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Efficacy of ImageJ in the Assessment of
Apoptosis
ObjectiveTo verify the efficacy of ImageJ 1.43n in determining the extent of apoptosis which is a complex and multistep process.Study Design: Cisplatin in different concentrations was used to induce apoptosis in cultured Hep2 cells. Cell viability assay and nuclear image analysis of stained Hep2 cells were used to discriminate apoptotic cells and cells suspected of undergoing apoptosis from control cells based on parameters such as nuclear area, circularity, perimeter and nuclear area factor (NAF), in association with visual morphology.
Results:
Image analysis revealed a progressive and highly significant decrease in nuclear area factor detected in apoptotic cells and in cells suspected of undergoing apoptosis compared to the control cells (P-values <0.01). Some of the other studied parameters showed also the same trend. This decrease was assumed to indicate DNA loss. Image analysis results correlated positively and significantly with the results obtained by cell viability assay (R=0.958, P-value= 0.042). NAF was the most reliable parameter in assessment of apoptosis.
Conclusion:
Nuclear area factor can be calculated using powerful free and open-source software. Consequently, a quantitative measure of apoptosis can be obtained that is linked to morphological changes. ImageJ 1.43n may therefore provide a useful tool for the assessment and discrimination of apoptotic cells.Virtual slides: The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/5929043086367338
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EGFR, CD10 and proliferation marker Ki67 expression in Ameloblastoma: possible role in local recurrence
Background:
Ameloblastoma is an odontogenic neoplasm characterized by local invasiveness and tendency towards recurrence.AimsStudying the role played by EGFR, CD10 and Ki67 in the recurrence of ameloblastoma.
Methods:
This study was carried out on 22 retrospective cases of mandibular ameloblastoma from the period from Jan 2002 to Jan 2008 with follow up period until Jan 2011 (minimal 3 years follow up). Archival materials were obtained from pathology department, Mansoura university. Paraffin sections of tumor tissue from all cases were submitted for routine H&E stains and immunohistochemistry using EGFR, CD10 and Ki67 monoclonal antibodies.Statistical analysis using of clinical data for all patients, tumor type, EGFR, CD10 and Ki67 expression in relation to recurrence were evaluated.
Results:
Among the 22 cases, 10 cases were males and 12 were females with sex ratio 1:1.2. Age ranged from 30 to 60 years old with a mean age 48 year. Five cases showed local recurrence within studied period and proved by biopsy. No statistically significant relation was found between local recurrence and patient age, tumor size, tumor type, EGFR expression. There was a significant relation between CD10 expression as well as Ki67 labelling index and recurrence (P value =0.003, 0.000 respectively).
Conclusion:
Evaluation of CD10 and Ki67 status together with conventional histological evaluation can help in providing more information about the biologic behavior of the tumor, while EGFR could be a target of an expanding class of anticancer therapies.Since ameloblastomas are EGFR-positive tumors, anti-EGFR agents could be considered to reduce the size of large tumors and to treat unresectable tumors that are in close proximity to vital structures.
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Worldwide variations in EGFR somatic mutations: a challenge for personalized medicine
Two studies recently reported around 10% of EGFR activating mutations in triple negative breast cancers from Asian patients. However, we did not find any EGFR activating mutation in a series of 229 breast tumor samples from European patients. Like in lung cancer, the EGFR mutation profiles seem to be related to the ethnical origin of patients. This is an important point that should be considered when developing anti-EGFR therapies.
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CD163 Versus CD68 in Tumor Associated Macrophages of Classical Hodgkin Lymphoma
Classical Hodgkin lymphoma (CHL) is a B-cell lymphoproliferative disorder with a relatively good prognosis. A small but significant percentage of patients, however, will respond poorly to therapy. A recent gene expression profiling study has identified a macrophage signature which has been correlated with primary treatment failure, and immunohistochemical tissue microarray for CD68 was shown to reflect the gene signature as a potentially clinically useful marker to predict adverse prognosis.We examined 44 cases of CHL, mostly nodular sclerosis subtype, in which the immunohistochemical stains for the histiocytic markers CD68 and CD163 were performed. The staining intensity was graded for each stain (<5, 5-25, and >25 percent of cells positive in the Hodgkin cell (HC) rich nodules) and background staining characteristics were recorded.CD163 staining was lower than CD68 in HC rich nodules, with lower background staining (p 0.03). There was no significant difference between either CD68 or CD163 and disease recurrence in a subset (N=41) of cases.In conclusion, we demonstrate that CD163 staining is lower than CD68, with less non-specific staining of background inflammatory cells and Hodgkin cells, therefore is a better marker for tumor associated macrophages. However, we did not identify a correlation between staining for CD68 or CD163 and recurrence of disease.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1460518258831620
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Brain MRI and biological diagnosis in five Tunisians MLD patients
Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is characterized by an accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA).we studied 5/200 cases of MLD and clearly distinguished three clinical forms. One of them presented the juvenile form; two presented the late infantile form; and two other presented the adult form. The Magnetic Resonance Imaging (MRI) of these patients showed a diffuse, bilateral and symmetrical demyelination. The biochemical diagnosis of MLD patients evidencing the low activity of ASA and sulfatide accumulation.Keys words: arylsulfatase A, urinary sulfatide, sulfatide, demylinisation, metachromatic leukodystrophy.Patients and methods: We studied 5/200 MLD patients addressed to us for behavioral abnormalities and progressive mental deterioration. All of them were diagnosed at first by brain MRI evidencing a bilateral demyelination, then the measurement of ASA activity using P-nitrocathecol sulfate as substrate, finally the sulfatiduria was performed using thin-layer chromatography using alpha-naphtol reagent.
Results:
In this study, from 200 patients presenting behavioral abnormalities and a progressive mental deterioration, we reported just 2patients were diagnosed as late-infantile form of MLD. Only1 case presented as the juvenile form; and 2 patients with the adult-type of MLD. The brain magnetic resonance imaging (MRI) of all patients showed characteristic lesions of MLD with extensive demyelination. Biochemical investigations of these patients detected a low level of ASA activity at 0degreesC and 37degreesC; the excess of sulfatide in sulfatiduria.
Conclusion:
MRI is required to orient the diagnosis of MLD patients; the latter must be confirmed by the biochemical investigations which is based on the measurement of ASA activity and the excess of sulfatide showed in the sulfatiduria.Virtual slides:The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1791578262610232
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Carcinoma involving the gallbladder: A retrospective review of 23 cases - pitfalls in diagnosis of gallbladder carcinoma
Background:
Carcinoma of the gallbladder (GBC) clinically mimics benign gallbladder diseases and often escapes detection until advanced stage. Despite the frequency of cholecystectomy, diagnosis of GBC remains problematic in many situations. We sought to identify pathologic features that contribute to the difficulty in recognition of GBC.
Methods:
We identified 23 patients (ranged from 45 to 86 years, male to female ratio 1:4.5) with carcinoma involving the gallbladder referred to an academic medical center over a period of 10 years for study. This includes 10 cases of primary GBC, 6 cases of metastatic tumor to gallbladder, 6 cases of directly invasive adenocarcinoma arising elsewhere in the biliary tree, and one case of unidentified origin adenocarcinoma. Primary tumors include adenocarcinoma not otherwise specified (NOS) in 6 cases, papillary adenocarcinoma in 2 cases, and single cases of undifferentiated carcinoma and combined adenocarcinoma and neuroendocrine carcinoma (NEC). Metastatic tumors to gallbladder were from a wide range of primary sites, predominantly the gastrointestinal tract.
Results:
These cases illustrate seven potential pitfalls which can be encountered. These include: 1) mistakenly making a diagnosis of adenocarcinoma of gallbladder when only benign lesions such as deeply penetrating Rokitansky-Aschoff sinuses are present (overdiagnosis), 2) misdiagnosing well-differentiated invasive carcinoma with minimal disease as benign disease (underdiagnosis), 3) differentiating between primary NEC of gallbladder and metastasis, 4) confusing primary mucinous adenocarcinoma of gallbladder with pseudomyxoma peritonei from a low grade appendiceal neoplasm disseminated to gallbladder, 5) confusing gangrenous necrosis related to cholecystitis with geographic tumoral necrosis, 6) undersampling early, grossly occult disease, and 7) misinterpreting extracellular mucin pools.
Conclusions:
Clinical history and a high index of suspicion are prerequisite to detecting GBC. Detection of GBC at an early stage is difficult because the symptoms mimic benign gallbladder diseases. Misinterpretation of subtle microscopic abnormalities contributes diagnostic failures in early cases. Careful attention to any evidence of mural thickening, thorough sampling, particularly in older patients, and close examination of any deeply situated glandular structures are critical. Correlations with radiographic and clinical findings are important helps to avoid misdiagnosis in this commonly resected organ.
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The value of CDX2 and cytokeratins 7 and 20 expression in differentiating colorectal adenocarcinomas from extraintestinal gastrointestinal adenocarcinomas: cytokeratin 7/20+ phenotype is more spesific than CDX2 antibody
Background/Objective: Metastatic adenocarcinoma from an unknown primary site is a common clinical problem. Determining the cytokeratin (CK) 7/CK20 pattern of tumors is one of the most helpful procedures for this purpose since the CK7/CK20+ pattern is typical of colorectal adenocarcinomas. CDX2, a critical nuclear transcription factor for intestinal development, is expressed in intestinal epithelium and adenocarcinomas. In the present study, we compared the sensitivity and specificity of CDX2 expression and the CK7/CK20+ phenotype in differentiating colorectal adenocarcinomas from pancreatic and gastric adenocarcinomas.
Methods:
CK7/CK20 staining pattern and CDX2 expression were evaluated in 118 cases of colorectal, 59 cases of gastric, and 32 cases of pancreatic adenocarcinomas. The sensitivity, specificity, and positive and negative predictive values of the CK7/CK20+ phenotype and of CDX2 expression were analyzed.
Results:
The CK7/CK20+ immunophenotype was expressed by 75 of 118 (64%) colorectal and 3 of 59 (5%) gastric tumors and was not observed in any pancreatic adenocarcinomas. The CK7+/CK20+ immunophenotype was expressed in 24/118 (20%) of colon, 28/59 (48%) of gastric and 7/32 (22%) of pancreatic adenocarcinomas. The CK7+/CK20 expression pattern was observed in only 2% (2 of 118) of colorectal carcinomas. CDX2 was expressed in 114 of 118 (97%) colorectal, 36 of 59 (61%) gastric, and 5 of 32(16%) pancreatic adenocarcinomas. There was no significant association between CDX2 expression and tumor differentiation in colorectal carcinomas. In gastric carcinomas, CDX2 expression was more common in intestinal type tumors than in diffuse type carcinomas. The CK7/CK20+ phenotype showed a specificity of 96.7% in predicting colorectal adenocarcinomas, which was superior to that of CDX2 expression. CDX2 expression at both cut-off levels (5% and 50%) had a higher sensitivity (96.6% and 78%) than the CK phenotype.
Conclusions:
Both the CK7/CK20+ phenotype and expression of the antibody CDX2 are highly specific and sensitive markers of colorectal origin. CDX2 expression should be a useful adjunct for the diagnosis of intestinal adenocarcinomas, particularly when better established markers such as CK7 and CK20 yield equivocal results. The CK7/CK20+ phenotype is superior in its specificity and positive predictive value and might be preferred.Virtual slides:The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/4851011866354821
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Concurrent hypermethylation of DNMT1, MGMT and EGFR genes in progression of gliomas
Background:
Gliomas are the most common neoplasm of the brain. High-grade gliomas often resist treatment even with aggressive surgical resection and adjuvant radiation and chemotherapy. Despite the combined treatment, they frequently recur with the same or higher-grade histology. Genetic instability is commonly associated with inactivation of the normal DNA repair function and tumour suppressor genes as well as activation of oncogenes resulting from alterations of promoter hypermethylation, but the molecular mechanisms of the histological and clinical progression of gliomas are still poorly understood.
Methods:
This study involved longitudinal analysis samples of primary and recurrent gliomas to determine whether the progression of low- and high-grade gliomas is associated with the promoter methylation of the DNMT1, MGMT and EGFR genes by PCR-based restriction enzyme assay. Epigenetic inactivation of these three important glioma-associated genes was analyzed in paired biopsy samples from 18 patients with tumour recurrence.
Results:
The methylation analysis of the CpG sites in the DNA methyltransferase (DNMT1) promoter revealed a total of 6 hypermethylations (6/18), the methylguanine-DNA methyltransferase (MGMT) promoter revealed a total of 10 hypermethylations (10/18) and the epithelial grow factor receptor (EGFR) promoter revealed a total of 12 (12/18) hypermethylations respectively in recurrent gliomas. The results demonstrated that DNMT1 promoter hypermethylation does not occur in low-grade gliomas, it was mainly observed in secondary glioblastomas. Additionally, the MGMT and EGFR promoter was hypermethylated in both low- and high-grade GLs and their corresponding histological transformed GLs.
Conclusion:
This study has provided further evidence that the histological transformation and progression of gliomas may be associated with the inactivation of the EGFR and MGMT genes. It seems that EGFR and MGMT promoter hypermethylations are early events in the clonal evolution of gliomas and this gene inactivation has proved to be stable even in tumour recurrence. However, the DNMT hypermethylation is a late part of glioma progression.Virtual slides: The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1935054011612460
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Secondary cutaneous Epstein-Barr virus-associated diffuse large B-cell lymphoma in a patient with angioimmunoblastic T-cell lymphoma: a case report and review of literature
Only a few cases of extranodal Epstein-Barr virus (EBV)-associated B-cell lymphomas arising from patients with angioimmunoblastic T-cell lymphoma (AITL) have been described. We report a case of AITL of which secondary cutaneous EBV-associated diffuse large B-cell lymphoma (DLBCL) developed after the initial diagnosis of AITL. A 65-year-old Chinese male patient was diagnosed as AITL based on typical histological and immunohistochemical characteristics in biopsy of the enlarged right inguinal lymph nodes. The patient initially received 6 cycles of chemotherapy with CHOP regimen (cyclophosphamide, vincristine, adriamycin, prednisone), but his symptoms did not disappear. Nineteen months after initial diagnosis of AITL, the patient was hospitalized again because of multiple plaques and nodules on the skin. The skin biopsy was performed, but this time the tumor was composed of large, polymorphous population of lymphocytes with CD20 and CD79a positive on immunohistochemical staining. The tumor cells were strong positive for EBER by in situ hybridization. The findings of skin biopsy were compatible with EBV-associated DLBCL. CHOP-R chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab) was then administered, resulting in partial response of the disease with pancytopenia and suppression of cellular immunity. To our knowledge, this is the first case of cutaneous EBV-associated DLBCL originated from AITL in Chinese pepole. We suggest the patients with AITL should perform lymph node and skin biopsies regularly in the course of the disease to detect the progression of secondary lymphomas.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1197421158639299
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Mechanisms of Cyst Formation in Metastatic Lymph Nodes of Head
and Neck Squamous Cell Carcinoma
Cystic change in metastatic lymph nodes occurs in certain types of tumors and mostly in squamous cell carcinoma of the head and neck. In the majority of cases, psuedocystic change is the mechanism of cyst formation. However, sometimes a true cyst cavity is formed. This occurrence is unexplained and some theories are introduced to explain it. In this paper, related articles and introduced concepts are reviewed and the best conclusions of present hypotheses are provided. Cystic SCC in cervical lymph node is now considered as a typical presentation of metastatic SCC arising in the oro/nasopharynx. True cystic cavities have eosinophilic fluid content and present active transport mechanism across the epithelium; Cytokeratin7 is also expressed in the lining of these cysts, which is an accepted marker of ductal differentiation. These are all strong evidences that show salivary gland type cells are present among tumor cells. In fact, some squamous cell carcinomas, especially those arising in Waldeyer's ring, originate from minor salivary glands. The other probability is that these tumors are cancers of transitional type and arise from transformed keratinocytes, which have intrinsic property for cyst formation. These malignant cells in lymph nodes, rather than primary sites, found the opportunity to express their parental property.Virtual slides: The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/6838476096250792
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