Observation of lymphatic vessels in orbital fat of patients with inflammatory conditions: a form fruste of lymphangiogenesis?

In the absence of antibodies specific for lymphatic vessels, analysis of lymphatic vessels within different tissues has been widely performed with light microscopic and, most importantly, electron microscopic techniques. In regard to lymphatic vessels in the ocular globe and the periocular structures, controversy remains about the specific distribution of lymphatic channels. It is postulated that bulbar and retrobulbar tissues are devoid of lymphatic vessels, but lymphatic vessels have been demonstrated in lacrimal gland and epibulbar conjunctiva. In this study, we analyzed orbital fat for the presence of lymphatic tissue using D2-40, a monoclonal antibody, specific for lymphatic vessels. We found lymphatic vessels present within bulbar conjunctiva extending to the level of the ciliary apparatus. No lymphatics were identified in healthy anterior orbital adipose tissue. In two cases of orbital mucor-mycosis and one case of panendophthalmitis, significant lymphovascular proliferation was present within granulation tissue associated with the acute inflammation. We conclude that lymph vessel proliferation may be induced in inflammatory conditions in tissues which are normally devoid of lymph channels.

Proliferation of D2-40-expressing intestinal lymphatic vessels in the lamina propria in inflammatory bowel disease.

Lymphatic vessels in the colon are normally distributed beneath the muscularis mucosae with rare branches reaching through the muscularis mucosae to the most basal aspect of the colonic crypts. In chronic inflammatory bowel disease demonstrating acute inflammation and architectural disarray, lymph vessel proliferation is seen within the lamina propria and within the submucosa. We analyzed the number and distribution of lymphatic vessels within the lamina propria and submucosa in chronic active and treated ulcerative colitis with restoration of architecture by immunostaining with D2-40, a specific monoclonal antibody against lymphatic vessels. We found significantly increased numbers of lymph vessels in chronic active ulcerative colitis both within the lamina propria and the submucosa as compared to normal mucosa. Numbers of lymph vessels in lamina propria were highest in severe chronic active ulcerative colitis and less in moderate and minimal residual disease with minimal architectural disarray (p<0.05). Lymph vessels in the submucosa were increased significantly above normal values in both severe, moderate and minimal residual disease. We conclude that lymph vessel distribution in chronic active ulcerative colitis extends into the lamina propria. With restoration of architectural morphology, the integrity of the lamina propria in regards to the distribution of lymph vessels is restored.

Identification of lymphatic vessels in malignant, adenomatous and normal colonic mucosa using the novel immunostain D2-40.

Distribution of lymphatic vessels in normal and neoplastic colon has been previously analyzed with electron microscopic techniques, as reliable antibodies have not been available for selective lymph vessel staining. A novel monoclonal antibody, D2-40, is recently available to differentiate lymphatic vessels from blood vessels. In this study, we analyzed the distribution of lymphatic vessels in normal colon, adenomas with and without superficial stalk invasion and invasive carcinomas without identifiable polypoid precursor lesions. In contrast to previous studies, we found lymphatic vessels in superficially misplaced stalk stroma in adenomas, and closely associated with early invasive epithelial nests in invasive lesions. Lymphatic vessels were identified within the lamina propria of the in situ aspect of in invasive tumors. We conclude that lymphatic vessel structures are seen more superficially in adenomas and invasive carcinomas than previously described. Since intramucosal carcinomas in adenomas do not metastasize, these lymph vessels may be immature or not communicate with deeper lymphatics. Proliferation and distribution of lymphatic vessels may be related to prognosis and early metastasis.

Evaluation of the c-Met immunostain to detect malignant cells in body cavity effusions.

The cytologic diagnosis of malignant cells in serous effusions can be difficult. A wide variety of immunostains and other diagnostic techniques have been studied but without widespread acceptance of one staining panel or technique. Expression of the tyrosine kinase c-Met has been associated with several malignancies but not with benign mesothelium. We investigated the diagnostic value of the c-Met immunostain in serous effusions. Cell block material from 76 cases of unequivocally benign or malignant effusions were studied. Cases were stained with c-Met using the avidin-biotin complex method following antigen retrieval. The presence of strong granular cytoplasmic staining that was distinct from background staining was considered positive. Positive cells were identified in 38 of 42 (90%) malignant cases and in 18 of 34 (53%) benign cases. Typically, benign cases contained only individual positive cells, but positive cell clusters were also identified. The c-Met immunostain lacks sufficient specificity to be clinically useful in this cytologic setting. The expression of c-Met in benign mesothelium may reflect mesothelial proliferation.

Diagnostic value of hepatocyte paraffin 1 antibody to discriminate hepatocellular carcinoma from metastatic carcinoma in fine-needle aspiration biopsies of the liver.

BACKGROUND: Diagnosing liver tumors by fine-needle aspiration biopsy is safe and accurate. However, there are cases that prove diagnostically difficult. Traditionally, immunostains for alpha-fetoprotein and polyclonal carcinoembryonic antigen have been used to distinguish adenocarcinomas from hepatocellular carcinomas (HCCs). In poorly differentiated tumors, these immunostains have limitations in both sensitivity and specificity. An hepatocyte-specific immunostain has been described in the surgical pathology literature. To the authors' knowledge, this hepatocyte antibody has not been studied in liver fine-needle aspiration biopsies. The authors examined the Hepatocyte Paraffin 1 (HP1) antibody for its diagnostic utility in this cytologic setting. METHODS: Cell-block material from 40 cases of HCC and 53 cases of metastatic adenocarcinoma were studied. Slides were stained for HP1 by the avidin-biotin complex method following antigen retrieval. The percentage of malignant cells that exhibited coarse granular staining in the cytoplasm was estimated for all cases of HCC, poorly differentiated HCC, and metastatic adenocarcinoma. RESULTS: HP1 was expressed in 83% of all HCCs but in only 56% of poorly differentiated HCCs. Only 2 of 53 (4%) of metastatic tumors expressed HP1. The overall sensitivity of HP1 was 79% and its specificity was 96%. CONCLUSION: HP1 was found to be a specific immunostain that may prove helpful in diagnosing all but the most undifferentiated liver tumors biopsied by fine-needle aspiration.

Glucose transporter Glut-1 is of limited value for detecting breast carcinoma in serous effusions.

Diagnosing breast carcinoma that has metastasized to body cavity fluids can be difficult. Recently, immunostaining for the facultative glucose transporter Glut-1 has been described as a sensitive and specific means of detecting carcinomas in effusions. However, only five cases of breast carcinoma were studied. We examined Glut-1 specifically as a means of detecting breast carcinoma in effusion cytology. Using avidin-biotin immunocytochemistry, cell block material from 31 cases of breast carcinoma metastatic to body cavity effusions and 33 cases of benign effusions were studied. All cases were immunostained with the Glut-1 antibody. An additional set of slides from these same cases was stained for mucin using the Mayer's mucicarmine technique. Slides were graded for percentage of cells exhibiting immunoreactivity for Glut-1 or for the presence of mucin. Results of staining for both Glut-1 alone and in combination with mucicarmine were compared between the benign and malignant groups. Of the breast cancer cases, 19 of 31 (61%) were immunoreactive for Glut-1, and 25 of 31 (81%) were positive for either Glut-1 or mucicarmine. One of the 33 (3%) benign cases was immunoreactive for Glut-1, and none were positive for mucin. These data suggest that using Glut-1 as a single immunostain or in conjunction with mucicarmine is a specific but modestly sensitive means of detecting breast carcinoma in this cytologic setting.

Expression of survivin, YB-1, and KI-67 in sporadic adenomas and dysplasia-associated lesions or masses in ulcerative colitis.

Sporadic adenomas are said to exhibit an orderly growth pattern with a reversal of proliferative and apoptotic cell distribution as compared with normal colonic crypts. Dysplastic polyps of patients with ulcerative colitis (UC) may represent dysplasia-associated lesions or masses (DALM) with a high associated cancer risk, or, alternatively, may represent sporadic adenomas. Histologic criteria to differentiate between sporadic adenomas and DALM have not focused on the balance between cell renewal and cell loss. The expression of the novel anti-apoptosis gene product, survivin, and the proliferation markers, Ki-67 and Y-box binding protein (YB-1), were investigated by immunohistochemical localization in sporadic adenomas and DALM lesions of patients with UC. In adenomas, KI-67 was expressed preponderantly at the luminal aspect of the polyp, whereas its expression was diffuse in DALM. Survivin was detected diffusely in both adenomas and DALM. YB-1 showed positive staining in the deep aspect of adenomatous glands but only to a minor degree at the surface, whereas both deep and diffuse expression patterns of YB-1 were seen in DALM. The authors conclude that DALM and sporadic adenomas exhibit different patterns of cellular proliferation and that molecular markers of cell proliferation, Ki-67 and YB-1, may be useful to distinguish sporadic adenomas from DALM. However, the similar expression of survivin suggests that the underlying mechanisms that regulate apoptotic cell death are uniform in these lesions.

Glut-1 expression in dysplastic and regenerative lesions of the colon.

Monosaccaride transporter proteins are responsible for transmembrane transport of monosaccarides into cells. Glucose transporter protein 1 (Glut-1) is most prevalent in the cell membranes of erythrocytes and facilitates transport of glucose in tissues with barrier functions, i.e. blood brain barrier. Expression of Glut-1 in malignant tumors is increased due to increased metabolic need of the proliferating cell populations. In colorectal adenomas and carcinomas, membranous expression of Glut-1 has been associated with higher grade of tumors and decreased survival time. We studied the expression of Glut-1 in dysplastic proliferations of the colon which included sporadic adenomas and dysplasia associated lesions (DALM) in patients with ulcerative colitis and reactive/regenerative proliferations of the colon, including non-dysplastic chronic colitis, acute colitis and ischemia. Two patterns of Glut-1 expression were detected. Most adenomas and DALMs showed at least focal membranous expression of Glut-1. In addition a second staining pattern was recognized which consisted of prominent supranuclear dots. This pattern of staining was not only seen in adenomas and DALM but also in non-dysplastic areas immediately surrounding sporadic adenomas, in regenerative chronic colitis and in areas surrounding acute inflammation. Areas away from dysplasia did not show any positive staining for Glut-1. We conclude that two distinct patterns of Glut-1 expression may be found in colonic epithelial proliferation: membranous staining, associated with dysplasia, and, heretofore not described, supranuclear staining which may be related to Glut-1 expression secondary to expression of specific growth factors and not necessarily related to dysplasia.

Dieulafoy's lesion of the small bowel causing massive gastrointestinal bleeding: two case reports and literature review.

Dieulafoy's lesions are an often unrecognized cause of obscure, massive GI hemorrhage. Their diagnosis may elude conventional investigations, including upper and lower endoscopy, arteriography, and even laparotomy. In this paper, we report two cases of small-bowel Dieulafoy lesions. The first, a jejunal lesion, occurred in a young patient and was discovered at laparotomy. The second was an ileal Dieulafoy's malformation in an older patient. An intraoperative endoscopy with surgical guidance may be needed for definitive localization of this lesion.

Diagnostic utility of BCA-225 in detecting adenocarcinoma in serous effusions.

OBJECTIVE: To determine the diagnostic value of the BCA-225 antibody in discriminating adenocarcinoma from benign mesothelium in body cavity effusions. STUDY DESIGN: One hundred four cases of unequivocally benign (34 cases) and malignant (70 cases) serous effusions with cell block material were immunostained for BCA-225 using the ABC method without antigen retrieval. The percentage of positively staining cells in each case was estimated in a blind fashion. RESULTS: BCA-225 stained at least 10% of morphologically malignant cells in 28 of 32 (88%) breast carcinomas and 58 of 67 (87%) adenocarcinomas overall. Neuroendocrine carcinomas (two cases) and one mesothelioma were positive in < or = 5% of their respective tumor cells. Of 34 benign cases, 6 (18%) exhibited positive staining, albeit in rare, morphologically benign cells. CONCLUSION: BCA-225 is able to discriminate adenocarcinoma from reactive mesothelium in cell block preparations and may prove useful as part of an antibody panel.

Diagnostic value of a second generation CA 15-3 antibody to detect adenocarcinoma in body cavity effusions.

BACKGROUND: Detecting malignant cells in body cavity effusions can be diagnostically challenging. Several monoclonal antibodies have been studied to improve the diagnostic yield of effusion cytology but without widespread acceptance. The CA 15-3 antibody has demonstrated high sensitivity but limited specificity for breast carcinoma in surgical pathology. A second generation CA 15-3 antibody has been developed that has not been studied in serous effusions to the authors' knowledge. The authors examined this second generation CA 15-3 antibody for its diagnostic utility in detecting adenocarcinomas in this cytologic setting. METHODS: Cell block material from 114 cases of unequivocally benign or malignant body cavity effusions were studied. Slides were stained for CA 15-3 by using the avidin-biotin complex method. The percentage of cells exhibiting strong staining was estimated both for breast carcinoma and for all adenocarcinomas as a group. These results were compared with CA 15-3 staining exhibited by benign mesothelium. RESULTS: CA 15-3 was expressed in at least 10% of tumor cells in 97% of breast carcinoma cases and in 90% of adenocarcinomas overall. The highest sensitivity was observed in carcinomas of the breast, ovary, and lung. Of 40 cases of benign mesothelium, only 4 (10%) were positive (P < 0.001). The sensitivity of CA 15-3 was 97% for breast carcinoma and 91% for adenocarcinomas overall. Specificity was 95% for breast carcinoma and 91% for adenocarcinomas. CONCLUSIONS: CA 15-3 is an immunostain with high specificity and sensitivity for adenocarcinomas in cell block material from effusions. The antibody holds particular promise for detecting breast carcinoma. Cancer (Cancer Cytopathol)

FHIT protein is expressed in benign mesothelium and has no clinical value in detecting carcinoma in body cavity effusions.

The fragile histidine triad (FHIT) protein is a suspected tumor-suppressor gene frequently expressed in adenocarcinomas of the lung and other organs. Its expression in benign mesothelium has not been reported. This study examined the expression of FHIT in mesothelium from benign body cavity effusions. FHIT expression was also examined in a diversity of malignant effusions to evaluate any value of the FHIT protein to discriminate carcinomas from benign mesothelium. Fifty-eight cases of benign and malignant effusions were immunostained using cell block material and two different antigen retrieval methods with the ABC method. Benign and malignant cases were scored for percentage of cells with strong immunoreactivity. Benign mesothelium exhibited strong immunoreactivity for the FHIT protein in 32 of 32 (100%) cases, with 26 of 32 (81%) cases having expression in at least 50% of cells. Seventeen of 21 (81%) cases of adenocarcinomas also exhibited the FHIT protein in at least 50% of tumor cells. FHIT expression was also noted in small numbers of other malignancies. The FHIT protein is expressed consistently in benign mesothelium as well as many types of adenocarcinomas. Immunostaining for this protein has no value in discriminating adenocarcinomas from reactive mesothelium.

Distinction between dysplasia-associated lesion or mass (DALM) and adenoma in patients with ulcerative colitis.

Polyps with epithelial dysplasia in ulcerative colitis (UC) represent either dysplasia-associated lesions or masses (DALMs) or sporadic adenomas. DALMs are frequently associated with associated carcinoma and are an indication for colectomy. Removal of the polyp is treatment of choice for sporadic adenomas. Differentiating between these 2 lesions is not always easy. The goal of this study was to distinguish DALMs from adenomas in patients with UC on a genetic basis. We evaluated genetic alterations in DALMs and compared them with a previously published set of dysplastic polyps in patients with UC that were considered adenomas for the following reasons: (1) polyps were located outside of current active disease; (2) polyps had histological features of sporadic adenomas; and (3) patients displayed a uneventful follow-up after polypectomy (UC-adenomas). In addition, adenomas not associated with UC were studied. Genetic alterations on chromosome 3p were assessed for the markers D3S1766, D3S2409, and D3S2387. LOH with or without microsatellite instability was found in 70%, 37%, and 57% of cases of DALM, respectively. In contrast, UC-adenomas lesions exhibited genetic alterations in 8.3%, 11.7%, and 15.3% for the respective markers. Spontaneous adenomas exhibited genetic alterations in 10.5%, 7.1%, and 0% of cases, which were not significantly different from the UC-adenoma results. These results indicate that UC-adenomas are genetically and biologically similar to sporadic adenomas and that UC-adenomas may biologically represent sporadic adenomas, supporting on a genetic basis the criteria chosen to diagnose adenomas in UC. Genetic markers on chromosome 3p may be useful in the differential diagnosis between DALM and UC-adenomas.

Cytologic atypia in a 53-year-old man with finasteride-induced gynecomastia.

Finasteride has been associated with the development of gynecomastia. Although cytoplasmic vacuolization has been noted in prostatic epithelium in men taking this drug, we found no documentation of the cytologic changes in finasteride-associated gynecomastia. We present the case of a 53-year-old man who developed unilateral gynecomastia following finasteride therapy for alopecia. A fine-needle aspiration biopsy of the mass was diagnosed as adenocarcinoma on the basis of nuclear atypia and particularly because of cytoplasmic vacuolization. Subsequent excisional biopsy revealed benign gynecomastia with no evidence of malignant change. The ductal epithelium did exhibit cytoplasmic vacuolization similar to that described in the prostate following finasteride therapy. We believe this is the first reported case documenting the cytologic changes seen in gynecomastia secondary to finasteride therapy. Cytoplasmic vacuolization in this setting should not be considered evidence of malignancy in men with gynecomastia. As with gynecomastia in general, extreme caution should be used before rendering a cytologic diagnosis of malignancy.

Ultra fast identification of Aspergillus species in pulmonary cytology specimens by in situ hybridization.

The cytologic diagnosis of pulmonary aspergillosis infection is typically made on a presumptive basis and later confirmed by fungal culture, which may take up to one week to complete. An in situ hybridization (ISH) probe specific for Aspergillus for use in surgical pathology specimens has been developed which has not been used on cytology preparations. We describe a supra-threshold adapted testing (STAT) in situ hybridization test for cytology specimens, which takes less than one hour to finish. We performed ISH on three cases of culture-proven pulmonary aspergillosis and one case with Aspergillus fungal forms but negative cultures to test the feasibility of using this same Aspergillus probe on cytology specimens. Four patients with pulmonary aspergillosis were initially diagnosed by cytologic examination of their respective specimens. The presumptive diagnosis was confirmed by culture to be Aspergillus fumigatus on three cases. ISH on both cytology cytospin and Thin-Prep specimens was performed using an rRNA Aspergillus specific probe. All four cytology specimens exhibited positive staining with the Aspergillus probe. Most, but not all, fungal hyphae were stained with the probe. Even though ISH is more expensive than culture, in situ hybridization can be performed in less than one hour on cytology specimens and may be beneficial for patients in selected clinical circumstances.

Diagnostic utility of sialosyl-Tn in discriminating carcinomatous cells from benign mesothelium in body cavity effusions.

OBJECTIVE: Detecting malignant cells in the setting of reactive mesothelium can be difficult. Several techniques have been tried but without widespread acceptance. Sialosyl-Tn (STn) is an aberrantly glycosylated precursor of the MN blood group antigen frequently expressed in carcinomas and dysplastic epithelium. We investigated the STn monoclonal antibody for its clinical utility as an isolated stain to discriminate benign mesothelium from malignant cells. STUDY DESIGN: Cell block material from 72 cases of body cavity fluids were immunostained for STn using the avidin-biotin complex method without antigen retrieval. Slides were incubated overnight at 4 degrees C in a humidified chamber. RESULTS: Strong immunoreactivity was noted in 31/40 (77%) carcinomatous cases. Only moderate staining was noted in 1 of 28 (4%) benign effusions and weak staining in 5 (18%) additional benign cases. Specificity was 100%, sensitivity 78%, positive predictive value 100% and negative predictive value 76%. No staining was noted in four noncarcinomatous malignant effusions. CONCLUSION: STn may have diagnostic value in this cytologic setting as part of a diagnostic panel but not as an isolated stain.

Clinical utility of chromosome 17 alpha satellite probe in distinguishing benign mesothelium from malignant cells: a pilot study using routinely fixed specimens.

Fluorescent in situ hybridization has shown promise in detecting malignant cells in body cavity effusions. However, this requires special preparatory techniques not used in many laboratories. We developed an in situ hybridization (ISH) procedure specifically for ethanol-fixed specimens, and using it tested the clinical utility of the chromosome 17alpha satellite probe (C17alpha). ISH with C17alpha was used in 12 malignant and 10 benign ethanol-fixed body cavity effusions. Cells were pretreated with protease K prior to ISH. The probe was detected by an anti-digoxigenin-horseradish peroxidase method. Signals were counted in 100 nuclei and the chromosome index (CI) and percent diploid cells calculated in each case. ISH was successfully performed in all cases. Malignant cells had an average CI of 2.23 with less than 44% diploid nuclei and 50% of specimens exhibited bizarre signals. Benign effusions had an average CI of 1.98 with over 84.6% diploid nuclei. Questionably bizarre signals were seen in two (20%) benign specimens. ISH can be performed on ethanol-fixed specimens. The C17alpha probe may prove valuable in detecting malignant cells in body cavity effusions.

Mixed malignant germ cell tumor of the fallopian tube.

Immature teratomas of the fallopian tube are exceedingly rare with only three reported cases in the English literature. Reported here is a case of primary mixed malignant germ cell tumor of the fallopian tube composed of immature teratoma and yolk sac tumor.

BCL-2 expression and inhibin-A in adrenal neoplasms: a comparative study.

Differential diagnosis between adrenal cortical and adrenal medullary lesions may be difficult in many cases. Different immunohistochemical, histochemical tools as well as ultrastructural diagnostic techniques have been employed to aid in differentiating between these lesions. Recently, both inhibin-A and BCL-2 have been shown to stain selectively adrenal cortical tissue and its derived neoplasms but not adrenal medulla or pheochromocytomas. In this study we compared the staining reactions of inhibin-A and BCL-2 in cases of adrenal cortical adenomas and carcinomas as well as pheochromocytomas. We found that both inhibin-A and BCL-2 stained cortical derived tissues, but not medullary derived tissues. Staining intensity for inhibin-A was significantly weaker than for BCL-2. We found that fixation techniques may influence the staining reactivity, as some cases did not immunoreact with any of the antibodies. We conclude that both inhibin-A, and, preferentially, BCL-2 are useful additions to a staining protocol to help in the differential diagnosis of cortical and medullary neoplasms.