A survey of immunohistochemical biomarkers for basal-like breast cancer against a gene expression profile gold standard.

Gene expression profiling of breast cancer delineates a particularly aggressive subtype referred to as 'basal-like', which comprises ∼15% of all breast cancers, afflicts younger women and is refractory to endocrine and anti-HER2 therapies. Immunohistochemical surrogate definitions for basal-like breast cancer, such as the clinical ER/PR/HER2 triple-negative phenotype and models incorporating positive expression for CK5 (CK5/6) and/or EGFR are heavily cited. However, many additional biomarkers for basal-like breast cancer have been described in the literature. A parallel comparison of 46 proposed immunohistochemical biomarkers of basal-like breast cancer was performed against a gene expression profile gold standard on a tissue microarray containing 42 basal-like and 80 non-basal-like breast cancer cases. Ki67 and PPH3 were the most sensitive biomarkers (both 92%) positively expressed in the basal-like subtype, whereas CK14, IMP3 and NGFR were the most specific (100%). Among biomarkers surveyed, loss of INPP4B (a negative regulator of phosphatidylinositol signaling) was 61% sensitive and 99% specific with the highest odds ratio (OR) at 108, indicating the strongest association with basal-like breast cancer. Expression of nestin, a common marker of neural progenitor cells that is also associated with the triple-negative/basal-like phenotype and poor breast cancer prognosis, possessed the second highest OR at 29 among the 46 biomarkers surveyed, as well as 54% sensitivity and 96% specificity. As a positively expressed biomarker, nestin possesses technical advantages over INPP4B that make it a more ideal biomarker for identification of basal-like breast cancer. The comprehensive immunohistochemical biomarker survey presented in this study is a necessary step for determining an optimized surrogate immunopanel that best defines basal-like breast cancer in a practical and clinically accessible way.

Heparanase expression in periapical granulomas and radicular cysts.

Heparanase is an endo-ß-D-glucuronidase enzyme which degrades heparan sulfate glycosaminoglycan side chains of proteoglycans in the extracellular matrix and in basement membranes. The aim of this study was to evaluate the expression of heparanase in periapical granulomas (PGs) and radicular cysts (RCs). Immunohistochemistry was used to assess heparanase expression in PGs and RCs. Parameters including stain intensity, location and cell type were used to characterize heparanase expression in the periapical lesions. Ordered categories (from weak to strong) were used to compare the level of heparanase staining in the PG and RC groups. Both epithelial cells and inflammatory cells were positive for heparanase. The relative staining of the epithelial cells was strong, whereas the relative staining of the inflammatory cells was weak. Significant differences in immunohistochemical staining of epithelial cells were observed between RCs and PGs (p = 0.002). The relative expression of heparanase in epithelial cells in RCs was strong. In PGs, lesions with few or no epithelial cells, heparanase was predominantly expressed weakly by inflammatory cells. PGs and RCs have the same infectious origin. Therefore, the different cellular sources of heparanase in these periapical lesions may imply that this enzyme has specific pathogenetic functions in RCs and PGs.

Efficient purification of eosinophils from human tissues: a comparative study.

BACKGROUND: Eosinophils are key effector cells in allergy and in other inflammatory diseases. Although they carry out their function in the tissues, no efficient method exists allowing for consistent purification of tissue eosinophils for culture. Rather, studies rely mainly on peripheral blood eosinophils. This study aimed to determine the most efficient protocol for purifying eosinophils from nasal polyp tissue. METHODS: Nasal polyps were obtained from patients undergoing surgical polypectomy. The polyps were minced and enzymatically digested. Surface receptor analysis was performed by flow cytometry. In order to obtain optimal purification, the nasal polyp cell suspension was subjected to two methods of purification: 1) positive magnetic selection of CCR3+cells, or 2) negative selection using CD3/CD14/CD16 magnetic beads. Enriched tissue eosinophils were cultured with or without IL-3, IL-5 or GM-CSF, and their survival was evaluated by flow cytometry. RESULTS: Tissue-derived eosinophils exhibited surface expression of NEC2, DNAM-1, NTBa, 2B4, and CD300a comparable to similarly prepared eosinophils obtained from the peripheral blood of the same patients. Positive selection consistently yielded eosinophils of high purity (>90%) with 63% viability. In contrast, negative selection yielded better viability (88%), reduced purity (66%), and could be utilized for in vitro activation experiments. CONCLUSION: Eosinophils can be purified from nasal polyps. Negative selection appears to be advantageous due to improved viability of the eosinophils, which may be cultured and activated in vitro. This methodology is an important advance in studying tissue eosinophils for further investigations on inflammatory tissue responses.

Heparanase expression: a potential ancillary diagnostic tool for distinguishing between malignant cells and reactive mesothelium in body cavity effusions.

OBJECTIVE: Heparanase, an endoglycosidase that cleaves heparan sulphate, is frequently expressed in carcinomas and was suggested to play a role in cell invasion and metastasis. We investigated whether heparanase expression may serve as a reliable marker to discriminate benign mesothelial cells from malignant cells shed into body cavities. METHODS AND RESULTS: Cytological smears of effusions from 51 hospitalized patients were immunostained for heparanase. Strong immunoreactivity was noted in 35 of 40 (88%) carcinoma samples and in all three malignant mesothelioma cases. Only rare (<3%) reactive mesothelial cells were noted showing a faint negligible staining. Specificity was 100%, sensitivity 88%, and positive and negative predictive values were 100% and 89% respectively. CONCLUSIONS: Our results suggest that heparanase may be of value as a complementary component in a diagnostic panel of markers, contributing to its reliability and accuracy.

Adoptive transfer of small numbers of DX5+ cells alleviates graft-versus-host disease in a murine model of semiallogeneic bone marrow transplantation: a potential role for NKT lymphocytes.

Natural killer T (NKT) lymphocyte cells are a subset of regulatory lymphocytes with important immunemodulatory effects. Our aim was to evaluate the effect of transplantation of NKT lymphocytes on graft versus host disease (GVHD) in a murine model of semiallogeneic BMT. GVHD was generated by infusion of 2 x 107 splenocytes from C57BL/6 donor mice into irradiated (C57BL/6 x Balb/c)F1 recipient mice. Adoptive transfer of increasing numbers of DX5+ cells was performed. Recipient mice were followed for histological parameters of GVHD-associated liver, bowel, and cutaneous injury. Intrahepatic and intrasplenic lymphocytes were isolated and analyzed by FACS for CD4+ and CD8+ subpopulations. It was seen that adoptive transfer of 4.5 x 106 DX5+ cells significantly alleviated GVHD-related hepatic, bowel, and cutaneous injury, and improved survival (85% survival on day 28). In contrast, depletion of DX5+ cells led to severe GVHD-associated multiorgan injury and 100% mortality. A direct correlation with the number of transplanted DX5+ cells was noted (maximal effect with transplantation of 4.5 x 106 DX5+ cells). Tolerance induction was associated with an increased peripheral CD4/CD8 ratio, intrahepatic trapping of CD8 lymphocytes and a shift towards a Th2-type cytokine profile, manifested by decreased IL-12/IL10, IL-12/IL-4, IFNgamma/IL-10, and IFNgamma/IL-4 ratios. Transplantation of DX5+ cells holds promise as a novel therapeutic measure for GVHD.

Induction of oral tolerance in bone marrow transplantation recipients suppresses graft-versus-host disease in a semiallogeneic mouse model.

Graft-versus-host disease (GVHD) is the major obstacle for successful allogeneic stem cell transplantation (SCT). Morbidity and mortality are high, and novel therapeutic strategies are required. Current therapy, which is based mainly on immunosuppression, is associated with a high degree of complications. Immune hyporesponsiveness induced by oral antigen administration has recently been shown to prevent the development of chronic GVHD (cGVHD) in a murine model. The aim of the present study was to evaluate whether it is possible to induce tolerance and to alleviate GVHD in a semiallogeneic transplantation model in mice. GVHD was generated by infusing 2 x 10(7) splenocytes from C57BL/6 donor mice into (C57BL/6 x Balb/c)F1 recipient mice, which received 7 Gy (60)Co total body irradiation (TBI) prior to transplantation. Oral tolerance was induced by feeding recipient F1 mice with five oral doses of proteins, 50 micro g/mouse, extracted from C57BL/6 splenocytes on alternate days following transplantation. In vitro mixed lymphocyte reaction (MLR) from tolerized and nontolerized mice was performed. Recipient mice were followed for chimerism, and for clinical and histological parameters of GVHD. Induction of tolerance was documented by a significant reduction in MLR response of tolerated vs nontolerated splenocytes. A significant alleviation of the clinical and pathological manifestation of GVHD was observed in the liver, small bowel, and skin. Tolerance induction did not jeopardize engraftment. These results may constitute a step towards reducing the frequency of GVHD via manipulation of the immune system.

CT-guided biopsy with cutting-edge needle for the diagnosis of malignant lymphoma: experience of 267 biopsies.

AIM: We performed a retrospective study of 267 core needle aspiration biopsies in order to estimate the accuracy of CT-guided aspiration core needle biopsies for the diagnosis and subsequent treatment of malignant lymphoma. MATERIALS AND METHODS: Between 1989 and 1999, 267 CT-guided core needle biopsies were performed in 241 patients with either primary or recurrent malignant lymphoma. Patients age ranged from 4--88 years. One hundred and sixty-six (62.2%) nodal and 101 (37.8%) extranodal aspiration biopsies were performed using either 18G or 20G Turner needles. Statistical method used was Chi-square analysis. RESULTS: An accurate histological diagnosis was made in 199 (82.5%) patients, the remaining 42 (17.4%) patients had non-diagnostic CT biopsies. Thirty-seven of them were diagnosed by a surgical biopsy, four by bone marrow biopsy and in one patient by paracentesis. One hundred and seventy-nine patients had non-Hodgkin's lymphoma (NHL) and 62 had Hodgkin's disease (HD); 23 (9.54%) patients underwent repeated CT biopsy which was diagnostic in 17 (73.9%) and non-diagnostic in six (26%). CONCLUSION: CT-guided aspiration core biopsies were sufficient to establish a diagnosis in lymphoproliferative disorders in 82.5% of cases. In the light of this experience we suggest that imaging-guided core needle biopsy be used as the first step in the work up of many patients with lymphoma.

Percutaneous image-guided needle biopsy in children--summary of our experience with 57 children.

BACKGROUND: Percutaneous image-guided needle biopsy in children has been slower to gain acceptance than in adults where it is regarded as the standard clinical practice in screening suspicious masses. OBJECTIVES: To report our experience with percutaneous image-guided needle biopsy in the pediatric population and assess its clinical use, efficacy and limitations. MATERIAL AND METHODS: Sixty-nine percutaneous image-guided needle biopsies were performed in 57 children. The age of the children ranged from 4 days to 14 years (mean 5.6 years). We used 16- to-20-gauge cutting-edge needles. Sixty-two biopsies were core-needle biopsies and 7 fine-needle aspiration biopsies. RESULTS: There were 50 malignant lesions, 10 benign lesions and 2 infectious lesions. In 55 (88.7 %) lesions the needle biopsy was diagnostic. In 7 (11.3 %) the biopsy was non-diagnostic and the diagnosis was made by surgery. Core-needle biopsy was diagnostic in 47 of 50 (94 %) of the malignant solid tumors. In 3 out of 5 children with lymphoma, an accurate diagnosis was obtained with needle aspiration. Seven children underwent a repeated core-needle biopsy, (5 for Wilms' tumor and 2 for neuroblastoma) that was diagnostic in all cases. All the biopsies were performed without complications. CONCLUSION: Percutaneous image-guided needle biopsy is a simple, minimally invasive, safe and accurate method for the evaluation of children with suspicious masses. These data suggest that image-guided needle biopsy is an excellent tool for diagnosing solid tumors in the pediatric population. Negative studies should be considered nondiagnostic and followed by excisional surgical biopsies when clinical suspicion of malignancy is high.

CT-guided core-needle biopsy in the diagnosis of mediastinal lymphoma.

The advent of radiologic guidance techniques for percutaneous biopsy has changed the approach to the routine diagnosis of mediastinal lymphoma. The aim of the present study was to evaluate the diagnostic accuracy of CT-guided percutaneous core-needle biopsy (PCNB) in the clinical management of patients with mediastinal lymphoma. The results of 49 CT-guided PCNB of mediastinal lymphoma performed under local anesthesia in 42 ambulatory patients were analyzed. A positive diagnosis of lymphoma was obtained in 30 of 42 patients, with an overall success rate of 71.5%. The technique was equally successful in the diagnosis of Hodgkin's and non-Hodgkin's lymphoma. There were no major complications. Percutaneous CT-guided CNB of mediastinal lymphoma is a quick, safe, accurate, and efficient alternative to open biopsy in the evaluation of mediastinal lymphoma, mainly at presentation. It should become the preferred initial diagnostic procedure for obtaining histologic samples in patients with suspected mediastinal lymphoma.

The role of BAL in the diagnosis of pulmonary mucormycosis.

Five patients with pulmonary mucormycosis diagnosed during life are described. All had underlying predisposing conditions: either posttransplant or hematologic malignancies. In all cases, the diagnosis was made using fiberoptic bronchoscopy. In three patients, BAL was diagnostic. In two of these patients, the diagnosis was made by identifying the typical hyphae of mucormycosis in the BAL fluid alone. Transbronchial biopsy was diagnostic in three patients. Treatment was based on IV antifungal chemotherapy together with surgical removal of involved lung tissue whenever feasible. The clinical outcome of these patients was dismal and was determined primarily by the underlying condition.

Histopathological study of the human submandibular gland in graft versus host disease.

Major salivary gland dysfunction and severe xerostomia is one of the manifestations of graft versus host disease (GVHD). The histopathological evaluation of the major salivary gland in patients with GVHD has never been reported. The pathological findings of the submandibular glands in a GVHD patient who succumbed to the disease are described. Lymphocytic infiltration, parenchymal destruction, and fibrosis were observed, which may provide the pathophysiological mechanism for the xerostomia and hyposalivation observed in GVHD.

Similarity measurement method for the classification of architecturally differentiated images.

A similarity measurement method for the classification of architecturally differentiated image sections is described. The strength of the method is demonstrated by performing the complex task of assigning severity grading (Gleason grading) to histological slides of prostate cancer. As shown, all that is required to employ the method is a small set of preclassified images. The images can be real world images acquired by means of a camera, computer tomography, etc., or schematic drawings representing samples of different classes. The schematic option allows a quick test of the method for a particular classification problem.

Immunostaining of Lewis X in cells from voided urine, cytopathology and ultrasound for noninvasive detection of bladder tumors.

PURPOSE: We examined the use of immunostaining of the Lewis X antigen in exfoliated cells from voided urine samples, cytopathology and bladder ultrasound for noninvasive detection of bladder tumors as a potential substitute for cystoscopy. MATERIALS AND METHODS: A total of 260 patients were included, of whom 80 were evaluated because of irritative symptoms or hematuria and 180 were examined during followup visits after resection of bladder tumors. Voided urine samples were obtained from each patient for immunocytology and cytopathology. Bladder ultrasound and cystoscopy were performed. Biopsies were obtained whenever a bladder tumor was seen or if carcinoma in situ was suspected. Indirect immunoperoxidase staining was done on cytocentrifuge slides, using the P12 monoclonal antibody against the Lewis X antigen. RESULTS: Cystoscopy and biopsies revealed bladder tumors in 84 patients. Immunocytology of 1 urine sample resulted in a sensitivity of 79.8% and a specificity of 86.4%. The diagnosis of primary carcinoma in situ by immunocytology was correct in 100% of the cases. The examination of 2 consecutive urine samples detected 95.1% of the tumors. False-negative results occurred in a few cases with small, superficial, low grade tumors. Cytopathology and bladder ultrasound resulted in a sensitivity of 47.6 and 66.7%, and a specificity of 97.7 and 97.2%, respectively. The results of immunocytology of 2 urine samples were equivalent to the combination of immunocytology of a single urine sample, cytology and ultrasound. CONCLUSIONS: Immunostaining of the Lewis X antigen is significantly more sensitive than cytopathology for the detection of low grade as well as high grade tumor cells in voided urine. Immunocytological evaluation of 2 consecutive voided urine specimens for the Lewis X antigen is the most sensitive method currently available for noninvasive detection of transitional cell tumors. This assay may replace cystoscopy for detection of bladder cancer.

Intravascular lymphomatosis--an indolent or aggressive entity?

Intravascular lymphomatosis (IVL) is a rare malignancy characterized by neoplastic proliferation of lymphoid cells within the lumens of arteries, small veins and capillaries. We report four patients with IVL and review the recent world literature, relating to incidence, clinical features and possible therapy. In these cases diagnosis was established coincidentally in one patient after prostatectomy. This patient eventually had central nervous system involvement. In two other patients IVL was diagnosed from skin lesions. In the fourth case the diagnosis was established at post-mortem examination, where involvement of most organs was evident but particularly kidneys, myocardium, gastrointestinal tract and lymph nodes. Therapy was given to three patients, but the disease progressed in two and they both died with evidence of central nervous system involvement, while the third patient has had a good partial response to combination chemotherapy but has relapsed within two months of completing chemotherapy. As evident from our patients and the literature review IVL has a variable clinical course and currently, there appears to be no effective therapy for this rare disorder.

Alveolar macrophages fat stain in early diagnosis of fat embolism syndrome.

The aim of this study was to assess the contribution of bronchoalveolar lavage (BAL) in the diagnosis of fat embolism syndrome (FES). The presence of fat droplets in alveolar macrophages was addressed in 13 trauma patients with bone fractures and 10 non-trauma patients with acute respiratory distress syndrome (ARDS). The control group was composed of 5 anesthesized patients with ischemic heart disease, immediately prior to cardiac surgery. Two patients with suggestive clinical and laboratory signs of FES had 40% and 24% fat-containing alveolar cells, respectively. The trauma patients without signs of FES displayed a wide variation in the percentage of fat-containing macrophages (from 3% to 95%). Most of the patients with ARDS who were receiving lipid emulsion as part of their parenteral nutrition, had a high percentage (> 85%) of fat-containing macrophages. Patients with normal lungs had no fat-containing macrophages. Our findings suggest that BAL Oil Red O-positive macrophages are frequently observed in trauma patients irrespective of the presence of FES. Therefore, estimation of the percentage of fat-containing macrophages from BAL is an unreliable marker of FES.

[CT-guided core needle biopsy of abdominal, pelvic and retroperitoneal masses].

CT-guided core needle biopsy of abdominal, pelvic and retroperitoneal masses is accurate and safe and can be performed on an outpatient basis. Between 1987 and 1995, 809 patients (age range 1-87 years) underwent 851 biopsies (minimal lesion diameter 1 cm). Cutting needles were always used, facilitating both cytological and histological diagnosis while minimizing risk of complications. A positive result (malignant, inflammatory or infectious) was obtained in 69% of the 809 and a negative result (normal tissue) in 17.4%, while in 13.6%, material for diagnosis was insufficient. Biopsy was repeated in 42 of them in whom radiological or clinical suspicion of malignancy was high. In 24 (60%) a positive result was obtained after the second biopsy. Significant complications occurred in 7 (0.8%). 1 hemo-rrhaged following liver biopsy and required blood transfusion. Pancreatitis occurred in 6 (2.6%) following pancreatic biopsy. An intraabdominal fluid collection in 1 necessitated percutaneous drainage. There was no mortality following the procedure and no documented case of needle-tract seeding of tumor. All outpatients were discharged within 3 hours of completion of the biopsy, without ill effects.

Image-guided core-needle biopsy in malignant lymphoma: experience with 100 patients that suggests the technique is reliable.

PURPOSE: In an initial evaluation of 1,500 computed tomography (CT)-guided core-needle biopsies performed at our institute during the period from 1989 to 1994, we encountered 100 patients with the diagnosis of lymphoma. Here, we review the clinical impact of 109 image-guided needle biopsies in these 100 patients with non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). PATIENTS AND METHODS: NHL was diagnosed in 71 patients, and 29 had HD. Among the NHL patients, 17 (24%) had proven lymphoma diagnosed before the biopsy was performed; in 54 (76%) core-needle biopsy was performed as the first diagnostic procedure. Of 29 HD patients, nine (31%) were already established cases of HD, and in 20 (69%) core-needle biopsy was the first diagnostic procedure attempted. Most of the biopsies were performed under CT control using a 20- or 18-gauge Turner biopsy needle. RESULTS: Eighty-six patients received therapy based on the results of the needle biopsy alone. Fourteen patients received therapy after undergoing surgical biopsy for a suspected diagnosis of lymphoma, which could not be established with certainty on the basis of an earlier core-needle biopsy alone. In 78% of the patients, the needle biopsy saved a further surgical procedure that may have been difficult to perform because of the primary location of the tumor. CONCLUSION: From our experience in this study, image-guided core-needle biopsies provide sufficient information for the diagnosis of and subsequent therapeutic decision to treat most cases of lymphoma.

Assessment of testicular spermatozoa morphology by image analysis.

OBJECTIVE: We objectively evaluated the testicular sperm cell morphology. METHODS: The spermatozoa head morphology was evaluated by image analysis as presented on testicular fine-needle aspiration cytology smears. RESULTS: 2,356 spermatozoa heads were classified into six groups, according to different morphology parameters. CONCLUSIONS: This objective method of morphologic assessment of testicular spermatozoa provides an important tool for the evaluation of testicular spermatozoa and can serve as a guide for therapy in male infertility.

Radiologic spectrum of localized Castleman's disease.

We report seven patients with the localized form of Castleman's disease, diagnosed by surgical biopsy; four had the plasma cell type and three the hyaline vascular type. A variety of nonspecific clinical and radiologic findings were identified in these patients. Precise clinical staging is important to separate the widespread from the localized form of Castleman's disease, as the localized form may be successfully treated surgically. Percutaneous core needle biopsy is not helpful in the diagnosis of Castleman's disease. Significant computerized tomography enhancement with intravenous contrast in the hyaline vascular type is indicative of increased vascularity which may cause surgical complications when resection is attempted.