[Quantitative methylation-specific PCR for the diagnosis of lung cancer]. / Quantitative methylierungsspezifische PCR zur Lungenkarzinomdiagnostik.

Efficient, preferably early diagnosis of lung cancer represents a major challenge. Under this aspect the sensitivity of conventional histomorphology and cytomorphology procedures is unsatisfactory. This review highlights technical aspects, possibilities and drawbacks of the application of aberrant promoter methylation as a biomarker for lung cancer diagnostics using specimens of pulmonary exfoliative cytology.

[Aberrant promoter methylation as biomarker for molecular cytological diagnosis of lung cancer]. / Aberrante Promotor-Methylierungen als Tumormarker für die molekularzytologische Diagnostik des Lungenkarzinoms.

Aberrant promoter methylation represents a main mechanism of tumor suppressor gene inactivation and may serve as a new source for biomarker discovery. This study investigated its applicability as a molecular tool for lung cancer diagnostics on bronchial aspirates. A methylation assay was developed applying a quantitative methylation specific real-time PCR (QMSP). A total of 552 patients with the differential diagnosis of lung cancer were investigated. The QMSP findings on bronchial aspirates were compared with the methylation status of respective genes investigated in microdissected tumor tissues (QMSP, cloning and sequencing of promoter regions after bisulfite conversion). Among the genes tested a marker panel consisting of APC, p16(INK4a) and RASSF1A proved to be the best suited for lung cancer diagnostics. This panel allowed for a correct diagnosis of lung cancer in cases with an ambiguous or false negative conventional cytology. In a cohort study on 247 patients, the combination of histology (sensitivity 59 %), cytology (sensitivity 44 %) and QMSP-assay (sensitivity 53 %) raised the sensitivity of a single bronchoscopy for the diagnosis of lung cancer up to 81%. The methylation assay yielded its major diagnostic surplus with respect to peripheral tumors representing 59 % of all primaries detected. In patients without antecedent lung cancer its specificity considering malignancy was >99 %. Therefore, the QMSP-assay is a promising technique which could enhance the sensitivity and diagnostic impact of conventional cytology. The assay is applicable to residual material of regular diagnostic cytology even in retrospect.

Spontaneous malignant transformation of conventional giant cell tumor.

Spontaneous malignant transformation of conventional giant cell tumor (GCT) of bone is exceedingly rare. We report on a case of GCT of the iliac crest in a 35-year-old woman with malignant change into a high-grade osteosarcoma 10 years after the first appearance of GCT on a radiograph. Since the patient refused therapy for personal reasons the tumor remained untreated until sarcomatous transformation occurred. Image cytometry showed DNA aneuploidy and a suspiciously high 2c deviation index (2cDI) in the primary bone lesion. A thorough review of the world literature revealed only seven fully documented cases of secondary malignant GCT which matched the definition of a "sarcomatous growth that occurs at the site of a previously documented benign giant cell tumor" and not treated by radiotherapy. These cases as well as the current one suggest that a spontaneous secondary malignant GCT presents as a frankly sarcomatous tumor in the form of an osteosarcoma or malignant fibrous histiocytoma. It usually appears at sites of typical GCTs-often without any recurrent intermediate state-and is diagnosed 3 or more years after the primary bone lesion. The prognosis is poor.

[Molecular cytopathology of lung cancer--prevalence of genetic alterations and their role in the development of molecular biomarkers]. / Molekulare Zytopathologie bösartiger Lungentumoren--Prävalenz der genetischen Alterationen und ihre Bedeutung für die Entwicklung molekularer Biomarker.

Carcinogenesis of lung cancer proceeds via a complex process that involves multiple genetic abnormalities, which do not necessarily have a linear progression. Genetic alterations include aneuploidy, deletions and amplifications of chromosomal regions, loss of heterozygosity (LOH), microsatellite alterations, point mutations and aberrant promoter methylation. There is considerable effort to use these genetic alterations as molecular biomarkers for early cancer diagnosis applying different approaches. An ideal tumor marker should be highly sensitive, tumor specific, easily to handle and non-cost intensive. While previous studies used screening for mutations, LOH and microsatellite alterations, more recent strategies concentrate on multicolor fluorescence in situ hybridization (FISH) and aberrant promoter methylation. Since in general the genetic alterations are prone to be more extensive in tumor cells as compared to non-tumor cells, methods that provide quantitative data (e.g., methylation specific real-time PCR) are likely to improve specificity. Consequently, molecular biomarkers could constribute to a more accurate risk assessment in carcinogen exposed individuals and early molecular cytologic diagnosis of precancerous lesions and cancers of the lung.

Prognostic significance of DNA cytometry in carcinoma of the uterine cervix FIGO stage IB and II.

OBJECTIVE: To assess the prognostic value of DNA-image cytometry in cervical carcinoma of the uterus and its relation to other established prognostic factors. STUDY DESIGN: The study included 116 cases of cervical carcinoma FIGO stages IB and II which were treated with radical abdominal hysterectomy. The median follow-up was 55 months (range 1-162 months). DNA image cytometry was performed on cytologic specimens prepared by enzymatic cell separation from formalin-fixed, paraffin-embedded tissues. DNA stemline ploidy, DNA stemline aneuploidy, 5c exceeding rate, 9c exceeding rate, 2c deviation index, and DNA malignancy grade were computed. DNA-variables as well as various clinical and histological variables were related to survival rates. RESULTS: In multivariate statistical analysis DNA stemline ploidy using 2.2c as a cut-off value and FIGO stage showed to be statistically significant available presurgery predictors of survival, whereas the postsurgical parameters lymphonodal status, tumor size and parametrial involvement were significantly correlated with survival. The synopsis of all parameters in a multivariate Cox model indicated that - with declining relevance - the number of positive pelvic lymph nodes, DNA stemline ploidy using a cut-off level at a modal value of 2.2c, largest pelvic lymph node, 5c exceeding rate, and ratio of carcinoma area to cervix area, were of predictive value for survival. CONCLUSIONS: Our results suggest that prognostic information deducted from classical staging parameters is successfully complemented by DNA image cytometry which can be applied pretherapeutically.

Overexpression of cathepsin B and urokinase plasminogen activator is associated with increased risk of recurrence and metastasis in patients with chondrosarcoma.

BACKGROUND: Deregulation of the cellular protease network has been shown to be responsible for aggressive clinical behavior in several common human malignancies. In the current study, the authors evaluated the expression patterns of proteases in patients with chondrosarcoma of bone and correlated these patterns with clinical outcome. METHODS: The expression levels of urokinase plasminogen activator; matrix metalloproteinase types-1, -2, and -9; and cathepsins B and L were determined immunohistochemically in 114 cases of chondrosarcomas of bone and were correlated with their clinicopathologic parameters as well as with long term follow-up data. RESULTS: Overexpression of cathepsin B was associated with a high rate of local recurrence (P = 0.006) and a decreased recurrence free survival (P = 0.005). Overexpression of urokinase plasminogen activator was associated with an increased rate of metastasis (P = 0. 013), a decreased metastasis free survival (P = 0.016), and a decreased 5-year overall survival rate (P = 0.048). The univariate Cox model showed that tumor extension into soft tissue, high histologic grade, and overexpression of cathepsin B were predictors of adverse outcome. Multivariate analysis showed only overexpression of cathepsin B and tumor extension into soft tissue to be independent predictors of local recurrence. CONCLUSIONS: Overexpression of cathepsin B and urokinase plasminogen activator can be used to identify those patients with chondrosarcoma of bone who have an increased risk of local recurrence and distant metastases.

Mutation of p53 with loss of heterozygosity in the osteosarcomatous component of a dedifferentiated chondrosarcoma.

We investigated a dedifferentiated chondrosarcoma of a 61-year-old woman with an osteosarcomatous high-grade component for p53 alteration. The low-grade cartilaginous and the high-grade osteosarcomatous components of the tumor were macrodissected and evaluated separately by immunohistochemistry and molecular biology. We used PCR-SSCP analysis and direct sequencing to screen exons 4-8 for p53 mutations. The p53 intron 1-polymorphism was investigated for loss of heterozygosity. A functionally relevant p53 missense mutation in codon 193 of exon 6 (A-to-T transversion) with loss of wild-type allele was detected only in the dedifferentiated component. Using the monoclonal antibody DO-1, immunohistochemistry failed to show p53 overexpression. This evidence of p53 mutation may be regarded as at least a co-factor that "switched" the preexisting low-grade conventional chondrosarcoma to a highly malignant dedifferentiated tumor.

Rosai-Dorfman disease and generalized AA amyloidosis: a case report.

We report on a patient who, at 31 years of age, was found to suffer from sinus histiocytosis with massive lymphadenopathy (SHML; Rosai-Dorfman disease) with nodal and extranodal involvement as described previously. Five years later the patient presented with nephrotic syndrome caused by a generalized AA amyloidosis, and he subsequently died from pulmonary thromboembolism owing to renal vein thrombosis. Retrospective analysis of serum levels of C-reactive protein (CRP) showed that during the last 3 years before his death, he had a persistently elevated CRP level ranging from 73 to 161 mg/L, despite antiinflammatory treatment with prednisolone, methotrexate, or 6-mercaptopurine. These figures indicate that the patient was probably suffering from a permanent acute phase response which, in the absence of any other evidence of a chronic inflammatory disease which commonly causes AA amyloidosis, was most likely owing to SHML.

Osseous manifestation of Rosai-Dorfman disease (sinus histiocytosis with massive lymphadenopathy). A case report and review of the literature.

Rosai-Dorfman disease (sinus histiocytosis with massive lymphadenopathy) is an unusual form of histiocytic disorder. Bone lesions are infrequent. We describe a 33-year-old man with involvement of multiple bones but without lymphadenopathy at the time of presentation. The literature on osseous manifestation in this condition is reviewed.

Intervertebral variation in trabecular microarchitecture throughout the normal spine in relation to age.

The vertebral bodies of the complete spine (C-3-L-5) were removed in 26 autopsy cases without evidence for primary or secondary bone disease (13 males aged 19-79 years and 13 females aged 17-90 years). A sagittal segment through the center of all vertebral bodies was embedded undecalcified in hydroxyethylmethacrylate and processed to so-called surface stained block grindings. Histomorphometric analysis of the complete segment was performed using a computer-assisted image analysis system (IBAS 2000). The structural parameters investigated were bone volume (BV/TV) and trabecular interconnection quantificated by trabecular bone pattern factor (TBPf). A close correlation of BV/TV and TBPf was found in all vertebral bodies irrespective of vertebral region (r = 0.8, p < 0.001). This indicates that the age-related decrease of trabecular bone mass is primarily the consequence of the transformation from plates to rods and the loss of whole trabecular structures. This basic principle is valid throughout the complete spine. However, the systematic analysis of vertebral trabecular bone from C-3 to L-5 revealed a significant intervertebral variation of trabecular microarchitecture. The density of trabecular structure of cervical vertebrae is much higher than that of thoracic and lumbar vertebrae (p < 0.001). The extent of age-related loss of trabecular bone mass and structure showed a decrease within the spine from the caudal to the cranial region (p < 0.05). The loss of bone volume in individuals between the ages of 30 and 80 years in the lumbar spine was 53%, whereas in the thoracic spine the decrease was 41%, and in the cervical spine only 24%.(ABSTRACT TRUNCATED AT 250 WORDS)

The microarchitecture of the axis as the predisposing factor for fracture of the base of the odontoid process. A histomorphometric analysis of twenty-two autopsy specimens.

The axis from twenty-two cadavera was removed at autopsy and was sectioned in the sagittal plane to a thickness of one millimeter with use of a surface-stained block-grinding technique. Combined two and three-dimensional analysis included an evaluation of the volume of the trabecular bone, the trabecular interconnection, and the cortical thickness as well as qualitative investigation of the structure of the cancellous bone. The body of the axis, the base of the odontoid process, and the odontoid process were analyzed separately. The base of the odontoid process is a region of least resistance for fractures because of its unique microarchitecture. The mean volume of trabecular bone of the base of the odontoid process is 55 per cent less than that of the axis and the odontoid process. The base also has a markedly poorer trabecular interconnection and a cortical thickness that is one-third that of the odontoid process. In all of the specimens, trabeculae that were disconnected from the trabecular lattice (trabeculae with free ends) were demonstrated in the base of the odontoid process. The formation of microcallus in six (27 per cent) of the specimens supports the hypothesis that microfractures occur as a result of stress peaks, mechanical fatigue, and the relative insufficiency of bone in the static condition. Therefore, the base of the odontoid process can be considered as a site of predilection for fractures.

Polyostotic heterogeneity of the spine in osteoporosis. Quantitative analysis and three-dimensional morphology.

It was the aim of this study to record quantitatively and qualitatively the distribution of the three-dimensional microarchitecture throughout the human spine in osteoporosis. Bone biopsies of the iliac crest and the complete spine of 26 autopsy cases without skeletal disease and 11 female patients with proven osteoporosis were removed. Grindings of all vertebrae by a technique which we developed allowed two- and three-dimensional measurements simultaneously. The analysis included an evaluation of trabecular bone volume, trabecular interconnection, and trabecular thickness, as well as a qualitative investigation of the structure of cancellous bone. The bone loss in osteoporosis is a loss of structure. The relative loss of the trabecular microarchitecture is greater in the iliac crest than in the lumbar spine. It is a gradual change from normal bone to osteoporosis. Transformation from plates to rods and the loss of whole trabeculae are caused by perforations. The polyostotic heterogeneity in osteoporosis is remarkable. Adjacent vertebrae may show differences of up to 100% in bone structure and bone volume. This explains the difficulties in early diagnosis of osteoporosis. Due to the polyostotic heterogeneity it is impossible to define a threshold mineral content for osteoporotic fractures.

[Structure of the axis--key to the etiology of the dens fracture]. / Die Struktur des Axis--Schlüssel zur Atiologie der Densfraktur.

Fractures of the dens are seen especially in young adolescents but also in individuals after the sixth decade of life. The etiology of these fractures and the occurrence of non-union after initial treatment is still discussed controversially. To address these issues, the axis was removed from thirty-seven autopsy cases for histomorphometric analysis. The base of the dens is a region of least resistance for fractures due to its reduced trabecular bone volume, a poorer trabecular interconnection and a cortical thickness one third that of the axis. In all of the cases, trabeculae disconnected from the trabecular lattice, and in 30% microcallus formations were demonstrated in the base of the dens. In osteoporotics the microarchitectural differences of cancellous bone between the base of the dens and the other regions of the axis are increased markedly. The obtained data suggest that the bone structure of the axis is responsible for the location, the distribution and the frequency of fractures of the odontoid process. The deficiency of bone mass within the base also offers a new explantation for the occurrence of non-unions even after treatment of fractures of the base of the dens.

[Micro-callus formation of spongiosa. An up to now underestimated repair mechanism of the skeletal system]. / Mikrokallusformationen der Spongiosa. Ein bisher unterschätzter reparativer Mechanismus des Skelettsystems.

Microcallus formations are demonstrable in nearly all cancellous bones by means of suitable preparation techniques. Histologically, these structures are immature fibrous bone formed in local overloaded parts of the trabeculae. Using a preparation technique that allows combined two- and three-dimensional analysis, 26 normal human spines and 11 osteoporotic spines were investigated for microcallus. Microcallus formations occur frequently in people over 45 years of age. They are mainly localized in the lower thoracic and lumbar spine and occur significantly more frequent in females than in males. The number of microcallus formations depends more on the microarchitecture of the cancellous bone than on individual trabecular parameters. In about 33% of cases microfractures are demonstrable in the centre of the microcallus formation. In non-invasive studies the bone mass could be misinterpreted due to microcallus. Although it indicates instability of the bone structure, microcallus formation is not a purely negative mechanism. It stabilizes and regenerates the bone tissue. Furthermore, complete new trabeculae can be formed due to bridges of microcallus between residual trabeculae. Osteoporosis is not the result of an inability to form microcallus.

Three-dimensional analysis of the spine in autopsy cases with renal osteodystrophy.

The spinal column was removed from nine autopsy cases with chronic maintenance dialysis and sectioned in the sagittal plane to a thickness of 1 millimeter using a surface-stained block grinding technique. A combination of two- and three-dimensional analysis included an evaluation of the spine deformity index (SDI), the bone volume (BV/TV), the trabecular interconnection (TBPf), the trabecular thickness (Tb.Th), the trabecular number (Tb.N) as well as a qualitative investigation of the structure of cancellous bone. The control group consisted of 26 autopsy cases with intact skeletons. An iliac crest biopsy made a direct comparison of the diagnostic biopsy location and the spinal column possible. The SDI showed vertebral fractures in renal osteodystrophy (ROD) of types I and II, in spite of a trabecular bone volume within normal limits. The trabecular bone volume showed mean values and a distribution throughout the spinal column familiar in the skeletally-intact control group. Those cases with a longer history of hemodialysis showed higher BV/TV values regardless of age and sex. A trabecular volume within normal limits did not mean physiological trabecular interconnection. Perforations were commonly observed with ROD. Classical, hidden and tunnelling perforations were distinguished. Microcallus formations were frequently seen in the periphery of vertebrae at rod nodes, rods and plate intersections. Three-dimensional analysis in ROD shows a greater alteration in architecture than can be assumed from the two-dimensional histological sections.

Enzyme-linked immunosorbent assay for detection of immunoglobulin A and G antibodies to Campylobacter pylori.

An enzyme-linked immunosorbent assay (ELISA) employing an acid-glycine extract was used to detect IgG and IgA antibodies to Campylobacter pylori in sera from 179 patients with upper gastrointestinal disease, 174 blood donors and 65 children. The incidence of positive ELISA results clearly increased with the severity of histopathologic findings in the antrum mucosa and was also high in patients with peptic ulcers and gastric cancer. The incidence in blood donors and children was much lower and increased with age. The results achieved with the ELISA were similar to those observed previously using the immunoblot method. Differences between whole cell preparation and acid-glycine extract with respect to their protein profiles and immunoblot reactivities were minor. IgM titres were very low and could not be related to histopathological findings, peptic lesions or culture findings. The ELISA may be particularly useful for monitoring the outcome of therapy aimed at eradication of Campylobacter pylori.

Immunoblot analysis of immune response to Campylobacter pylori and its clinical associations.

Systemic immune response to Campylobacter pylori was detected by the immunoblot technique in serum samples from 200 patients, 129 blood donors, and 96 children. The results of the IgG immunoblot test showed excellent correlation with the detection of C pylori by culture and also with histopathological examination of the antrum, as well as with peptic ulcer disease. An IgA response also occurred and gave results comparable with those of the IgG immunoblot test, although on a quantitatively lower scale. The IgM immunoblots were of no help in the serodiagnosis of C pylori infection. The protein bands that seemed to be the most specific for C pylori and which were consistently observed in patients positive for C pylori were a 110 kilodalton and a 63 kilodalton band on the IgG immunoblot and an 89 kilodalton band on the IgA immunoblot. A 94 kilodalton and a 28 kilodalton band were also included in the evaluation. While immunoblot analysis may be used effectively for the serodiagnosis of C pylori infection and can distinguish between patients with normal antrum mucosa and those with gastritis, the test does not help to distinguish between those patients with antrum gastritis who subsequently develop peptic ulcers and those who do not.