Juxta-articular myxoma of the knee in a 5-year-old boy: a case report and review of the literature (2009: 12b).

Juxta-articular myxoma (JAM) is a relatively rare variant of myxoma that occurs in the vicinity of large joints. It is composed of fibroblast-like cells that produce an excessive amount of glycosaminoglycans rich in hyaluronic acid. The peak incidence is between the 3rd and 5th decades of life. In this report we describe an extremely rare case of JAM in the knee of a 5-year-old child. The clinical presentation, radiological features and histopathologic findings are described, and the relevant literature is reviewed.

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer.

AIM: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. METHODS: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 microm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100,000 cells were analysed on the flow cytometer. RESULTS: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple non-SLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. CONCLUSION: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.

Androgen, estrogen and progesterone receptor expression in the human uterus during the menstrual cycle.

Cyclic changes in steroid receptor expression in endometrial cells are considered a reflection of its differential functions. Besides estrogen and progestogens, androgens have also been suggested to affect the biological function of the female reproductive tract. We investigated the distribution and intensity of immuno-cytochemical estrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) staining in the various cell types of human endometrium and myometrium during the different menstrual cycle phases in 30 paraffin-embedded sections.AR expression in endometrial stromal cells decreased gradually from early proliferative till mid secretory phase. In the late secretory phase, AR expression in all cell types distinguished. Staining of epithelial cells was moderate. The disappearance of AR expression before cyclic separation of endometrial tissue may be causally related or just an epiphenomenon. Due to local competition for 5alpha-reduction of testosterone and the excess of progesterone in the secretory phase, the level of dihydrotestosterone (DHT) will be diminished. Hypothetically, if AR synthesis in endometrium would be DHT-dependent, it would explain the lack of AR expression in the late secretory phase.

Improved pre-operative mediastinal staging in non-small-cell lung cancer by serial sectioning and immunohistochemical staining of lymph-node biopsies.

OBJECTIVE: Mediastinal staging of non-small-cell lung carcinoma (NSCLC) by mediastinoscopy suffers from a low sensitivity, leading to a number of patients with unforeseen N2 disease at thoracotomy. This study was undertaken to assess whether pre-operative staging could be improved by serial sectioning and immunohistochemical staining of mediastinoscopy biopsies. METHODS: In 183 consecutive patients with NSCLC, a thoracotomy was performed after a thorough mediastinal staging by computed tomography scan and cervical mediastinoscopy. In 158 patients (88%), a mediastinal node dissection was performed, revealing unforeseen N2 disease in 24 cases (15%). The preserved mediastinoscopy biopsies of these patients were retrospectively serially sectioned and stained with MNF 116. RESULTS: Metastases could be identified in seven cases (30%), reducing unforeseen N2 disease from 15 to 10%. The number of patients who could theoretically benefit from neo-adjuvant therapy would have been increased by at least 10%. CONCLUSIONS: Pre-operative mediastinal staging can be improved considerably by serial sectioning and immunohistochemical staining of mediastinoscopic biopsy specimens.

Paraneoplastic pemphigus as the initial presentation of chronic lymphocytic leukemia.

The case history of a 61-year-old male patient is described, who presented with severe stomatitis, conjunctivitis and leukocytosis. The diagnosis chronic lymphocytic leukemia (CLL) stage A (0) was made, for which no treatment was necessary. Progression of stomatitis and conjunctivitis and erythosquamous skin lesions with bullae and vesiculae formation developed. Under the diagnosis of bullous pemphigoid the patient was treated with corticosteroids. The histologic and immunofluorescence examination of a skin biopsy was compatible with this diagnosis, and antibodies to skin could not be detected in a first serum sample. Pseudomonas was cultured from all lesions, the corticosteroids were stopped and antibiotic treatment was started, without clear effect. Because of progression of skin lesions and debilitation, the patient finally declined all treatment and died five weeks after admission. Post-mortem examination showed enlarged lymphnodes in the cervical, aortal en iliacal areas, with histology confirming the diagnosis of CLL. Indirect immunofluorescence with the second serum sample showed auto-antibodies in high titer directed against the intercellular epithelial substance. Immunoblot studies showed binding with the classic target antigens in paraneoplastic pemphigus. Re-examination of the histologic skin specimen and the result of direct immunofluorescence were in retrospect compatible with the diagnosis of paraneoplastic pemphigus.

A novel flow cytometric steroid hormone receptor assay for paraffin-embedded breast carcinomas: an objective quantification of the steroid hormone receptors and direct correlation to ploidy status and proliferative capacity in a single-tube assay.

Semiquantitative estimation of steroid hormone receptors by immunohistochemistry applied to paraffin sections is common practice in surgical pathology. Flow cytometric (FCM) analysis of estrogen receptor (ER) and progesterone receptor (PR) levels provides a faster and more objective quantitative assay. However, a major problem in such FCM analyses of solid tumor samples is the admixture of tumor cells with normal epithelial, stromal, and inflammatory cells. The aim of the underlying study was to investigate the applicability of a recently developed multiparameter flow cytometric methodology for the accurate estimation of the fraction of steroid hormone receptor-positive tumor cells and to explore whether this multiparameter approach allows the detection of specific, clinically relevant subsets of tumors, based on a combination of ploidy level, steroid hormone receptor status, and cell cycle characteristics. For this purpose, samples of 42 breast cancer patients, from which routine immunohistochemistry for ER and PR also was available, were analyzed. From each case, a cell suspension was prepared from the paraffin block by applying a heating and short pepsin digestion step to 50-microm-thick sections. These cell suspensions were double-immunostained for cytokeratin to identify the epithelial cells, and ER or PR, whereas DNA was quantitatively stained with propidium iodide using an optimized protocol. In the entire group of breast tumors, the percentages of ER- and PR-positive cells were registered in the epithelial subfraction, in combination with DNA ploidy and S phase fraction (SPF). A significant correlation was found between the fraction of hormone receptor-positive cells as found by the immunohistochemical and FCM procedures. For ER, a correlation coefficient of r = .87 was found, and for PR r = .62, both P < .0001. It became clear that all the diploid breast tumors had more than 30% tumor cells positive for ER with a SPF lower than 10%, whereas aneuploid tumors contained on average a smaller percentage of steroid hormone receptor-positive cells, and simultaneously an SPF greater than 10%. Our results show that this multiparameter FCM analysis allows an objective and reproducible quantification of the fraction of steroid hormone receptor-positive cells in the relevant epithelial cell compartment in relation to DNA ploidy status and proliferative capacity in a single-tube assay.

Clonality assessment of lymphoproliferative disorders by multiparameter flow cytometry of paraffin-embedded tissue: an additional diagnostic tool in surgical pathology.

A major drawback of immunohistochemical detection of monoclonality in B-cell lymphoproliferative disorders is the lack of contrast between surface-immunoglobulin staining and extracellular immunoglobulin staining. To bypass this drawback, immunophenotyping of single-cell suspensions by flow cytometry is commonly used. Although the expression of immunoglobulin light chain subtype can be quantified rapidly and reliably, the technique is hampered by the requirement of fresh unfixed material. We applied a recently developed technique for the isolation of single cells from formalin-fixed, paraffin-embedded material to measure clonality in B-cell lymphoproliferative disorders (lymphoid tissue (n = 10) and non-Hodgkin's B-cell lymphoma (n = 10). Immunocytochemistry indicated that common cell surface markers as well as the immunoglobulin light chains could be detected in the cell suspensions derived from archival material. In addition, the technique also allowed combined high-resolution DNA flow cytometric analysis. To investigate the effect of formalin fixation on cross-linking of extracellular immunoglobulins to lymphocytes, a double-immunostaining experiment for both light chain immunoglobulins (kappa and lambda) was performed. This experiment showed that this cross-linking was minimal (less than 2%). All cases of reactive lymphoid hyperplasia were DNA diploid and showed a polyclonal expression of immunoglobulin light chains. In contrast, in 9 of 10 non-Hodgkin's B-cell lymphomas, monoclonality was established on the basis of light chain expression, whereas only 6 of 9 cases were conclusive by immunohistochemistry. Four of the 9 cases were DNA aneuploid. One case did not show light chain expression at all by both techniques. However, this case could be classified as malignant by flow cytometric analysis because of the DNA-aneuploid nature of the B-cell subpopulation. The average S-phase fraction (SPF) of the B cells in the reactive lymphoid tissues was 3.5%. The mean SPF values for B cells in DNA-diploid cases of lymphomas was 3.0%, whereas the mean SPF of B cells in DNA-aneuploid cases was 6.1%. The presented technique is superior to immunohistochemistry for the detection of monoclonality in B-cell lymphoproliferative disorders and therefore provides a powerful tool to support the diagnosis of malignant lymphoma in routinely processed archival samples of lymphoid tissues.

Evaluation of the membrane attack complex of complement for the detection of a recent myocardial infarction in man.

The diagnosis of an acute myocardial infarction (MI) can be cumbersome for pathologists. Even with a positive nitroblue tetrazolium (NBT) reaction, haematoxylin and eosin (H&E) evaluation of the myocardial tissue can remain inconclusive. Early signs presumed diagnostic for myocardial infarction, such as hypereosinophilia, waviness, and contraction band necrosis, have to be considered non-specific and are probably reversible signs of ischaemia. Several studies implicate the complement system, and especially complement factor C9, as part of the membrane attack factor (MAC), in cardiomyocyte damage during MI. In a post-mortem study on well-documented cardiological autopsies, we evaluated the use of complement factor C9 immunostaining as a marker for the detection of very recent MI. Forty-three tissue samples from 40 patients were obtained from the left ventricular free wall only, a region that can be specifically attributed to one corresponding coronary artery. As some patients presented with MIs of various stages in that perfusion area, in total 57 observations were possible. C9 immunostaining specifically detected irreversibly damaged (=infarcted) cardiomyocytes, as is implied by the lytic activity of C9/MAC binding to cell membranes. Most interesting was the group of clinically suspected, NBT-positive MIs resulting from very recent myocardial ischaemia. In this population, where H&E evaluation by (cardio-) experienced pathologists was not conclusive, C9 immunostaining clearly pointed towards myocardial infarction in 47% of the cases. In conclusion, C9 immunostaining, routinely practicable in the pathology laboratory, has an additional value in discriminating between reversible ischaemia and infarcted cardiomyocytes in very early MIs.

A case of herpes-like Sweet's syndrome in acute myelogenous leukemia during treatment with G-CSF.

A 49-year-old patient with refractory acute myelogenous leukemia (AML) is described who developed fever and herpes-like skin lesions during treatment with G-CSF. Skin biopsies revealed dermal neutrophilic infiltrates compatible with the diagnosis of Sweet's syndrome. The fever and skin lesions disappeared completely after treatment with corticosteroids.

Squamous cell carcinoma of suprapubic cystostomy tract without bladder involvement.

This report describes a third case of squamous cell carcinoma of the suprapubic cystostomy tract. The first case reported in 1993 concerned a squamous cell carcinoma arising adjacent to the suprapubic cystostomy site and extending anteriorly to the abdominal wall in a 80-year-old man, 5 years after suprapubic urinary diversion for urethral stricture. A second case published in 1995 described a 50-year-old paraplegic man (T11-T12 spinal cord injury) in whom a suprapubic cystostomy tract squamous cell carcinoma developed after 25 years of urinary diversion. The tumour involved the cystostomy tract primarily with extension into the bladder but did not penetrate the bladder wall muscle. Our patient is in fact the second one to have a suprapubic cystostomy tract squamous carcinoma not involving the bladder.

Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue.

BACKGROUND: Flow cytometry of single-cell suspensions prepared by enzymatic digestion from formalin-fixed, paraffin-embedded tissue suffers from several major drawbacks. The most important factors that influence the results are the high and unpredictable coefficients of variation (CVs) of the G0/G1 peak in the DNA histogram and reduction of propidium iodide (PI) intercalation with DNA, resulting from protein cross-linking by formalin. METHODS: In this study we introduce a heating step (2 h incubation in citrate solution at 80 degrees C) prior to a brief pepsin digestion of tissue sections in the protocol for DNA content analysis of formalin-fixed and paraffin-embedded tissue. This new method is compared with established methods for the preparation of cell suspensions from frozen and paraffin-embedded tissues with respect to cell yield, DNA histogram resolution, DNA dye saturation kinetics, cell cycle parameters, and antigen retrieval in various epithelial and nonepithelial tissues. RESULTS: The recovery of single cells from the paraffin sections was doubled by the heat treatment step, while the limited time of proteolysis resulted in decreased cell debris. Furthermore, an increased fraction of cells became cytokeratin-positive, while these immunocytochemically stained cells also exhibited a higher mean fluorescence intensity. The DNA histograms prepared from cell suspensions obtained according to this new protocol showed a significantly improved resolution, leading to a better identification of peridiploid cell populations. Heat pretreatment of paraffin-embedded archival tissue sections showed PI saturation kinetics similar to, or even better than, those of fresh unfixed tissues, independent of duration of fixation. CONCLUSIONS: This new method, making use of routinely available antigen retrieval principles, thus allows high-resolution DNA analysis of routinely fixed and paraffin-embedded tissue samples. Using external reference cells, inter- and intralaboratory standardization of DNA histograms can be achieved.

Chylothorax or leakage of total parenteral nutrition?

The diagnosis chylothorax is based on a chemical analysis of the pleural effusion. According to the literature, this analysis can be rather straightforward, comprising measurements of triglycerides, chylomicrons, and cholesterol. In this report we present an autopsy case that alerted us to interpret these results critically. Although the laboratory tests of the pleural effusion in this patient with parenteral nutrition suggested chylothorax, additional tests (potassium (11.3 mmol.L(-1)) and glucose (128 mmol.L(-1)) proved otherwise. Comparison of the pleural effusion analysis and the content of the parenteral nutrition led to the final conclusion that the effusion was due to a leakage of parenteral nutrition instead of chylothorax. We therefore suggest adding glucose and potassium measurements to the biochemical work-up of a patient under suspicion of chylothorax.

Detection of K-ras point mutations in sputum from patients with adenocarcinoma of the lung by point-EXACCT.

PURPOSE: Kirsten ras (K-ras) point mutations are found in 30% to 56% of pulmonary adenocarcinomas by means of highly sensitive techniques. Recently, the Point-EXACCT (point mutation detection using exonuclease amplification coupled capture technique) method was described, which detected one cell with a mutation in 15,000 normal cells. The aim of this study was to examine whether K-ras point mutations could be found with this rapid method in the sputum of patients with adenocarcinoma of the lung. PATIENTS AND METHODS: DNA from paraffin-embedded adenocarcinoma and corresponding sputum samples were analyzed for mutations of the K-ras gene. Twenty-eight biopsy specimens and 54 sputum samples of 22 patients were used for amplification and K-ras codon 12 point mutation detection. RESULTS: In 11 of 22 patients (50%), a mutation in K-ras codon 12 was shown in the tumor sample. In five of 11 patients (45%) with a K-ras mutation in the tumor, the same type of mutation was identified in at least one sputum sample. A mutation could not be detected in any of the sputum samples from patients with a K-ras-negative tumor. Time between K-ras point mutation detection in sputum and clinical diagnosis of lung cancer varied from 1 month to almost 4 years. In two of the five patients with K-ras-positive sputum specimens, malignant cells were found with cytologic examination. CONCLUSION: Point-EXACCT is suitable for the detection of K-ras point mutations in sputum samples of patients with adenocarcinoma of the lung. This approach may be an important adjunct to cytology in the early diagnosis of lung cancer.

Validation of the Point-EXACCT method in non-small cell lung carcinomas.

K-ras point mutations are often detected in part of the lung carcinomas. For the validation of a highly sensitive and rapid assay for known point mutations, Point-EXACCT (Biochim Biophys Acta 1998; 1379:42-52), we analyzed 89 non-small cell lung carcinomas and compared the results with two sequencing methods. No point mutations were found with double-stranded sequencing. Single-stranded sequencing detected six patients positive for K-ras codon 12. When Point-EXACCT was used, K-ras codon 12 mutations were detected in 8 of 52 patients with squamous cell carcinomas, 10 of 29 patients with adenocarcinomas, and 3 of 8 patients with large cell carcinomas. The finding of K-ras mutations in squamous cell carcinomas is explained by the high sensitivity of the method. Therefore, Point-EXACCT may be applicable to detection of those alterations occurring at a low frequency among an excess of cells with wild-type DNA.

Reduced E-cadherin expression is associated with increased lymph node metastasis and unfavorable prognosis in non-small cell lung cancer.

E-cadherin is a calcium-dependent, epithelial cell adhesion molecule whose reduced expression has been associated with tumor dedifferentiation and increased lymph node metastasis in clinical studies involving several carcinomas. In this study, 111 patients who had previously undergone complete resection and systematic mediastinal lymph node dissection for non-small cell lung cancer (NSCLC) were studied retrospectively. In the primary tumor, as well as in the lymph node metastases, E-cadherin expression was detected by immunohistochemistry using a monoclonal antibody (HECD-1; Takara, Otsu, Japan). There was a significant inverse correlation between E-cadherin expression and lymph node stage (Pearson correlation coefficient -0.52, p = 0.0001) as well as tumor differentiation (Pearson correlation coefficient -0.27, p = 0.005). Moreover, Kaplan and Meier survival estimates showed a significant correlation between E-cadherin expression and patient survival in log rank testing (p = 0.006). In the patient group with the highest proportion of E-cadherin positive tumor cells, 60% of the patients were still estimated to be alive at 36 mo, versus 32% of the patients in the group classified as showing negative E-cadherin expression. Our findings provide clinical evidence that reduced E-cadherin expression is associated with tumor dedifferentiation, increased lymphogenous metastasis and poor survival. It seems therefore that E-cadherin expression might be an important prognostic factor in NSCLC.

E-cadherin and calretinin: a useful combination of immunochemical markers for differentiation between mesothelioma and metastatic adenocarcinoma.

AIMS: To assess the diagnostic accuracy of combinations of antibodies in the differential diagnosis of metastatic carcinomas and mesotheliomas in pleural lesions. METHODS AND RESULTS: The specificity and sensitivity of the commercially available antibodies Ber-EP4, MOC-31, CEA, B72.3, CD15, E-cadherin and calretinin were evaluated using an indirect immunoperoxidase staining technique. This technique was applied to formalin-fixed, paraffin-embedded tissue blocks of pleural lesions. Twenty-one patients with metastatic carcinoma (MC) and 20 patients with malignant mesothelioma (MM) were included. The combination E-cadherin/calretinin had the highest specificity (MC 100% and MM 91%) and sensitivity (MM 100% and MC 91%) considering both categories of tumours. CONCLUSIONS: The combination of E-cadherin/calretinin appears to be a suitable panel for distinguishing metastatic carcinomas and mesotheliomas in pleural lesions. The additional value of other markers is limited.

Relationship between ovarian production of estrone, estradiol, testosterone, and androstenedione and the ovarian degree of stromal hyperplasia in postmenopausal women.

OBJECTIVE: To study the relationship between ovarian production of estrone (E ), estradiol (E2), testosterone (T), and androstenedione (A) and the ovarian degree of stromal hyperplasia in postmenopausal women. DESIGN: In 18 postmenopausal women, the ovarian vein hormone levels of E1, E2, T, and A were compared with the degree of ovarian stromal hyperplasia. The degree of stromal hyperplasia was assessed by histological analysis (group 1: atrophic ovaries, n = 8; group 2: slight stromal hyperplasia, n = 8; group 3: moderate or severe stromal hyperplasia, n = 2). RESULTS: The ovarian levels of E1 and E2 did not correlate with the ovarian degree of stromal hyperplasia. The ovarian levels of A in group 3 were significantly higher than those in groups 1 and 2 (p < 0.02 and p < 0.01, respectively). The ovarian levels of T in group 3 were significantly higher than those in group 1 (p < 0.01) but did not differ significantly from those in group 2. CONCLUSIONS: The amount of stromal hyperplasia in postmenopausal ovaries is correlated with the ovarian vein levels of A and T. Morphological characteristics of the postmenopausal ovary determine the local (pelvic) endocrine status and may play a role in the etiology of hormone-dependent diseases of the internal genitals.

The expression of beta-catenin in non-small-cell lung cancer: a clinicopathological study.

AIMS: To investigate the expression of beta-catenin in non-small-cell lung cancer (NSCLC) and its clinical significance. METHODS: 101 patients were surgically treated for NSCLC by lobectomy or pneumectomy with systematic lymph node dissection. Follow up was available in all patients, ranging from 24 to 110 months. Immunostaining of tissue sections from primary tumours and (when present) their lymph node metastases was performed and evaluated using a monoclonal antibody against beta-catenin. Correlations were investigated between beta-catenin immunostaining in primary tumours and E-cadherin immunostaining (data available from a previous study), lymph node stage, and survival. RESULTS: There were significant correlations between scores for beta-catenin immunostaining and E-cadherin immunostaining in primary tumours (p = 0.007), and between the beta-catenin immunostaining score in primary tumours and in their lymph node metastases (p = 0.006). An inverse correlation was found between the beta-catenin immunostaining score in primary tumours and lymph node stage N0, N1, or N2 (p = 0.03). According to the Kaplan-Meier survival estimate, the level of beta-catenin expression in primary tumours was a statistically significant prognostic factor (p = 0.01). CONCLUSIONS: Reduced beta-catenin expression in surgically treated NSCLC is clearly associated with lymph node metastasis and an infavourable prognosis. The existence of a functional relation between E-cadherin and beta-catenin is supported by the results of this clinicopathological study.

[Pseudomyxoma peritonei]. / Pseudomyxoma peritonei.

Pseudomyxoma peritonei was diagnosed in 3 men aged 38, 66 and 54 years with weight loss and distension of the abdomen. Pseudomyxoma peritonei results from seeding of the peritoneal cavity with mucus-producing epithelium. The disease is traditionally characterized by accumulation of huge amounts of mucinous ascites, relatively long survival and absence of distant, extraperitoneal metastases. Mostly, the primary tumour is an appendicular adenoma or adenocarcinoma. Sometimes, the primary tumor is localized in the ovaries. Extensive surgical debulking with postoperative intraperitoneal chemotherapy appears to be the treatment of choice.