Molecular Pathological Analysis Research Articles

Role of tumor markers in oral squamous cell carcinoma: Review of literature and future consideration

Sensitive and reliable early diagnostic markers for oral squamous cell carcinoma (OSCC) remain unavailable. Early identification of recurrence for OSCC is also a challenge. This article reviews the recently identified biomarkers for OSCC such as cytokeratins, p53, tumor necrosis factor alpha (TNF-α), etc., which are of great utility in early diagnosis. In addition, the biomarkers that have been correlated with OSCC tumor malignancy by molecular pathology analysis are also described. This article speaks about selected reaction monitoring (SRM) which might even be applied to monitor differential expression of tumor proteins in blood, saliva, or fresh frozen tissue materials. SRM technique may complement or possibly replace western blotting for biomarker verification and for selection of potential biomarker candidates. This article may help to identify the potential biomarkers for screening and the molecular pathology analysis for high-risk patients of OSCC. Effective screening to identify high-risk patients will allow clinicians to provide an early and appropriate treatment to patients without delay and also reduce the risk of recurrence of OSCC.

Donor-transmitted metastasis of colorectal carcinoma in a transplanted liver

A 62-year-old man with alcoholic liver cirrhosis underwent liver transplantation. The transplantation went uneventful and the ultrasound imaging of the liver performed after transplantation did not show any abnormalities. Eighteen months later, an intra-hepatic focal lesion was found on ultrasound. A contrast-enhanced ultrasound revealed a lesion with a malignant pattern of contrast uptake. The histo-pathological and subsequent molecular-pathological analysis concluded a colorectal metastasis of donor origin. The donor had no history of malignancy but no complete autopsy had been performed which illustrates the importance of the meticulous donors` screening. Transplanted patients carry a high risk of developing malignancy in general but donor related-tumors are very rare. The therapeutic considerations differ substantially between recipient- and donor-related malignancies. Therefore, considering the possibility of donor-related tumor by raising suspicion of malignant lesion with appropriate imaging and distinction from recipient-related malignancy by molecular analysis are crucial for proper therapeutic decision.

ENDOSCOPICALLY TREATED CRONKHITE-CANADA SYNDROME ASSOCIATED WITH MINUTE INTRAMUCOSAL GASTRIC CANCER: AN ANALYSIS OF MOLECULAR PATHOLOGY

There have been no reports of Cronkhite-Canada syndrome (CCS) associated gastric cancer resected with endoscopy because it is very difficult to identify small cancers that are candidates for endoscopic resection. We report a case of CCS with gastric cancer treated with endoscopic submucosal dissection, and we evaluate the molecular pathological analysis of malignant transformation in patients with CCS. A 74-year-old man had an advanced rectal cancer and gastrointestinal polyposis after presenting with hypoproteinemia, partial hair loss and atrophic nails as well as hyperpigmentation on the hands. He was diagnosed as having CCS. On upper endoscopy, a 7 mm discolored polyp with an irregular microvascular pattern revealed by magnified narrow-band imaging (NBI) was identified in gastric diffuse CCS polyposis. This lesion was treated with endoscopic submucosal dissection and diagnosed as a flat, elevated-type, mucosal well-differentiated tubular adenocarcinoma without lymphatic or venous infiltration, and with tumor-free margins. Microsatellite instability was detected in both the cancer and the surrounding CCS polyps. Mucin-histochemical analysis of the cancer area showed the complete intestinal type, and thus may have differentiated the CCS polyps from that of the common gastric hyperplastic polyps. This case illustrates that a clue to detecting small cancers may be to look for the discolored lesion among reddish CCS polyposis and thereafter to observe the irregular vascular pattern with NBI endoscopy. From the viewpoint of genetic alterations, patients with CCS polyps are considered to be at high risk for developing gastric cancer, and therefore careful follow-up examinations are necessary for the early detection of malignancies.

Biomarkers predicting development of metachronous gastric cancer after endoscopic resection: an analysis of molecular pathology of Helicobacter pylori eradication.

Metachronous gastric cancer (MGC) after endoscopic resection (ER) of gastric cancer still occurs to some degree even after Helicobacter pylori (H. pylori) treatment. We evaluated whether two biomarkers related to carcinogenesis expressed in intestinal metaplasia (IM) become predictors for MGC development after eradication. We performed a hospital-based, case-control study of 75 patients, including 50 mucosal cancer patients who had undergone ER (Group DYS), and 25 age- and sex-matched chronic gastritis patients for whom H. pylori had been successfully eradicated (control). Additionally, Group DYS patients were divided into two groups: 25 successfully H. pylori-eradicated (eradicated group) and 25 un-eradicated patients (persistent group). All patients were followed for 1 year. We analyzed microsatellite instability (MSI) and immunoperoxidase assays using a monoclonal antibody for the colonic phenotype (Das-1). Both MSI and Das-1 reactivity in IM were significantly higher in Group DYS than in the control (p < 0.05 and p < 0.01, respectively). MSI and Das-1 reactivity were strong and independent predictors for gastric cancer (OR = 7.09, 95% CI 1.27-39.6, p = 0.03 for MSI and OR = 4.96, 95% CI 1.64-15.0, p = 0.005 for Das-1 reactivity). The incidence of MSI tended to decrease in the eradicated group (p = 0.07), but not in the persistent group. The Das-1 immunoreactivity in IM also declined in both the eradicated group and the control. Interestingly, all MGCs after ER were positive for MSI or Das-1 reactivity. MSI or Das-1 reactivity in IM strongly predicts the development of MGC. Patients in whom these biomarkers persist after eradication may therefore have a high risk of developing MGC.

Primary Ewing’s Sarcoma of the Kidney in a 73-Year-Old Man

Objective. Ewing's sarcoma of the kidney is rare and is usually found in young adults. We present here a single case study of Ewing's sarcoma found in an elderly man. Material and methods. A 73-year-old man underwent routine surgery for hydrocoele of the testis. He developed urinary obstruction symptoms, and radiological examinations revealed a tumour in the right kidney. Results. Microscopical, immunohistochemical, and molecular pathological analysis of the tumour was consistent with Ewing's sarcoma. FISH showed rearrangement of chromosomes 22q12 (EWSR1). The patient subsequently underwent nephrectomy followed by 6 adjuvant chemotherapy cycles. Follow-up after 7 months shows no recurrence. Conclusion. This case report presents not only the rare finding of Ewing's sarcoma in the kidney, but also the occurrence of this tumour entity in an elderly patient. Treatment options for the different types of renal tumours are vastly different and the need for a correct diagnosis is, therefore, vital.

Neues aus dem Jenaer Weichteiltumor-Register

The Jena Institute of Pathology has been serving as a consultation and reference center for soft tissue tumors in Germany since 1978. The present study provides an overview of the clinicopathological data from a two-year period and an update on diagnostics and research. Retrospectively, 7043 cases sent to the institute in the years 2006 and 2007 were analyzed. The majority of cases (>77.7%) were soft tissue tumors, of which 49% were categorized as malignant, 11.4% as intermediate, 35% as benign and 4.6% as tumors of uncertain biological potential. Neoplasms with fibroblastic differentiation were the most frequent. The mean age of patients with a sarcoma was 63 years. The molecular pathological analysis of soft tissue tumors has attained a major role in diagnosis. This is further advanced at the Jena institute in the context of a German Federal Ministry of Education and Research (BMBF) project for molecular sarcoma diagnosis with the aim of developing and validating DNA probes for in situ hybridization detection of translocations and their associated chromosomal breaks on the one hand, and DNA chips for the detection of fusion transcripts on the other. Research projects relate to the analysis of specific biomarkers in large tumor collectives and the pathomechanisms in several sarcoma entities.

Indole-3-carbinolのラット肝発がんプロモーション作用についての分子病理学的解析

Indole-3-carbinolのラット肝発がんプロモーション作用についての分子病理学的解析

Clinical, molecular pathological and genetic analyses of a Chinese family with congenital muscular dystrophy type 1A

To analyze and characterize the clinical, molecular pathological and genetic features of a Chinese family with congenital muscular dystrophy type 1A (MDC1A). Clinical data of the proband and her family members were collected. Immunohistochemistry staining was performed on muscular biopsy tissues with anti-merosin, alpha-dystroglycan, beta-dystroglycan and dystrophin antibodies. Genomic DNAs from the patient and her parents were extracted using standard procedures from the peripheral blood leukocytes. PCR and DNA direct sequencing were employed to analyze all of the 65 exons of the LAMA2 gene to determine the gene mutation, and the relationships between genotype and phenotype were analyzed. The proband presented with delayed motor development and a myopathic face. Her midrange elevated serum creatine kinase (CK) levels and white matter signal intensity changes are consistent with MDC1A, and was clinically diagnosed as MDC1A. The immunohistochemistry analysis for the proband exhibited complete loss of merosin staining. Further test with PCR detected a homozygous mutation of c.817A>T in exon 5, while her parents were heterozygotes for the mutation. The authors have defined the clinical manifestation of the Chinese family with MDC1A. The proband carried a homozygous nonsense mutation c.817A>T, and her parents were heterozygous carriers, consistent with autosomal recessive inheritance.

Pediatric giant cell glioblastoma: New insights into a rare tumor entity

Little is known about giant cell glioblastoma (GCG) in pediatric patients. The present study identified 18 pediatric patients with centrally reviewed GCG from the HIT-GBM database of the Gesellschaft für Paediatrische Onkologie und Haematologie in Germany, Austria, and Switzerland. Clinical and epidemiological data were compared with those of 178 pediatric patients with centrally reviewed glioblastoma multiforme (GBM) from the same database. In this unique series, median age, male preference, and median clinical history did not differ significantly between pediatric GCG and GBM patients. GCG showed a stronger predilection for cerebral hemispheres than did GBM, which may only partly explain the higher percentage of gross total tumor resections in GCG patients. Most surprising, the widely distributed hypothesis that GCG may imply a better prognosis than GBM could not be substantiated for our pediatric series. Future studies with larger patient numbers and molecular pathological analyses are still needed to corroborate the present findings and further elucidate the biology of GCG in children.

Progression of Epididymal Maldevelopment Into Hamartoma-like Neoplasia in VHL Disease

Inactivation of the von Hippel-Lindau (VHL) gene and activation of the hypoxia-inducible factor (HIF) in susceptible cells precedes formation of tumorlets and frank tumor in the epididymis of male VHL patients. We performed detailed histologic and molecular pathologic analysis of tumor-free epididymal tissues from VHL patients to obtain further insight into early epididymal tumorigenesis. Four epididymides from two VHL patients were serially sectioned to allow for three-dimensional visualization of morphologic changes. Areas of interest were genetically analyzed by tissue microdissection, immunohistochemistry for HIF and markers for mesonephric differentiation, and in situ hybridization for HIF downstream target vascular endothelial growth factor. Structural analysis of the epididymides revealed marked deviations from the regular anatomic structure resulting from impaired organogenesis. Selected efferent ductules were represented by disorganized mesonephric cells, and the maldeveloped mesonephric material was VHL-deficient by allelic deletion analysis. Furthermore, we observed maldeveloped mesonephric material near cystic structures, which were also VHL-deficient and were apparent derivatives of maldeveloped material. Finally, a subset of VHL-deficient cells was structurally integrated in regular efferent ductules; proliferation of intraductular VHL-deficient cells manifests itself as papillary growth into the ductular lumen. Furthermore, we clarify that that there is a pathogenetic continuum between microscopic tumorlets and formation of tumor. In multiple locations, three-dimensional reconstruction revealed papillary growth to extend deeply into ductular lumina, indicative of progression into early hamartoma-like neoplasia. We conclude epididymal tumorigenesis in VHL disease to occur in two distinct sequential steps: maldevelopment of VHL-deficient mesonephric cells, followed by neoplastic papillary proliferation.

Patterns of liver infiltration in lymphoproliferative disease

Liver involvement is a common finding in patients suffering from lymphoproliferative disease, and histopathological patterns of infiltration vary according to lymphoma subtype. Data correlating the form of liver involvement with distinct lymphoma subtypes is, however, scarce. The aim was to review 89 liver biopsies diagnosed with lymphoma infiltration and evaluate the infiltration patterns. In equivocal cases, additional immunohistochemical and molecular pathology analyses were performed to differentiate between neoplastic and reactive cell infiltrates and to classify the lymphoma subtypes. Diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukaemia (CLL), Hodgkin's lymphoma (HL) and Burkitt lymphoma (BL) were the most prevalent subtypes in our series, which included 14 different lymphoma entities in total. Whereas DLBCL and BL predominantly demonstrated tumour nodules deranging the normal hepatic architecture, CLL and HL mostly showed infiltration of the portal fields. Interestingly, distinct lymphoma entities, particularly marginal zone B-cell lymphomas (MZL) and HL, commonly revealed lympho-epithelial lesions of bile ducts, which were observed in 10% of all investigated cases. Four cases, initially interpreted as T-cell lymphomas, proved to be reactive T-cell lesions. Distinct lymphoma subtypes show characteristic patterns of liver infiltration. Additional molecular analyses can support diagnosis by verification of clonality or detection of characteristic genetic aberrations.

Plasma cell granuloma of the thyroid gland mimicking carcinoma: A case report and review of the literature

Plasma cell granulomas (PCG) are rare tumor-like lesions consisting of sheets of polyclonal plasma cells admixed with numerous lymphocytes and other inflammatory cells surrounded by fibrous stroma. They usually appear in the lung, but involvement of diverse extrapulmonal sites has been described. PCGs occurring in the thyroid are very uncommon. Since 1981, only 11 cases have been described in the English literature. Here, we present the case of a 50-year-old Arabic man who noticed an enlargement of his thyroid gland during the previous 2 years, and he developed swallowing disturbances and a feeling of narrowness in the neck. A nearly total resection of the thyroid gland was made because of clinical suspicion of carcinoma. On histologic examination, PCG of the thyroid associated with Hashimoto's thyroiditis (HT) was diagnosed. This is the first case in which molecular pathological analyses for EBV and HHV8 DNA were made. As these were negative, distinct etiological features were suggested.

Origins of breast cancer subtypes and therapeutic implications

This Review summarizes and evaluates the current evidence for the cellular origins of breast cancer subtypes identified by different approaches such as histology, molecular pathology, genetic and gene-expression analysis. Emerging knowledge of the normal breast cell types has led to the hypothesis that the subtypes of breast cancer might arise from mutations or genetic rearrangements occurring in different populations of stem cells and progenitor cells. We describe the common distinguishing features of these breast cancer subtypes and explain how these features relate both to prognosis and to selection of the most appropriate therapy. Recent data indicate that breast tumors may originate from cancer stem cells. Consequently, inhibition of stem-cell self-renewal pathways should be explored because of the likelihood that residual stem cells might be resistant to current therapies.

ICon-TMA ® for control and quantitation of immunohistochemistry and in situ molecular pathology analysis

Reliable assessment of in situ pathology analyses like immunohistochemistry, RNA in situ hybridization, and FISH are critically important in diagnosis and treatment decisions. We describe the development of small tissue microarrays (TMA) for the internal control and quantitation of these techniques, the iCon-TMA ®. In its simple variant, these iCon-TMA ® (abbreviated from internal control TMA) consist of 2 tissue cylinders, one being clearly positive and the other clearly negative for a specific marker. In its more elaborate format, the iCon-TMAs ® carry additional tissue spots providing a scale for quantitatively assessable markers, e.g. representing scores of negative, weak, moderate, or strong positivity. The specimen with the yet unknown reactivity is then applied to the iCon-TMA ® section, and both are simultaneously processed and analyzed, thus providing a convenient measure for in situ quality control and quantitation.

Computertomografisch gesteuerte Interventionen bei Kindern

In pediatric CT-guided interventions specific features have to be taken into account. Due to a lack of cooperation or limited ability to cooperate, procedures are often performed using analgosedation or general anesthesia. To provide radiation protection, justified indication for CT-guided intervention is necessary and sonography and MRI are to be preferred whenever possible. CT examinations also need to be dose-adapted with sequential scanning and a tube voltage and tube current reduction compared to pediatric diagnostic CT studies must be ensured. Gonad shields are recommended for male patients. Biopsy device selection depends on the assumed tumor entity since histology and also immunohistochemical, molecular pathological and cytogenetical analysis are necessary to differentiate pediatric tumors (small, round, blue cell tumors). In addition to diagnostic procedures, therapeutic interventions (drainage, injection therapies, neurolysis, and radiofrequency ablation) can also be used in children and can provide an alternative to surgery in selected cases. With justified indications and precise performance, CT-guided interventions can be successful in pediatric patients with limited risks.

Systematic collection of tissue specimens and molecular pathological analysis of newly diagnosed solid cancers among atomic bomb survivors

To elucidate precise mechanism of radiation-induced cancers, it is important to analyze the genetic and epigenetic alterations in cancer cases among atomic bomb (A-bomb) survivors. Stored tissue samples are damaged in a certain extent, and fresh tissue samples are suitable for molecular analyses. We have established a network system comprising major hospitals in Hiroshima area, Hiroshima University and Radiation Effects Research Foundation (RERF) to conduct systematic collection and storage of fresh tissue samples of newly diagnosed solid cancers among A-bomb survivors. The project is carried out as “The Ministry of Health, Labor and Welfare of Japan Group Study on A-bomb Diseases.” The subjects of this study are RERF Life Span Study (LSS) cohort members (> 0 dose) who have been diagnosed with cancer of the stomach, colorectum, esophagus, breast, or lung, and have undergone surgery. As controls, LSS cohort members (= 0 dose) and non-LSS members matched by sex, age, and medical institution are selected. Using the tissue samples thus collected, we are searching for genetic and epigenetic events involved in the development of solid cancers. We have developed custom-made 3-dimension oligo-DNA microarray with a total of 207 genes including those related to DNA damage response and repair. We identified 10 genes whose expression levels in the tumors were significantly different between A-bomb survivors (LSS: > 0 dose) and control subjects. They might be candidate genes which participate in radiation-induced carcinogenesis and possible genetic markers for radiation-induced solid cancer.

Retrospective sequence analysis of the humanPRNP gene from the formaldehyde-fixed paraffin-embedded tissues: Report of two cases of creutzfeldt-jakob disease

The definitive diagnosis of the CJD (Creutzfeldt-Jakob disease; very rare neurodegenerative disorder) can be established only on the basis of post-mortem examination of the central nervous system tissue. Formaldehyde-fixed paraffin-embedded (FFPE) tissue samples may thus constitute the only material available for molecular pathology analyses. We performed post-mortem analysis of the coding region of the prion-protein gene (PRNP)-sequence variations in two definite CJD cases suggestive of genetic form. Only FFPE tissues were available for molecular analyses. The PRNP gene open reading frame was amplified from the genomic DNA (FFPE isolated) in four overlapping, two round semi-nested PCR products that were directly sequenced. We found known pathogenic sequence variation g.532 G>A (Asp178Asn) in patient 1 but we did not find any pathogenic sequence variation in patient 2 despite her origin from the Slovak Orava region. Based on these results, we were able to discriminate between genetic and sporadic form of CJD in patient 1 and 2, respectively. The established method was found to be efficient for the sequence-variation analysis of the entire PRNP gene coding region using the genomic DNA isolated from the FFPE tissues; it can be employed in other retrospective molecular studies.

Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice

Piperonyl butoxide (PBO), α-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes—cytochrome P450 ( Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase ( Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.

Epididymal cystadenomas and epithelial tumourlets: effects of VHL deficiency on the human epididymis

Although epididymal cystadenomas (ECAs) are among the most frequent VHL disease-associated tumours, fundamental questions about their pathogenesis have remained unanswered. Classification of ECAs is controversial, and the cell of origin is unknown. It is also unknown whether ECAs-like other VHL disease-associated tumours-arise as a result of VHL gene inactivation, and whether ECAs exhibit subsequent activation of hypoxia-inducible factor HIF. Moreover, the morphological spectrum of earliest ECA formation is unknown. In a detailed molecular pathological analysis of a series of epididymides collected from VHL patients at autopsy, we found that ECAs are true neoplasms that arise secondary to inactivation of the wild-type copy of the VHL gene, followed by early and simultaneous activation of HIF1 and HIF2 associated with up-regulation of downstream targets, including CAIX and GLUT-1. The observations also indicate that ECA formation evolves from a variety of microscopic epithelial tumourlets, and that these tumourlets are confined to the efferent ductular system. Although genetic and immunohistochemical analysis of precursor structures consistently revealed VHL gene inactivation and activation of HIF in the precursor lesions, only a small subset appears to progress into frank cystadenoma. Thus, ECA tumorigenesis in VHL disease shares fundamental principles with tumorigenesis in other affected organ systems.

Molecular Pathological Analysis of Apoptosis Induced in Testicular Germ Cells after a Single Administration of Mono-(2-ethylhexyl) phthalate to F344 Rats

Mono-(2-ethylhexyl) phthalate (MEHP) is known to cause germ cell apoptosis in the testis of rats as a consequence of Sertoli cell damage. In the present study, a single dose of 2,000 mg/kg body weight MEHP was orally administered to Fischer 344 rats and the molecular apoptosis pathway was surveyed. Histopathological examination, TUNEL assay and immunohistochemical analysis of cleaved caspase-3 were performed to assess the reproducibility of apoptosis. In addition, immunohistochemical analysis and RT-PCR to determine the parameters of the apoptosis pathway were performed. Histopathological examination showed that from 6 to 12 hours after MEHP treatment there was detachment of germ cells that had eosinophilic cytoplasm and pyknotic nuclei. A TUNEL assay detected a significant increase in TUNEL-positive cells at 12 hours after treatment. Immunohistochemistry showed some of the cytoplasm of germ cells was positive for cleaved caspase-3 at 12 hours after treatment. In immunohistochemistry of the Fas/Fas ligand (FasL) apoptosis pathway, the cytoplasm of germ cells was found to be occasionally positive for Fas at 12 hours after treatment, and the expression of the Fas gene in RT-PCR had significantly increased at 12 hours after treatment. RT-PCR of the mitochondrial apoptosis pathway revealed a significant increase in the expression of Bax and Bcl-2 genes in the treated group at 12 hours after treatment. These results strongly suggest that both the Fas/FasL and mitochondrial pathways are involved in germ cell apoptosis induced by MEHP.