPV genome with NISH, of which 3 were PCR-positive. Integrated HPV 16 DNA was demonstrated in 4 of the 6 NISH positive cases. Two cases showed integrated HPV 33. HPV DNA was not detected in the three remaining cases. These results show that the integrated high-risk HPV, in particular type 16, is associated with this uncommon cervical tumor.

Common association of HPV 2 with anogenital warts in prepubertal children.

Anogenital (AG) warts in 31 prepubertal children were HPV typed by nonisotopic in situ hybridization (NISH) using digoxigenin-labeled probes for human papilloma virus (HPV) types 1-5, 6, 11, 16, 18, 31, and 33. Mode of transmission was determined from historical, clinical, and laboratory data independent of HPV typing. HPV 2 was detected most commonly (13/31 warts) followed by HPV 6 (7/31), HPV 11 (5/31), and HPV 16 (1/31). Although not reaching statistical significance, our results suggested that a mucosal HPV type (6, 11, 16) in a child's AG warts implied transmission from mucosal warts and conversely cutaneous HPV 2 transmission from warts at a cutaneous site. HPV typing provided no helpful information regarding actual mode of transmission of AG warts in these children. The high prevalence of HPV 2 in children's AG warts and the low prevalence of sexual abuse (2 of 31 children) found in this study suggest innocent auto- or heteroinoculation from cutaneous warts may be a common means by which children acquire AG warts.

A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation.

AIM: To determine the optimum form of labelling and the most efficient reporter molecule for non-radioisotopic in situ hybridisation (ISH). METHODS: Nine deoxyoligonucleotides complementary to histone mRNA were synthesised and...

IN SITU DETECTION OF KAPOSI'S SARCOMA HERPESVIRUS / HUMAN HERPESVIRUS 8 (KSHV / HHV 8) AND EXPRESSION OF THE CYCLIN D HOMOLOG IN EARLY PATCH-STAGE LESIONS OF KAPOSI'S SARCOMA

Background: KSHV/HHV 8 has been demonstrated in most lesions of KS, in primary effusion (body-cavity-based) lymphomas (PEL), and in a significant percentage of multicentric Castleman's disease. KSHV/HHV 8 is present in each type of KS including AIDS-related (epidemic), classical (Mediterranean), endemic, and post-transplant-associated (iatrogenic). In these tissues, KSHV/HHV 8 is present in a very low copy number thereby confounding detection unless sensitive polymerase chain reaction (PCR)-based assays are used. In situ hybridization(ISH) after PCR amplification of KSHV/HHV 8-specific DNA sequences or using radioactively-labeled probes has demonstrated this virus in plaque-stage and nodular/tumor-stage lesions, however, the in situ demonstration of KSHV/HHV 8 in cutaneous early patch-stage lesions of KS has not been documented. Methods: Formalin-fixed, paraffin-embedded tissues from 12 early patch-stage lesions of KS were evaluated for KSHV/HHV 8-specific sequences using a nonisotopic ISH procedure based on colorimetric detection after catalyzed signal amplification. The KSHV/HHV 8 cyclin D homolog gene was selected as a target for ISH since this gene is expressed at significant levels during latent viral infection in KS thus allowing detection of both viral cyclin mRNA and episomal DNA. PEL cell lines known to harbor KSHV/HHV 8 by Southern blot analysis were used as a positive control for the ISH assay. NHLs which do not harbor KSHV/HHV 8 and benign cutaneous hemangiomas were used as negative controls. Results: KSHV/HHV 8 cyclin-specific sequences were detected in all 12 early patch-stage lesions of KS. Hybridization signals were present in the spindle cells forming the atypical vascular spaces and in endothelial cells in some well-formed blood vessels in the perilesional dermis. Most of the hybridization signals could be eliminated by RNAse digestion prior to ISH. PEL cell lines contained many hybridization signals, however NHLs not associated with KSHV/HHV 8 and benign hemangiomas were negative. Conclusions: The combination of this sensitive, non-isotopic ISH method with a probe for viral cyclin D allowed the detection of KSHV even in the earliest histologically-recognizable stages of KS providing further evidence for KSHV/HHV 8 in the pathogenesis of KS. Demonstration of other genes which are expressed during the early stages of tumor progression will further define the pathobiology of KSHV/HHV 8.

Human papillomavirus 16 and 18 infection is absent in urinary bladder carcinomas.

The role of human papillomavirus (HPV) in the pathogenesis of bladder neoplasia remains controversial. Studies to date have mainly utilized polymerase chain reaction and DNA blot techniques to detect HPV DNA. The detection rates have varied from 0 to 81%. One of the major limitations of these techniques is that they lack topographic information. We have used a highly sensitive in situ hybridization (ISH) technique which detects single copies of viral genome and provides topographic information for investigation of HPV infection in bladder carcinomas. Thirty-one samples of formalin-fixed paraffin-embedded bladder carcinomas (4 adenocarcinomas, 5 squamous cell and 22 transitional cell carcinomas) were examined using non-isotopic ISH with biotin-labelled DNA probes of HPV 16 and 18 subtypes. HPV-positive skin and anal carcinoma samples were used as controls. No positive signals for HPV 16 or 18 were detected in any of the bladder carcinoma samples. Given the sensitivity of the technique, the result indicates that HPV 16 and 18 are not implicated in the development of bladder neoplasia compared to certain other epithelial malignancies. The possibility that other subtypes of HPV contribute to bladder epithelial carcinogenesis remains to be clarified.

Human papillomavirus DNA in squamous cell carcinoma of the lung.

To compare the incidence of squamous cell carcinoma (SCC) of the lung in Okinawa with that in Niigata on the mainland. All patients presenting with SCC of the lung in Okinawa and Niigata in 1993 were included in the study. Diagnoses were confirmed by conventional histological examination of paraffin wax sections. Human papillomavirus (HPV) was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for the E6 and E7 regions of the HPV genome. PCR products were analysed by Southern and dot blotting. The incidence of well differentiated SCC of the lung was high in patients from Okinawa compared with moderately and poorly differentiated types, and compared with the incidence of SCC in patients from Niigata. This is despite similar patterns of age, sex (predominatly male), and smoking habit. More patients from Okinawa, however, were positive for HPV DNA by PCR (79%) and NISH (53%). Many patients haboured HPV types 6, 16, and 18. Only 30% of patients from Niigata were positive for HPV DNA by PCR and 20% by NISH. These patients all harboured one HPV type only. Surprisingly large numbers of patients from Okinawa were positive for HPV DNA. The detection of HPV DNA was strongly associated with well differentiated SCC. This was particularly true for HPV types 6 and 16. There was no correlation between either smoking and detection of HPV DNA, or smoking and histological differentiation.

Detection of integrated high risk human papillomavirus in adenoid cystic carcinoma of the uterine cervix.

To investigate the role of human papillomavirus (HPV) in adenoid cystic carcinoma of the uterine cervix. Eleven archival, paraffin wax embedded specimens were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, and 33 using digoxigenin labelled probes. The polymerase chain reaction (PCR) was carried out on each of the cases using consensus primers to HPV. A total of eight adenoid cystic carcinomas harboured the HPV genome by NISH, of which five were PCR positive. Integrated HPV 16 DNA was demonstrated in seven of the eight NISH positive cases. One adenoid cystic carcinoma showed integrated HPV 31. HPV DNA was not detected in the three remaining cases. Integrated high risk HPV genome, in particular type 16, is associated with this uncommon type of primary cervical cancer.

LABELLING PATTERN OBTAINED BY NON-ISOTOPICIN SITU HYBRIDIZATION AS A PROGNOSTIC FACTOR IN HPV-ASSOCIATED LESIONS

In the study of infection of the lower female genital tract caused by human papillomavirus (HPV), one of the main concerns is the search for prognostic factors to predict the evolution of premalignant low- and high-grade squamous intraepithelial lesions. This study has evaluated the prognostic usefulness of the patterns of positive reaction obtained by non-isotopic in situ hybridization (NISH), referred to as diffuse, punctate, or mixed 'labelling patterns'. The study examined 141 vulvar and uterine cervical biopsy specimens that were positive for HPV by a NISH screening technique and that had the following histological diagnoses: low-grade squamous intraepithelial lesion (LSIL; n = 87); high-grade squamous intraepithelial lesion (HSIL, n = 40); and squamous cell carcinoma (SCC n = 14). Typing of all the specimens was carried out by NISH with DNA probes specific for HPV types 6/11 (low risk), 16/18 (high risk), and 31/33/51 (intermediate risk), and the labelling pattern observed in each specimen was recorded. Statistical analysis of the results showed that there was a significant difference in the distribution of labelling patterns, both by lesion diagnosis (P < or = 0.004) and by infecting viral type (P < or = 10(-6)). Lesions with a punctate or mixed pattern are considered more likely to undergo malignant evolution and consequently have a worse prognosis than lesions with a diffuse pattern.

Molecular Cloning and Assignment of FAK2, a Novel Human Focal Adhesion Kinase, to 8p11.2–p22 by Nonisotopicin SituHybridization

This report describes the localization of a novel human focal adhesion kinase, FAK2, to human chromosome 8p11.2-p22 using in situ hybridization. The focal adhesion kinases are a subfamily of protein tyrosine kinases and aid in the transduction of ligands. 6 refs., 2 figs.

Evaluation of Non-Isotopic in situ Hybridization for mRNA in Reactive and Neoplastic Lymphoid Cells

An evaluation of nonisotopic in situ hybridization (NISH) for mRNA in archival lymphoid tissue was carried out and an analysis of factors affecting the final outcome was performed. A modification of the in situ reverse transcription method for RNA preservation assessment has been used and described. We have shown that, for frozen samples mRNA detection is optimal within 3 months of the biopsy being taken, while preservation declines after 1 year of storage.

Increased dimeric IgA producing B cells in the bone marrow in IgA nephropathy determined by in situ hybridisation for J chain mRNA.

To investigate the possible role of the systemic IgA immune system in the pathogenesis of IgA nephropathy J chain mRNA expression in the IgA cells of the bone marrow was studied. Bone marrow trephine biopsy specimens from seven patients with IgA nephropathy and seven matched controls were examined by (1) non-isotopic in situ hybridisation (ISH) and (2) combined immunofluorescence and non-isotopic ISH to identify the plasma cell type. Serum polymeric IgA was also determined using standard high pressure liquid chromatography and sandwich enzyme linked immunosorbent assay. Non-isotopic ISH revealed a similar number of J chain mRNA positive cells/unit length in biopsy specimens from patients (16.5 +/- 2.7 cells/mm) and controls (17.7 +/- 2.4 cells/mm). Combined immunofluorescence and ISH revealed a greater proportion of J chain mRNA positive IgA cells in patients (7.6 +/- 1.45%) compared with controls (3 +/- 0.8%). Serum polymeric IgA was similar in both patients (91 +/- 22 mg/l) and controls (77 +/- 24 mg/l). These data suggest that excess production of dimeric IgA occurs in the bone marrow in IgA nephropathy.