Flow cytometry significantly improves the diagnostic value of fine needle aspiration cytology of lymphoproliferative lesions of salivary glands.

OBJECTIVE: Lymphoid proliferations of the salivary glands can be either reactive or malignant. Diagnosis based solely on fine needle aspiration (FNA) cytology may be troublesome in view of the difficulty in distinguishing low-grade B-cell and mucosa-associated lymphoid tissue (MALT) lymphomas from reactive lymphoid proliferations. We report our experience with FNA cytology combined with flow cytometry (FC) immunophenotyping for the diagnosis of lymphoproliferative processes affecting the salivary glands. METHODS: Sixty-one FNA specimens, obtained from salivary glands over a 10-year period, were analysed by cytology and FC. The results were correlated with histological follow-up if available. RESULTS: A diagnosis of lymphoma was given in 37 of 61 (61%) specimens; 22 of 61 (36%) specimens were considered as benign/reactive or non-lymphomatous processes; two of 61 (3%) specimens were considered as suspicious for lymphoma on cytological analysis and negative on FC. Histological control was available in 23 malignant, four non-lymphomatous and one cytologically suspicious case. Data obtained by the combination of cytology and FC were confirmed in all but one case: the case suspicious on cytology received a histological diagnosis of carcinoma. Four of seven cases with small populations of clonal cells (less than 15%) were histologically confirmed as lymphoma, whereas two remain under surveillance and one was reactive. Correlation with histological data showed a sensitivity of 100% and a specificity of 83% for the combination of cytology and FC. CONCLUSIONS: FC is fundamental for the diagnosis of lymphoproliferative lesions of the salivary glands. It may solve cytologically suspicious cases and detect the presence of neoplastic B or T cells. This combined approach reduces the time to therapy and may prevent unnecessary surgical biopsies.

Diagnosis of deep-seated lymphomas by endoscopic ultrasound-guided fine needle aspiration combined with flow cytometry.

OBJECTIVE: Although endoscopic ultrasound combined with fine needle aspiration (EUS-FNA) is rapidly becoming the preferred diagnostic approach for the sampling and diagnosis of gastrointestinal and mediastinal malignancies, there are limited data as to its use in the diagnosis of lymphoproliferative disorders. Therefore, we carried out a retrospective evaluation of the performance of EUS-guided FNA combined with flow cytometry (FC) as a tool to improve overall sensitivity and specificity in the diagnosis of lymphoma. METHODS: Of 1560 patients having EUS-guided FNA during the period of the study, a total of 56 patients were evaluated by cytology with FC after EUS-FNA. There was adequate material to perform FC analysis for all but one case. RESULTS: EUS-FNA-FC gave a diagnosis of lymphoma in 11 cases and of reactive lymphadenopathy in 20. A specific histological type was defined by FC alone in eight cases. The remaining cases were diagnosed later by cytology and cell block sections: 13 carcinomas, nine granulomatous lymphadenopathies and one mediastinal extramedullary haematopoiesis. One case was considered only suspicious for lymphoma on cytology and FC but was not confirmed on molecular analysis and one had insufficient material for FC. CONCLUSIONS: Our results show that a combination of EUS-FNA-FC is a feasible and highly accurate method, which may be used for the diagnosis and subtyping of deep-seated lymphoma, providing a significant improvement to cytomorphology alone both for diagnosis and treatment planning, as long as immunocytochemistry is available for non-lymphoma cases.

Endoscopic ultrasound-fine needle aspiration (EUS-FNA) for pancreatic lesions: effectiveness in clinical practice.

BACKGROUND: Diagnosis of pancreatic masses is often difficult. Endoscopic ultrasound-fine needle aspiration has been proposed as the best single-step strategy. AIMS: To prospectively evaluate feasibility, effectiveness and safety of endoscopic ultrasound-fine needle aspiration of pancreatic masses in a consecutive study of unselected patients. METHODS: Two hundred ninety-three patients were enrolled in two referral Hospitals in Northern Italy. All patients were referred either due to the presence of imaging test abnormalities (suspected or evident masses, or features indirectly suggesting the presence of a mass) or due to clinical or biochemical findings suggesting pancreatic cancer in the absence of positive imaging. All patients underwent linear array endoscopic ultrasound and, when indicated, fine needle aspiration. All procedures were recorded prospectively. The final diagnosis was established at the end of follow-up or when the patients underwent surgery or died. RESULTS: Fine needle aspiration was indicated in 246 of 293 cases (84%), considered technically feasible in 232 of 246 cases (94%) and gave adequate samples for histopathological diagnosis in 204 of 232 cases (88%). Endoscopic ultrasound sensitivity, specificity and accuracy were 79, 60 and 72%, respectively; the corresponding figures for endoscopic ultrasound-fine needle aspiration were 80, 86 and 82%. There was good agreement with final diagnosis for endoscopic ultrasound-fine needle aspiration (kappa 0.673, 95%CI 0.592-0.753), greater than that for endoscopic ultrasound alone (kappa 0.515, 95%CI 0.425-0.605). There was one case of intracystic haemorrhage and one case of transient hyperthermia (0.3%). CONCLUSIONS: Endoscopic ultrasound-fine needle aspiration of pancreatic masses seems to be feasible, effective and safe in this consecutive study of patients.

[On-site evaluation and triage for endoscopic ultrasound-guided fine needle aspiration cytology. The Turin experience]. / Valutazione on-site e triage per la citologia agoaspirativa sotto guida ecoendoscopica. L'esperienza torinese.

AIM: Evaluation of the importance of the on-site presence of a skilled cytopathologist during endoscopic ultrasound-guided fine needle aspiration at determining samples' adequacy and performing ancillary techniques which can be helpful for the diagnosis. METHODS: A retrospective analysis of our institute's experience with EUS-FNA sampling is presented. From January 2001 to May 2007, 404 patients underwent the EUS-FNA evaluation. From 2003 a cytopathologist was present during the procedure and started making an extemporary evaluation of the samples' adequacy. RESULTS: Before 2003, a final cytological diagnosis was available in only 70% of the cases (without an on-site cytopathologist). After 2003, in 90% of the cases (with an on-site cytopathologist). It is possible planning and performing: immunocytochemistry on cell block material including evaluation of the proliferation index; to obtain a sample for the flow cytometry in cases of lymphomas or a microbiologic workup in cases of infective lesions. CONCLUSION: The quality of the specimens and the proper handling of the aspirated sample are very important to succesfully obtain a definitive cytological diagnosis in EUS-FNA. On-site evaluation and triage of the material allow to improve the accuracy of the diagnosis.

Detection of hepatitis delta virus RNA by a nonradioactive in situ hybridization procedure.

A digoxigenin-tailed, synthetic oligodeoxynucleotide was used to detect genomic hepatitis delta virus (HDV) RNA in formalin-fixed, paraffin-embedded liver sections by a nonisotopic in situ hybridization (NISH) procedure. Twenty-three liver samples from chronically HDV-infected patients were studied. Eight liver specimens from humans and chimpanzees without markers of active HDV infection served as negative controls. In three samples, the NISH findings were compared with characteristic nuclear features and with the distribution of the HDV encoded antigen, HDAg, as detected by direct immunofluorescence. All samples from HDV-infected patients were positive for HDV RNA by NISH. The viral genome was exclusively observed within the hepatocytic nuclei. No enzymatic reaction was detected after hybridization with the negative controls. "Sanded" nuclei, a cytopathologic change associated with HDV infection, were HDV RNA-positive, but only a small percentage of infected cells showed that feature. Hepatocytes containing the HDV RNA were sometimes binucleated or exhibited giant nuclei. When HDAg and HDV RNA were sequentially detected within the same sections, the localization of the viral antigen almost completely overlapped with the expression of the HDV transcripts, and vice versa. In conclusion, detection of intrahepatic HDV RNA by NISH is a rapid, sensitive, and specific technique that is easily applicable to routine histopathology and allows correlation of HDV with the morphology of hepatocyte nuclei.

Progression from superficial to invasive carcinoma of the bladder: genetic evidence of either clonal heterogeneous events.

Until now, no definitive molecular evidence proving or disproving a true progression from superficial to invasive bladder tumors has been reported. A total of 36 lesions from 6 patients affected by invasive bladder cancer after multiple superficial recurrences were analyzed for loss of heterozygosity on 8 loci of chromosome 9 and 2 loci of chromosome 17. In addition, the clonal composition of the tumors from two female patients was examined using the human androgen receptor assay. Our data suggest that papillary bladder lesions can and sometimes do make a true progression into invasive life-threatening tumors; however, this progression is not an invariable sequence because it was definitely proven in 2 but not confirmed in 3 of the cases we examined.

Epstein-Barr virus detection by in situ hybridization in fine-needle aspiration biopsies.

Metastasis to neck lymph nodes is often the presenting symptom of occult head and neck tumors, including undifferentiated nasopharyngeal carcinoma (UNPC). The diagnosis of the primary site of the tumor by conventional cytological analysis of tissue obtained by fine-needle aspiration (FNA) may be difficult. As Epstein-Barr virus (EBV) infection is consistently associated with UNPC, we evaluated the diagnostic significance of EBV detection using a nonradioisotopic in situ hybridization assay. The data obtained by FNA from metastatic head and neck tumors was correlated with the histology of the corresponding surgical specimens. In a series of 25 FNA specimens of cervical lymph node metastases of tumors of unknown origin, EBV was found expressed in all seven metastases of UNPC but in none of 18 metastases of tumors of different types. Therefore, detection of EBV in cervical metastatic adenopathy may be successfully used to identify the presence of occult UNPC.

Serum and salivary CEA and GICA levels in oral cavity tumours.

The gastrointestinal cancer-associated antigen (GICA) is recognised by a monoclonal antibody in both serum and tissues of patients with neoplasm of the GI tract. This study compared the serum and saliva values of carcinoembryonic antigen (CEA) and GICA in 19 healthy subjects, 43 patients with benign oral cavity lesions and 26 with histologically confirmed squamous-cell carcinomas. Serum CEA levels were much the same in all three groups, whereas salivary values were significantly higher (p less than 0.001) in both patient groups than in the controls. Serum GICA gave the opposite result: lower in carcinoma than in controls (p less than 0.001) and benign lesions (N.S.), while salivary GICA was significantly lower in carcinoma than in both the other two groups (p less than 0.001). The meaning of this difference between the values for the two antigens is discussed.

Pharmacokinetics of an antibody-ricin conjugate administered intraperitoneally to mice.

Immunotoxins have been extensively studied for the treatment of neoplasias; their intracavitary administration could be useful for the therapy of tumors confined to the pleural or peritoneum spaces. To study the feasibility of this "locoregional" treatment, a pharmacokinetic study of immunotoxins delivery is necessary. Ricin, a plant toxin extracted from the seeds of Ricinus communis, has often been used in immunoconjugates for its high activity; nevertheless, appropriate strategies have been necessary to limit the aspecific toxicity. We previously prepared a AR-3-ricin immunotoxin lacking the ability to bind galactosidic cell surface residues, a so-called sterically blocked immunotoxin. The monoclonal antibody AR-3, an IgG1 specific to the CAR-3 antigen, was able to recognize human colorectal adenocarcinomas. Preclinical trials in nude mice, intraperitoneally grafted with the target neoplasia, showed that this immunotoxin suppressed tumor growth without showing any undesirable ricin toxicity. In the present work we report the pharmacokinetic properties of this immunotoxin, showing the in vivo stability and a relatively long blood survival. With a biodistribution study in tumor-bearing mice, we demonstrate that in tumor-invaded tissues, the concentration of the specific AR-3-ricin immunotoxin was higher and progressively increased in a multiple-dose regimen. In contrast, an irrelevant immunotoxin behaved differently because it did not show specific tumor uptake. Moreover the pharmacokinetic data reported in this work improve the potential for "locoregional" treatment of malignancy with blocked immunotoxins.

Antitumoral activity of liposomes and immunoliposomes containing 5-fluorouridine prodrugs.

Liposomes and immunoliposomes containing cytotoxic agents may be highly efficacious in intracavity therapy of malignancies confined principally to the peritoneal cavity. To assess the feasibility of this locoregional treatment, we prepared two derivatives of 5-fluorouridine (5-FUR), a highly cytotoxic metabolite of 5-fluorouracile, and incorporated them into REV liposomes, prepared with the reverse phase evaporation method. Encapsulation efficiency, drug leakage, and stability were determined, and size analysis and differential scanning calorimetry were carried out to evaluate the drug delivery potential of liposomes containing 5'-palmitoyl-5-FUR, 5'-succinyl-5-FUR, or the parent drug 5-FUR. The most suitable drug for encapsulation, in terms of minimum leakage and encapsulation efficiency, was 5'-palmitoyl-5-FUR, which differential scanning calorimetry indicated as being firmly anchored to the lipid bilayer. Thus, 5'-palmitoyl-5-FUR was chosen to prepare a chemotherapeutic liposome-monoclonal antibody conjugate (immunoliposome). The covalent linkage between antibody and liposome was realized by coupling the thiolated monoclonal antibody AR-3 with REV liposomes, containing N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine. The cytotoxic activity of drug-bearing liposomes and immunoliposomes was evaluated on the HT-29 human colon adenocarcinoma cell line; the immunoliposomes had higher cytotoxicity than liposomes or 5-FUR. To explore the potential of these drug formulations in anticancer therapy, we ip injected liposomes or immunoliposomes into athymic mice ip grafted with human HT-29 cell line. In this mouse model, the immunoliposome containing 5'-palmitoyl-5-FUR displayed the best antitumoral activity, since on day 27 postgraft only 5% of residual tumor mass was present, compared to control mice; there was a close relationship between exposure time of tumor tissue to the drug and antitumor potency.

In situ hybridization and the detection and localization of HCV RNA.

The procedure described below was originally reported to detect the hepatitis C virus RNA (genomic strand) by nonradioisotopic in situ hybridization in formalin-fixed, paraffin-embedded liver tissue of two acutely infected chimpanzees, in a collaborative study conducted with R. H. Purcell, at the National Institute of Allergy and Infectious Diseases, Bethesda, MD (1). Briefly, a synthetic DNA 50-mer was end-labeled with a digoxygenin-conjugated dUTP (2) and hybridized to liver sections. After washing, hybrides were detected by a specific antidigoxigenin antibody and the antigen-antibody reaction revealed by alkaline phosphatase-based enzymatic reaction, through a signal amplification procedure (3). Although the probe represented only 0.5% of the target sequences, the procedure was sensitive enough to detect the low amounts of genomic HCV RNA present in the acutely infected livers, probably owing to both the multistep amplification of the enzymatic reaction and to the prehybridization treatment of the tissue sections, intended to facilitate the diffusion of both the probe and the revealing system molecules.

Prostatic duct carcinoma with combined prostatic duct adenocarcinoma and urothelial carcinoma features: report of a case.

We report a unique case of prostatic duct carcinoma (PDC) featuring both prostatic duct adenocarcinoma (PDA) and high-grade urothelial carcinoma (HG-UC). An 84-year-old man presenting with hematuria showed at ultrasonography and cystoscopy a papillary neoplasia located near to the verumontanum. Histopathologic examination of specimens from transurethral resection revealed a tumor originating from large prostatic ducts showing 2 different components: PDA with endometrioid features (main pattern) and HG-UC (minor part). Immunohistochemically, the areas of PDA were positive for prostatic acid phosphatase (PAP), prostatic specific antigen (PSA), and androgen receptors (AR), while negative for estrogen (ER) and progesterone receptors (PGR). Prognostic factors evaluation pointed out a low proliferation index (10%) and focal expression of p53 (6%); c-erb-B2 was not overexpressed. The HG-UC areas were negative for all previous markers, while positive for thromobomodulin. The proliferation index was high (60%), and p53 was diffusely expressed (55%). The incidence and significance of PDC with combined features is discussed with reference to literature data.

Staining reaction for beta-galactosidase in immunocytochemistry and in situ hybridization.

beta-galactosidase, revealed by an indigo blue reaction product, represents a valid tracer in immunohistochemistry. Observations on the instability of the indigo precipitate led us to investigate this phenomenon. We conclude that avoiding of xylene, and mounting of the preparates in Histoclear (a xylene substitute) and Canada balsam (instead of synthetic resin mountants) yields a sharp and stable indigo precipitate. In addition, we propose a sensitive alternative staining procedure for beta-galactosidase, based on TNBT reduction and precipitation. These reactions can be used both in immunohistochemistry and in in situ hybridization.

Apocrine cells in a fine needle aspirate of gynecomastia. A case report.

BACKGROUND: Apocrine cells are a common finding in female mammary cysts, while only rare cases of apocrine metaplasia in gynecomastia have been found in surgical specimens. CASE: A 65-year-old male presented with painful, monolateral gynecomastia. Fine needle aspiration biopsy showed sheets of large, eosinophilic epithelial cells. On immunocytochemistry these cells were positive for apocrine marker GCDFP-15. The patient had ischemic heart disease and was under treatment with spironolactone. CONCLUSION: Apocrine cysts in gynecomastia are rare histologic findings, and this is the first case diagnosed by fine needle aspiration. The finding of apocrine cells confirms the nonneoplastic nature of the lesion, avoiding surgical excision.

Pancreatic oncocytic endocrine tumors. Cytologic features of two cases.

BACKGROUND: Oncocytoma designates a usually benign tumor consisting of oncocytes (cells rich in mitochondria). Rarely do endocrine pancreatic tumors show oncocytic transformation, and consequently their liver metastases may resemble a hepatocellular carcinoma. CASES: Case 1, a 36-year-old male, presented with an 8-cm pancreatic mass with multiple liver metastases. Fine needle aspiration (FNA) biopsy was performed on the liver. The cytologic features were highly cellular material; numerous isolated cells and irregular, loose cellular aggregates; rare mitoses; round or polygonal cell shape; rosette formation; and large, granular, eosinophilic cytoplasm (suggestive of poorly differentiated hepatocellular carcinoma). Case 2, a 57-year-old female with hypoglycemia, had a 13-cm pancreatic mass. FNA material showed the same cytologic features as case 1. In situ hybridization to detect albumin mRNA was negative in both cases, while immunocytologic reactions for glandular epithelial cytokeratin and chromogranin A were positive. Case 2 was also positive for insulin. CONCLUSION: Oncocytic transformation in endocrine tumors of the pancreas is a rare occurrence and must be kept in mind in the diagnostic workup of FNA material from tumors of the hepatopancreatic region.

Peri and intratumoral macrophage infiltration in laryngeal carcinoma. An immunohistochemical study.

The role of macrophages infiltrating tumor tissue is still poorly explained; the data available are sparse and, to the best of our knowledge, there is no study relating the characteristics of such infiltration to tumor evolution and final outcome. We decided to investigate the spatial relationships between macrophages and neoplastic tissue and their possible prognostic significance on a series of laryngeal carcinomas (98 patients) all of which were operated in our Department and had a follow-up of at least 3 years. In order to identify the macrophage infiltration, formaline-fixed/paraffin-mounted specimens of the tumor were treated with MoAb HAM56. No statistically significant relationship could be established between the degree of macrophage infiltration and the clinical outcome (recurrence), even when the N status was taken into account (N- vs. N+). However, a clear trend was observed when the analysis was confined to grade 3 cases, where the presence of a significant number of macrophages was correlated with a higher probability of recurrence.

[Perirenal immunoblastic lymphoma. Description of a case]. / Linfoma immunoblastico perirenale. Descrizione di un caso.

The primary perirenal localization of non-Hodgkin lymphomas is rare and normal methods of image diagnosis do not enable a reliable preoperative diagnosis. In the majority of cases renal function is not affected and this pathology is often presented as an occasional finding. The pathologies included in the differential diagnosis are renal neoplasias, abscess and inflammatory processes in a perirenal site. Echotomography shows the lesion as an hypoanechoic zone surrounding the kidney. Computed tomography show it as isodense with the renal parenchyma. Histological tests together with immunohistochemical tests identified a malignant large B cell immunoblastic-type lymphoma in the case described here, with plasmoblastic-plasmocytic differentiation and high malignancy according to the Working Formulation. The pathogenesis of this rare localisation is controversial. We maintain that lymphomatous proliferation may be triggered off by lymphatic follicles present in the perirenal space. The concomitant presence of other clinical signs, such as splenomegalia and adenopathies, may contribute to the diagnosis. On the contrary, monolateral involvement in the absence of other signs, as in this case, raises considerable problems of differential diagnosis. Perirenal lymphoma must therefore always be borne in mind in the diagnosis of renal or perirenal masses.

DNA flow cytometry and 67Ki proliferating index as prognostic factors of early recurrence and progression in G1-G2/Ta-T1 and G3/Ta-T1 transitional cell carcinoma of the bladder.

Using flow cytometry, gross genomic alterations, defined as DNA ploidy, and the fraction of S-phase cells, can be calculated in bladder cancer cells. In aneuploid superficial bladder cancer the recurrence rate has been reported to be three times higher than in diploid forms. A correlation between the S-phase fraction and progression has been reported for G1-G2/Ta-T1 tumours, but not for G3/Ta-T1. The aim of our study is to evaluate whether the traditional cytometric parameters can be used as valid predictors of early recurrences and progression in G1-G2/Ta-T1 and G3/Ta-T1 bladder cancer patients and to compare the proliferation indexes as defined by S-phase fraction and 67Ki monoclonal antibody in the two groups of patients.