Correction of uneven illumination (vignetting) in digital microscopy images.

BACKGROUND: Many digital microscopy images suffer from poor illumination at the peripheries (vignetting), often attributable to factors related to the light path between the camera and the microscope. A commonly used method for correcting this has been to use the illumination of an empty field as a correction filter (white shading correction). AIMS: To develop an alternative shading correction method that does not require this additional image. METHODS: / RESULTS: This report outlines an alternative shading correction method that is based upon the intrinsic properties of the image, which are revealed through Gaussian smoothing. The technique can be implemented within Adobe Photoshop or other graphics editing software and works well with both haematoxylin and eosin and immunohistochemical images. CONCLUSIONS: This technique provides an effective means of optimising digital microscopy image appearances for printing, image analysis, or telepathology.

Robotic telepathology: efficacy and usability in pulmonary pathology.

Robotic telepathology is well established in the USA as a method of case referral, but is less frequently used in the UK. Using cases covering a broad spectrum of pulmonary pathology, this study assessed its application in primary diagnosis and its functionality in terms of accuracy of diagnosis and time per case, for both small biopsies and open lung biopsies/resections. Forty cases (20 bronchoscopic and 20 surgical lung biopsy/resection specimens) were reviewed in blinded fashion by a single pathologist using robotic telepathology. Connection between the John Radcliffe and Royal Brompton Hospitals was via 10 Mb/s LAN to the Internet (supported by the Joint Academic Network). The cases were then randomized and reviewed a second time with conventional light microscopy. Diagnosis, initial time to reach diagnosis, and overall time per case were recorded. In two bronchoscopic biopsy cases, there were clinically significant differences between telepathology and conventional light microscopy, one probably attributable to user inexperience and the other to either speed of image capture or digital image quality. In the surgical lung biopsies and resections, there was one variation of opinion: with telepathology a case was considered to be probably mesothelioma, whereas this was thought less likely on light microscopy. In both instances, immunohistochemistry was requested prior to clinical management. Telepathology was 14 times slower than conventional light microscopy when examining bronchoscopic biopsies. The average time spent per slide was 7 min 21 s, compared with 32 s per slide with conventional light microscopy. When assessing open lung biopsies and resections, telepathology was five times slower, at 6 min 13 s compared with 1 min 10 s with conventional light microscopy. This study showed that robotic telepathology is accurate for primary diagnosis in pulmonary histopathology, but modifications in both laboratory protocols and telepathology hardware are needed to decrease the time difference between telepathology and conventional light microscopy, for telepathology to be usable within the framework of a busy referral practice.

Automated complete slide digitization: a medium for simultaneous viewing by multiple pathologists.

Developments in telepathology robotic systems have evolved the concept of a 'virtual microscope' handling 'digital slides'. Slide digitization is a method of archiving salient histological features in numerical (digital) form. The value and potential of this have begun to be recognized by several international centres. Automated complete slide digitization has application at all levels of clinical practice and will benefit undergraduate, postgraduate, and continuing education. Unfortunately, as the volume of potential data on a histological slide represents a significant problem in terms of digitization, storage, and subsequent manipulation, the reality of virtual microscopy to date has comprised limited views at inadequate resolution. This paper outlines a system refined in the authors' laboratory, which employs a combination of enhanced hardware, image capture, and processing techniques designed for telepathology. The system is able to scan an entire slide at high magnification and create a library of such slides that may exist on an internet server or be distributed on removable media (such as CD-ROM or DVD). A digital slide allows image data manipulation at a level not possible with conventional light microscopy. Combinations of multiple users, multiple magnifications, annotations, and addition of ancillary textual and visual data are now possible. This demonstrates that with increased sophistication, the applications of telepathology technology need not be confined to second opinion, but can be extended on a wider front.

Detailed deletion mapping at chromosome 11q23 in colorectal carcinoma.

Loss of heterozygosity (LOH) is frequent at the chromosomal region 11q22-q23 in several types of tumours of diverse cell origin. Previous investigations of LOH at this chromosomal region in colorectal carcinoma have been contradictory in their findings, and have only included between 1-4 loci. In order to define any regions of LOH on 11q23, we investigated 16 loci between D11S940 and D11S934 on the long arm of chromosome 11 using microsatellite analysis. Of 57 colorectal carcinomas specimens, 36 (63.2%) demonstrated LOH at one or more marker, with the highest frequencies of LOH at D11S1340 (41.0%), located between 105.13-111.97 Mb from the centromere, and D11S924 (37.1%) and D11S4107 (40.5%), both located approximately 113 Mb from the centromere. No statistically significant associations between LOH and age-of-presentation or Dukes' stage were found. LOH was observed in colorectal tumours of all Dukes' stages, including Dukes' stages A and B, suggesting that the inactivation of a tumour suppressor gene(s) on 11q23 occurs in the early stages of colorectal carcinoma. These results confirm the presence of putative tumour suppressor gene(s) at chromosome 11q23, involved in the carcinogenesis of colorectal carcinoma, and will facilitate future identification of candidate genes.

Clinical trial of telepathology as an alternative modality in breast histopathology quality assurance.

Telepathology is a potential alternative to conventional histopathology. A clinical trial using a robotic telepathology system was conducted to assess the clinical and technical utility and effectiveness of telepathology in the U.K. breast screening pathology quality assurance program. Eighty-seven cases of breast disease were chosen at random from a series of 192 cases from the U.K. Breast Screening Pathology National Quality Assurance Scheme (NEQAS) collection. There were 20 benign, 23 carcinoma in situ (CIS), and 44 invasive malignant cases. The diagnostic accuracy of telepathology (TP) compared with conventional light microscopic (LM) diagnosis was 98.8%; this included a single case deferred for LM examination. The figure was similar when compared with expert consensus diagnosis (CD). In invasive tumor typing, TP accuracy was 95.4% (42/44 cases), the difference being attributable to slide color fading and would have had no impact on patient management. The accuracy of TP versus LM and expert consensus in tumor grading was 91.3% for carcinoma in situ (21/23 cases), a discordance with no relevance to patient management. TP grading of invasive tumor compared with LM diagnosis, had an accuracy of 86.4% (38/44) with a clinically significant accuracy of 97.7% (43/44). The time taken for TP diagnosis averaged 3.9 minutes per case by the end of the study. This data demonstrates that telepathology diagnostic accuracy is comparable to conventional microscopy and may therefore be envisaged as an alternative to conventional light microscopy for more rapid proficiency testing in breast screening (and perhaps other) quality assurance schemes.

Heat shock cognate 70 mutations in sporadic breast carcinoma.

Heat Shock Cognate 70 (HSC70) is a constitutively expressed molecular chaperone, functions of which regulate the structure, subcellular localization, and turnover of cell proteins. It is also a component of the centrosome facilitating rearrangements during mitotic/meiotic spindle formation and cytoplasmic microtubule organization. We localized HSC70 to 11q23.3, a region deleted in 40% of sporadic breast and other cancers. Sequencing demonstrated mutation in the NH2-terminal ATPase domain of HSC70 in 2 of 15 sporadic breast carcinomas examined. In both cases, mutation was coincident with allelic imbalance, suggesting that HSC70 is a target of somatic mutation and deletion in a fraction of breast carcinomas.

CD40 upregulation is independent of HHV-8 in the pathogenesis of Kaposi's sarcoma.

AIMS: Human herpesvirus 8 (HHV-8) is now acknowledged as the infective cofactor in the pathogenesis of Kaposi's sarcoma. The mode by which HHV-8 causes Kaposi's sarcoma is unresolved and it is probable that it acts in conjunction with other factors including cytokines, anti-apoptosis proteins, and cell surface receptors. CD40, a cell membrane receptor belonging to the tumour necrosis factor receptor super family, promotes B cell survival and is expressed constitutively on endothelial cells. It is upregulated on cytokine treatment and has been documented recently in Kaposi's sarcoma. Because the HHV-8 genome contains cytokine homologues, this study investigated whether CD40 expression in Kaposi's sarcoma correlated with HHV-8 status, using a unique set of HHV-8 positive and negative specimens. METHODS: Twenty one paraffin wax embedded samples of Kaposi's sarcoma were selected, of which 18 were screened for the presence of HHV-8 using both conventional solution phase and TaqMan polymerase chain reaction (PCR). CD40 immunohistochemistry was assessed using a biotinylated amplification system. Staining was scored semiquantitatively. RESULTS: The results indicated that the expression of CD40 is independent of viral status, being present in both HHV-8 positive and negative specimens. CONCLUSIONS: This suggests that HHV-8 promotes Kaposi's sarcoma cell survival following infection by mechanisms other than those involving CD40.

Chromosomes, 11Q and cancer: a review.

This review aims at providing a general understanding of how the multiple cytogenetic aberrations in cancer cells arise and exemplifies this by considering the specific role of chromosome 11q loci in carcinogenesis. Section I provides a theoretical molecular and structural framework for understanding the cytogenetic aberrations described in cancer. Given this background, Section II describes advances in the identification and localization of cancer susceptibility genes on chromosome 11q, highlighting ongoing areas of investigation.

11q23.1 and 11q25-qter YACs suppress tumour growth in vivo.

Frequent allelic deletion at chromosome 11q22-q23.1 has been described in breast cancer and a number of other malignancies, suggesting putative tumour suppressor gene(s) within the approximately 8 Mb deleted region. In addition, we recently described another locus, at the 11q25-qter region, frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this approximately 2 Mb deleted region. An 11q YAC contig was accessed and three YACs, one containing the candidate gene ATM at 11q23.1, and two contiguous YACs (overlapping for approximately 400-600 kb) overlying most of the 11q25 deleted region, were retrofitted with a G418 resistance marker and transfected into murine A9 fibrosarcoma cells. Selected A9 transfectant clones (and control untransfected and 'irrelevant' alphoid YAC transfectant A9 clones) were assayed for in vivo tumorigenicity in athymic female Balb c-nu/nu mice. All the 11q YAC transfectant clones demonstrated significant tumour suppression compared to the control untransfected and 'irrelevant' YAC transfected A9 cells. These results define two discrete tumour suppressor loci on chromosome 11q by functional complementation, one to a approximately 1.2 Mb region on 11q23.1 (containing the ATM locus) and another to a approximately 400-600 kb subterminal region on 11q25-qter.

Cyclin D1 expression and HHV8 in Kaposi sarcoma.

BACKGROUND: Human herpesvirus 8 (HHV8) appears to be the agent responsible for Kaposi sarcoma. The mechanism remains undetermined but may involve cell cycle regulating genes including D type cyclins which are pivotal in cell cycle progression. Recent HHV8 genetic analysis has revealed the presence of a v-cyclin which is homologous to D type cyclins. AIMS: First, to assess whether there is an independent relation between endogenous cyclin D1 expression in Kaposi sarcoma and HHV8 status; second to determine whether v-cyclin mRNA expression varies with Kaposi sarcoma stage. METHODS: Cyclin D1 immunohistochemistry was performed on 17 paraffin embedded Kaposi sarcoma samples from 16 patients. HHV8 status was assessed in 15 of these using nested polymerase chain reaction (PCR) to ORF 26 and the newly described technique of TaqMan PCR. An additional 10 fresh Kaposi sarcoma samples (early and nodular) were examined for HHV8 v-cyclin RNA. RESULTS: One case, which did not contain amplifiable HHV8, showed strong cyclin D1 staining. The remaining cases were negative or weakly staining; v-cyclin transcript load was higher in early Kaposi sarcoma. CONCLUSIONS: While endogenous cyclin D1 expression is independent of HHV8 status, v-cyclin transcription is higher in early lesions, supporting the "viral hit" hypothesis.

Human herpes virus 8 (HHV-8) in Kaposi's sarcoma: lack of association with Bcl-2 and p53 protein expression.

AIMS: Human herpes virus 8 (HHV-8) is the infectious agent implicated in the pathogenesis of Kaposi's sarcoma, although its mode of action is unclear. Recent work indicates that the HHV-8 genome encodes a viral Bcl-2 homologue (v-Bcl-2). The aim of this study was to explore Bcl-2 expression in Kaposi's sarcoma using a unique set of HHV-8 positive and negative cases, and to determine whether there is a relation with p53 expression. METHODS: Up to 18 specimens from 17 patients were selected. HHV-8 status was determined using nested polymerase chain reaction (PCR) to the open reading frame (ORF) 26, with further confirmation by TaqMan PCR. In addition, Bcl-2 and p53 immunohistochemistry were performed using standard protocols. RESULTS: The results suggest that Bcl-2 and p53 expression is independent of HHV-8 status. In addition, there does not appear to be a direct correlation with disease stage. CONCLUSIONS: HHV8 histopathogenesis is likely to be a multifactorial complex process, which may be mediated in part by viral genes and apoptosis regulating homologues.

Human osteoclast formation from blood monocytes, peritoneal macrophages, and bone marrow cells.

Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)2D3 and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)2D3, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS.

Allelic deletion at 11q23.3-q25 is an early event in cervical neoplasia.

Twenty per cent of cervical intraepithelial neoplasias-III (CIN-III) progress to invasive cancer. Human papillomavirus (HPV) infection alone does not determine progression. CIN-III lesions were collected from 161 women. Each tissue was microdissected into a maximum of 32 contiguous units and assayed at multiple microsatellite loci on chromosome 11q, a region frequently deleted in invasive cervical and other cancers. Eight of 108 informative cases (7%) had 11q23.3-q25 deletions; focally intra-lesional in six (one with focal loss of alternate alleles), and pan-lesional in two cases. Hence, 11q deletion can occur early in cervical neoplasia, and possibly predisposes to invasion.

Identification of HHV8 in early Kaposi's sarcoma: implications for Kaposi's sarcoma pathogenesis.

AIMS: Kaposi's sarcoma is a vascular tumour of uncertain pathogenesis possibly caused by an infectious agent, identified in high risk groups. Accumulating solution phase polymerase chain reaction (PCR) and seroepidemiological data suggest that a previously undescribed herpes DNA virus (human herpesvirus 8 (HHV8)) is the causative agent. Using a unique cohort of early Kaposi's sarcoma, the precise cell type infected with HHV8 in such lesions was identified to elucidate further the role of HHV8 in the pathobiology of Kaposi's sarcoma. METHODS: Sixteen cases of early Kaposi's sarcoma (derived from skin and lymph node) were assessed for the presence of HHV8 using both standard solution phase PCR and TaqMan PCR to the KS330 Bam region of HHV8. In situ amplification was also performed on a selected group in an attempt to identify the candidate infected cells. RESULTS: Using both conventional solution phase and TaqMan PCR, 87% of cases were positive. In addition, HHV8 amplicons were localised in situ to endothelial and spindle cell proliferations in early Kaposi's sarcoma. The HHV8 viral load varied from lesion to lesion. CONCLUSIONS: The presence of HHV8 in early lesions supports a role for HHV8 in the pathogenesis of Kaposi's sarcoma. Coupled with recent seroepidemiological studies, these results suggest that HHV8 is the aetiological agent of Kaposi's sarcoma. Its precise interaction with other factors known to be involved in the development of Kaposi's sarcoma, including cytokines and anti-apoptosis genes, requires elucidation.

Human tumour-associated macrophages differentiate into osteoclastic bone-resorbing cells.

Macrophages are commonly found within osteolytic secondary carcinomas in bone, but the manner in which these cells contribute to malignant bone resorption is uncertain. Macrophages isolated from primary breast carcinomas were co-cultured for up to 21 days with UMR 106 rat osteoblast-like cells on bone slices and glass coverslips in the presence and absence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and human macrophage colony stimulating factor (M-CSF). Cell cultures were then assessed for the presence of phenotypic markers of macrophage and osteoclast differentiation. Isolated cells were negative for osteoclast markers including tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and the ability to carry our lacunar bone resorption, but were positive for CD11b and CD14, macrophage markers which are not present on osteoclasts. In 21-day co-cultures of breast carcinoma tumour-associated macrophages (TAMs) and UMR 106 cells, incubated in the presence of 1,25(OH)2D3 and M-CSF, numerous TRAP- and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed. Contact with UMR 106 cells and the presence of 1,25(OH)2D3 and M-CSF were absolute requirements for differentiation of human breast carcinoma TAMs into mature functional osteoclasts. TAM-osteoclast differentiation may represent an important cellular mechanism of osteolysis in metastatic skeletal carcinomas.

Microsatellite analysis in human disease.

Microsatellites (or simple sequence length polymorphisms [SSLPs]; and short tandem repeat polymorphisms [STRs]) are tandemly repeated arrays of short stretches of nucleotide sequences, typically (dA-dC/dT-dG), where n is between 15 and 30 (1,2) The human genome is estimated to contain approximately 12,000 (dA-dC)n microsatellites with a polymorphic information content (PIC) > 0.5 (3) Further information on microsatellite orgamzation, distribution, and origin can be obtained from a recent review (4). For the purposes of this chapter, it suffices to state that owing to their high heterozygosity, Mendelian codominant inheritance, ubiquity through the genome, and ease of polymerase chain reaction (PCR) typability, microsatellites are the markers of choice in the construction of human (and other model organisms) linkage maps. For instance, a recent human microsatellite linkage map consists of over 5264 (dA-dC)n loci with a heterozygosity >70% distributed along the genome at an average interval of 1.6 centiMorgans (CM) (5) This forms an invaluable resource for the genetic dissection of complex traits (e.g., type I diabetes).

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.

Induction of myocardial heat shock protein 70 during cardiac surgery.

Animal experiments have shown that members of the heat shock protein (HSP) family have cytoprotective properties against ischaemia. In experimentally induced cardiac ischaemia, the induction of HSP70s correlates with reduced infarct size and enhanced myocardial function and endothelial recovery. Direct evidence that increased myocardial HSP70 expression result in cytoprotection during ischaemia has also been obtained using transgenic mice overexpressing either rat or human HSP72. This study examined the induction and expression of myocardial HSP70s after an obligatory period of ischaemia in patients during cardiac surgery. The level of HSP72/HSC73 protein in Tru-cut biopsies of the myocardium, taken before and after an acute ischaemic insult, was examined using a polyclonal antibody. The amount of HSP72 mRNA in the biopsies was also determined by reverse transcriptase polymerase chain reaction (RT-PCR) and correlated HSP72/HSC73 protein expression. In four patients subjected to brief alternating periods of normothermic ischaemia and reperfusion, the amount of myocardial HSP72/HSC73 protein was increased several fold after ischaemic insult. This was accompanied by increased expression of HSP72 mRNA. In contrast, the amounts of myocardial HSP72/HSC73 protein and HSP72 mRNA were unchanged in a patient subjected to a single prolonged period of hypothermic ischaemia. Given the proven myocardial protective properties of HSP72 in experimental models, it is postulated that the observed induction of HSP72 may have a similar function in man.

HHV8 and Kaposi's sarcoma: a time cohort study.

AIMS: The recent finding that human herpes virus 8 (HHV8) is found in the majority of Kaposi's sarcoma (KS) cases supports the epidemiological observation that the tumour may be caused by an infectious agent. This study aimed to address when and how HHV8 evolved. METHODS: A cohort of African endemic KS (49 samples from 45 patients) and European KS (18 samples from 13 patients), spanning 27 years, was assessed for the presence of HHV8 by both standard solution phase polymerase chain reaction (PCR) and the newly described technique of TaqMan PCR. RESULTS: HHV8 was present in approximately 49% (24 of 49 tissue samples) of the African cases and in more than 90% (16 of 18 tissue samples) of the European cohort, in keeping with recent seroepidemiological data. CONCLUSIONS: HHV8 is strongly linked to the development of KS; however, in some patients, other factors may operate. The utility and flexibility of TaqMan PCR in detecting low copy viral target in human tissues was demonstrated.