Investigation of mechanisms underlying chaotic genetic patchiness in the intertidal marbled crab Pachygrapsus marmoratus (Brachyura: Grapsidae) across the Ligurian Sea.

BACKGROUND: Studies on marine community dynamics and population structures are limited by the lack of exhaustive knowledge on the larval dispersal component of connectivity. Genetic data represents a powerful tool in understanding such processes in the marine realm. When dealing with dispersion and connectivity in marine ecosystems, many evidences show patterns of genetic structure that cannot be explained by any clear geographic trend and may show temporal instability. This scenario is usually referred to as chaotic genetic patchiness, whose driving mechanisms are recognized to be selection, temporal shifts in local population dynamics, sweepstakes reproductive success and collective dispersal. In this study we focused on the marbled crab Pachygrapsus marmoratus that inhabits the rocky shores of the Mediterranean Sea, Black Sea and East Atlantic Ocean, and disperses through planktonic larvae for about 1 month. P. marmoratus exhibits unexpectedly low connectivity levels at local scale, although well-defined phylogeographic patterns across the species' distribution range were described. This has been explained as an effect of subtle geographic barriers or due to sweepstake reproductive success. In order to verify a chaotic genetic patchiness scenario, and to explore mechanisms underlying it, we planned our investigation within the Ligurian Sea, an isolated basin of the western Mediterranean Sea, and we genotyped 321 individuals at 11 microsatellite loci. RESULTS: We recorded genetic heterogeneity among our Ligurian Sea samples with the occurrence of genetic clusters not matching the original populations and a slight inter-population divergence, with the geographically most distant populations being the genetically most similar ones. Moreover, individuals from each site were assigned to all the genetic clusters. We also recorded evidences of self-recruitment and a higher than expected within-site kinship. CONCLUSIONS: Overall, our results suggest that the chaotic genetic patchiness we found in P. marmoratus Ligurian Sea populations is the result of a combination of differences in reproductive success, en masse larval dispersion and local larval retention. This study defines P. marmoratus as an example of marine spawner whose genetic pool is not homogenous at population level, but rather split in a chaotic mosaic of slightly differentiated genetic patches derived from complex and dynamic ecological processes.

Sensitivity of hypogean and epigean freshwater copepods to agricultural pollutants.

Widespread pollution from agriculture is one of the major causes of the poor freshwater quality currently observed across Europe. Several studies have addressed the direct impact of agricultural pollutants on freshwater biota by means of laboratory bioassays; however, as far as copepod crustaceans are concerned, the ecotoxicological research is scarce for freshwater species and almost nonexistent for the hypogean ones. In this study, we conducted a comparative analysis of the available literature data on the sensitivity of freshwater copepods to agricultural pollutants. We also assessed the acute and chronic sensitivity of a hypogean and an epigean species, both belonging to the Crustacea Copepoda Cyclopoida Cyclopidae, to two N-fertilizers (urea and ammonium nitrate) and two herbicides (ARIANE(TM) II from Dow AgroSciences LLC, and Imazamox), widely used for cereal agriculture in Europe. According to the literature review, freshwater copepods are sensitive to a range of pesticides and N-fertilizers. Ecotoxicological studies on hypogean species of copepods account only one study. There are no standardized protocols available for acute and chronic toxicity tests for freshwater copepods, making comparisons about sensitivity difficult. From our experiments, ionized ammonia proved to be more toxic than the herbicide Imazamox, in both short and chronic bioassays. Urea was the less toxic chemical for both species. The hypogean species was more sensitive than the epigean one to all chemicals. For both species and for all tested chemicals, acute lethality and chronic lethality were induced at concentrations higher than the law limits of good water body quality in Europe, except for ionized ammonia, which provoked the chronic lethality of the hypogean species at a lower concentration. The hazardous concentration (HC) of un-ionized ammonia for 5 % of freshwater copepods, obtained by a species sensitivity distribution, was 92 µg l(-1), significantly lower than the HC computed for traditional test species from freshwater environments.

Oncocytic papillary renal cell carcinoma: potential pitfall in small enucleation.

OBJECTIVE: We describe an emerging entity, recently recognized as a pitfall in the diagnostic practice among eosinophilic renal cell tumours. METHODS: A 60-year-old male underwent enucleation of a 1.2 cm nodule. Immunohistochemistry and FISH analysis were performed. RESULTS: Histology revealed a neoplasm composed of large cells with eosinophilic cytoplasm, Fuhrman grade 3, arranged in papillae. At the immunohistochemical level, cells showed positivity for AMACR and CD10. Fluorescence in situ hybridization (FISH) demonstrated gains of chromosomes 7 and 17 and loss of Y. A diagnosis of oncocytic papillary renal cell carcinoma was made. CONCLUSIONS: The distinction between renal oncocytoma and oncocytic papillary renal cell carcinoma is of substantial importance because of their different behaviour and prognosis, since the latter has malignant potential. Although the available evidence supporting tumour enucleation as the surgical treatment for renal cortical tumours < or = 4 cm, due to aforementioned clinicopathological features such tumours need to be evaluated using appropriate immunophenotypical and cytogenetic analyses.

[Perianal Paget's disease: five cases report]. / Malattia di Paget perianale: presentazione di 5 casi.

AIM: Extramammary Paget's disease is a rare neoplastic condition, often associated with a synchronous or metachronous underlying skin or visceral malignancies. The aim of this study was to evaluate retrospectively the results we got in 5 cases of perianal Paget's disease and to revise what literature have reported about this issue. PATIENTS AND METHODS: Five patients with perianal EMPD were consecutively treated in our Division between March 1996 and December 2006. In 3 cases the disease was limited to the epidermidis, in one case with multiple recurrences there was dermal infiltration, and one patient had a low rectal adenocarcinoma with pagetoid phenomenon. The surgical treatment we performed in all patients was a wide perianal excision, followed by reconstruction with cutaneous grafts; the resection of rectal adenocarcinoma was carried out using the transanal approach. In two cases we performed a temporary stoma with the sigmoid colon to help the wound healing. RESULTS: We didn't record any complication neither postoperative nor at long time. Two patients developed a local recurrence, but none of our patients showed distant metastases. Four patients are alive and free from disease and one, who developed a multiple local recurrences, died for heart failure. DISCUSSION: The studies available in literature clearly distinguish between a primary EMPD (intraepidermal/intradermal) and secondary disease which is associated with anorectal adenocarcinomas and is thought to be a pagetoid phenomenon, while few informations can derive from those cases in which the disease is associated with an underlying cutaneous adnexal carcinoma. Disease tend to relapse even after a radical surgery and can have metastatic spread, also in intraepidermal form. CONCLUSIONS: Perianal EMPD is a complex disease, difficult to recognize and the association with synchronous or metachronous malignancies imposes long term clinical and instrumental follow up.

Independent prognostic value of fascin immunoreactivity in stage I nonsmall cell lung cancer.

Fascin-1, the most expressed form of fascin in vertebrate tissues, is an actin-bundling protein that induces cell membrane protrusions and increases motility of normal and transformed epithelial cells. Very few data are available on the role of this protein in nonsmall cell lung cancer (NSCLC). Two hundred and twenty patients with stage I NSCLC and long-term follow-up were evaluated immunocytochemically for fascin expression. Overall, variable fascin immunoreactivity was detected in 98% of 116 squamous cell carcinomas, in 78% of 96 adenocarcinomas, in 83% of six large cell carcinomas, and in the two adenosquamous carcinomas under study. Neoplastic emboli were commonly decorated by the antifascin antibody (P<0.001), also when the surrounding invasive carcinoma was unreactive. Fascin immunoreactivity correlated with high tumour grade (P=0.017) and, in adenocarcinomas, with high Ki-67 labelling index (P=0.021). Adenocarcinomas with a prevalent bronchiolo-alveolar in situ component were less commonly immunoreactive for fascin than invasive tumours (P=0.005). Contralateral thoracic or distant metastases were associated significantly with diffuse (>60% immunoreactive tumour cells) fascin expression in adenocarcinomas (P=0.043), and marginally with strong fascin immunostaining in squamous cell carcinomas (P=0.13). No associations were noted with any other clinicopathological variables tested. Patients with tumours showing diffuse (>60% immunoreactive neoplastic cells) and/or strong immunoreactivity for fascin had a shorter survival (P=0.006 for adenocarcinomas and P=0.026 for squamous cell carcinomas), even after multivariate analysis (P=0.014 and 0.050, respectively). The current study documents for the first time that fascin is upregulated in invasive and more aggressive NSCLC, being an independent prognostic predictor of unfavourable clinical course of the disease. Targetting the fascin pathway could be a novel therapeutic strategy of NSCLC.

[Peutz-Jeghers syndrome: case report and update on diagnosis and treatment]. / Sindrome di Peutz-Jeghers. Caso clinico ed attualità diagnostico-terapeutiche.

Peutz-Jeghers syndrome is a rare autosomal dominantly inherited condition with an incidence of 1/120.000 liveborns, characterized by the presence of hamartomatous gastrointestinal polyps and mucocutaneous pigmentation. This syndrome predisposes to various clinical problems such as intussusception and cancer development in different loci (gastrointestinal tract, breast and ovary). For this reason, PJS patients should undergo a surveillance protocol of the genital and gastrointestinal apparatus. Therefore, the early diagnosis of PJS in at-risk family members is very important in preventing cancer development. Germline mutations within the LKB1 or Serine Threonine Kinase (STK11) gene, located on chromosome 19p13.3, are responsible for most cases of PJS so far studied. The existence of a second locus is suspected on chromosome 19q13.4 in a minority of families. The LKB1 gene, recently cloned, encodes the Serine Threonine Kinase LKB1 and is ubiquitously expressed. The identification of the disease-causing mutation in each family makes it possible to perform a presymptomatic diagnosis; therefore, only the mutation carriers will undergo the clinical surveillance program. In this paper, the case of a PJS patient who has been surgically treated is presented. The DNA screening of the LKB1 gene in this patient has led to the identification of the causing mutation. A critical review of the literature and is also presented as well as the proposal to establish an Italian Registry of PJS.

[Current problems of the surgical treatment of rectal cancer: analysis of the literature and personal experience]. / Problematiche attuali nella terapia chirurgica del cancro del retto: analisi della letteratura ed esperienza personale.

The surgical procedure is a crucial factor in preventing local recurrence in rectal cancer, and total mesorectal excision (TME) particularly is widely accepted as being associated with a decreased local recurrence rate. In this study, concerning 187 patients with rectal cancer, we compare conventional surgery, performed in 140 patients from 1979 to 1993, with a standardised TME procedure in 47 patients over the period from 1994 to 1998. The first group not treated by TME were operated on for 56 (40%) tumours of the upper rectum and 84 (60%) of the lower rectum; 35 (25%) were Dukes' A, 77 Dukes' B and 28 (20%) Dukes' C. 42 abdominoperineal amputations (30%) and 98 anterior resections (70%) were performed. The second group in which TME was performed comprised 17 (36%) tumours of the upper rectum and 30 (64%) of the lower rectum, 8 (17%) in stage I AJCC (Dukes' A), 16 (34%) in II (B) and 23 (49%) in III (C). 9 abdominoperineal amputations (19%) and 38 anterior resections (81%) were performed, 8 (21%) with an ultra-low anastomosis. In the first group of patients we observed 28 local recurrences (20%) and a 5-year disease-free survival in 50% of cases. In the second group we achieved a decreased rate of local recurrence (10.6%) which is about half that observed after conventional surgery, but there was no significant difference in survival. These data confirm the effectiveness of TME in reducing local recurrence rate, according to the literature; in future this procedure can get to reconsider the role of adjuvant therapy in the management of rectal cancer.

Immunoreactivity for thyroid transcription factor-1 in stage I non-small cell carcinomas of the lung.

Thyroid transcription factor-1 (TTF-1) is a nuclear protein regulating the transcriptional activity of lung-specific genes in the normal and neoplastic bronchioloalveolar cells. It has been implicated in the normal growth and development of the lung, and the disruption of the TTF-1 locus leads to neonatal death with pulmonary hypoplasia. We evaluated retrospectively the prevalence and clinical significance of TTF-1 immunoreactivity in 222 patients with stage I non-small cell lung carcinoma (NSCLC) with a follow-up time of at least 5 years, and we investigated its relationship with other markers of tumor growth, namely cell proliferation and angiogenesis. TTF-1 immunoreactivity was documented by using the commercially available monoclonal antibody 8G7G3/1 in 72% of 97 adenocarcinomas, 5% of 119 squamous cell carcinomas, and in the glandular component of two adenosquamous carcinomas. Four large cell carcinomas were completely unreactive. In adenocarcinomas, but not squamous cell carcinomas, TTF-1 immunoreactivity correlated significantly with microvessel density (p = 0.04) and inversely with the tumor proliferation fraction assessed by Ki-67 immunostaining (p = 0.03). Also, TTF-1-immunoreactive adenocarcinomas showed a trend for a size less than 3 cm (p = 0.08). TTF-1 expression was not related to specific growth patterns, tumor grade, or tumor cell typing. TTF-1 immunoreactivity did not significantly affect patient survival, although patients with more than 75% immunoreactive neoplastic cells showed a trend for longer overall and disease-free survival. Our findings suggest that TTF-1 could be involved in the development of small pulmonary adenocarcinomas, but it has not prognostic implications in patients with stage I NSCLC.

A phase II study of biochemotherapy for the treatment of metastatic malignant melanoma.

The use of interleukin-2 (IL-2) and interferon-alpha (IFNalpha) in combination with chemotherapy for the treatment of advanced malignant melanoma has generated considerable interest. In particular, the relatively high number of durable complete responses has suggested this may be a significant advance in the treatment of malignant melanoma. We report our experience at the University of Colorado in 43 patients, including many with poor prognostic factors. Patients received cisplatin 20 mg/m2 on days 1-4, vinblastine 1.6 mg/m2 on days 1-4, dacarbazine 800 mg/m2 on day 1, IL-2 9 x 10(6) IU/m2 per day intravenously over 24h on days 1-4 and IFNalpha 5 x 10(6) IU/m2 per day subcutaneously on days 1-5 every 3 weeks. The median follow-up for all patients was 34 months. Responses were seen in 20 patients (47%, 95% confidence interval 31-62%) and comprised five complete responses (CRs) (12%) and 15 partial responses (PRs) (35%). Two patients achieving a CR remain disease free at 45 and 47 months follow-up. In addition three patients who obtained a surgical CR and another with only minor residual changes on computed tomography scan have not progressed at 27, 30, 40 and 27 months, respectively. Toxicity was manageable, but all patients had at least one grade 3 or 4 toxicity, predominantly hypotension and neutropenia. There were no treatment-related deaths. In conclusion, the response rate and duration is within the range previously reported for biochemotherapy. The results of ongoing randomized studies are awaited to better define the value of biochemotherapy in the treatment of advanced melanoma.

Immunocytochemical assessment of bone marrow aspirates for monitoring response to chemotherapy in small-cell lung cancer patients.

Recent reports have suggested that tumour cell immunodetection in bone marrow of small-cell lung cancer patients is by far more frequent than found cytohistologically and may have clinical relevance. This study evaluates primarily the efficacy of chemotherapy as method of in vivo purging, but also the relationship of marrow involvement with survival. A total of 112 bone marrow aspirates from 30 chemo-naïve patients were stained twice using anti-NCAM antibodies, first at diagnosis and then after chemotherapy (24 patients) or at disease progression (six patients). Marrow contamination was associated with lower survival (P = 0.002), and was also detected in 7/17 patients conventionally staged as having limited disease. At multivariate analysis, marrow involvement was an independent factor of unfavourable prognosis (P = 0.033). The amount of tumour contamination, before and after chemotherapy, remained unchanged also in responders and even in the subset of patients with apparent limited disease. Following chemotherapy, bone marrow became tumour negative only in 25% of initially positive responders and in none of non-responders. Our results indicate that (i) chemotherapy is not effective in purging bone marrow even in chemo-responsive patients and (ii) a subset of patients with limited disease and negative bone marrow aspirates might have a more favourable prognosis.

Fibro-osseous lesions of the central nervous system: report of four cases and literature review.

Fibro-osseous lesions, also reported as calcifying pseudoneoplasms of the neural axis, are uncommon lesions of the CNS. We report four additional cases: two extraaxial and two intraaxial, in patients ages 33, 47, 49, and 59 years at presentation. Fibro-osseous lesions involving the CNS demonstrate variable proportions of fibrous stroma, bone, palisading spindle to epithelioid to multinucleated cells in association with a highly distinctive, perhaps pathognomonic, chondromyxoid-like matrix often distributed in a nodular pattern. This histopathologically distinctive lesion can be seen in many regions of the neuraxis, often with a dural association, and most commonly along the vertebral column. It appears to be a slow-growing lesion and, with wide excision, the prognosis is excellent. The etiology remains unclear, but the preponderance of data favors a reactive rather than neoplastic process. If this putative pseudotumor is not recognized histopathologically, a neoplastic or infectious differential might result in inappropriate investigations and potentially harmful therapies.

Effects of different immunolabeling techniques on the detection of small-cell lung cancer cells in bone marrow.

Recent reports have suggested that the immunodetection of tumor cells in bone marrow of small-cell lung cancer (SCLC) patients is by far more effective than traditional cytohistological methods and that this may be clinically relevant. This study aimed to evaluate whether the level of detection of tumor cells in bone marrow is affected by different immunostaining methods. Using two anti-NCAM monoclonal antibodies (MAbs), we compared four different "sandwich" methods on cytospin preparations of the N592 human SCLC cell line and of bone marrow aspirates from 37 SCLC patients. Our data indicate that the combination of the alkaline phosphatase-anti-alkaline phosphatase and streptavidin-biotin-alkaline phosphatase complex methods provides the best results in terms of sensitivity and specificity, and of intensity of immunoreaction and absence of staining background. Moreover, bone marrow micrometastases detected by this method were prognostically relevant and identified, among patients with apparently limited disease according to conventional staging procedures, a subgroup with shorter survival. We suggest that the choice of a sensitive immunostaining technique may significantly increase the detection rate of SCLC cells in bone marrow, mirroring the biological aggressiveness of the disease.

Surgical treatment of pancreatic metastases from renal cell carcinomas.

Pancreatic metastases from a renal cell carcinoma are rare and may occur long after manifestation of the primary disease. Resection of the metastases should be regarded as the best treatment. In our center, owing to the slow evolution of these secondaries, we perform resections capable of limiting the destruction of the pancreatic parenchyma as far as possible. The use of 'atypical' resections of the pancreas is characterized by a higher incidence of postoperative complications, particularly fistulas. Despite this, we believe that adjusted resection is to be advocated because of the possibility of additional remote secondaries, the shorter duration of surgery, the preservation of the glandular parenchyma and intact adjacent organs, such as duodenum, stomach, and spleen, and the fact that there have been no reports on local recurrences.

Differential inhibitory effects of antidiabetic drugs on arterial smooth muscle cell proliferation.

We compared three drugs representing different classes of antidiabetic pharmacology (glyburide, a sulfonylurea; pioglitazone, a thiazolidinedione; and metformin, a biguanide) in terms of their direct effects on proliferation of cultured arterial smooth muscle cells (SMC). Rat aortic SMC were seeded at 4 x 10(4)/35 mm well. After 24 h, they were treated every 2 to 4 days for 2 weeks with 5% fetal bovine serum (FBS) in normal culture medium containing either drug vehicles or a low and a high but nontoxic level of glyburide (0.5 and 2.5 mumol/L), pioglitazone (1 and 5 mumol/L), and metformin (20 and 100 mumol/L). Vehicle-treated cells increased from 2 +/- 0 to 6 +/- 1 to 42 +/- 3 to 210 +/- 14 (cells per well x 10(4); 5 wells each) from day zero to 4 to 9 to 14. From day 9 to 14 these cell numbers were decreased an average of 20% by the 2.5 mumol/L glyburide (P < .05) and 43% by the 5 mumol/L pioglitazone (P < .05). The low levels of glyburide and pioglitazone and both the low and high levels of metformin failed to influence cell numbers. In a second experiment, even half the abovementioned high level of pioglitazone (2.5 mumol/L) still exerted a markedly greater antiproliferative effect on aortic SMC than a high level of 2.0 mumol/L glyburide (P < .05). In addition, neither drug's antiproliferative effect was influenced by a high level of insulin added to the medium (10 mU/mL). Similarly, a small but significant stimulatory effect of this high insulin on cell proliferation (P < .05) was not significantly affected by these two drugs (although pioglitazone tended to inhibit it). These results suggest that thiazolidinediones may be more useful antidiabetic agents than sulfonylureas and biguanides in inhibiting abnormal arterial SMC proliferation associated with atherosclerosis and postangioplastic restenosis which are common in diabetic patients.

Solid and cystic papillary neoplasm of the pancreas: a clinico-cytopathologic and immunocytochemical study of five new cases diagnosed by fine-needle aspiration cytology and a review of the literature.

We report here on five new cases of solid and cystic papillary neoplasm (SCPN) of the pancreas diagnosed by fine-needle aspiration cytology (FNAC). All cytologic samples were obtained by ultrasonography, and the smears were conventionally fixed and stained. Special histochemical and immunocytochemical stains were also performed in some samples. Cytology revealed in all but one case numerous pseudopapillary structures composed of fibrovascular stalks lined with one or more layers of bland-appearing, uniform tumor cells. The tumor cells had round-to-oval euchromatic nuclei with frequently folded smooth contours and one or two small nucleoli. Their cytoplasm often contained eosinophilic, PAS-positive, and diastase-resistant inclusions. Foamy cells, psammoma bodies, blood, and cellular debris were found in the background. The criteria for the differential diagnosis versus other pancreatic lesions are discussed in some detail, as is the role of immunocytochemistry (ICC). In the literature, only 28 cases of cytologically investigated SCPN have been reported to the best of our knowledge. The most helpful criteria for the conclusive identification of SCPN by FNAC include the pseudopapillary arrangement with bland-appearing tumor cells, and, especially, the finding of acidophilic, PAS-positive, and diastase-resistant cytoplasmic granules.

Effects of oral calcium and potassium on endothelium-dependent relaxation in hypertensive rats.

High oral potassium (K) decreases stroke incidence in aging high salt-fed stroke-prone spontaneously hypertensive rats (SHRSP). We have seen high oral Ca increase stroke incidence in aging high salt-fed SHRSP without increasing blood pressure (BP) but with signs of K wasting. Therefore, we sought to determine whether high oral Ca suppresses the previously reported oral K-related enhancement of arterial endothelium-dependent relaxation as seen in younger high salt-fed SHRSP before the appearance of strokes. Four groups of female SHRSP were fed high-salt diets containing either low (0.4%) or high (1.6%) K with low (0.4%) and high (1.6%) Ca from age 1 to 4 mos. High oral K decreased BP independent of Ca intake (P < 0.05). High oral Ca did not affect BP. In contrast to aging SHRSP, high oral Ca neither increased urinary excretion nor decreased plasma concentration of K in these young adult SHRSP. However, high (vs. low) oral K only significantly reduced the half-maximal effective dose for acetylcholine-induced relaxation of aortic rings from rats fed low (18 +/- 3 vs. 38 +/- 6 nM, P < 0.05) not high Ca (25 +/- 5 vs. 31 +/- 3 nM). Neither oral K nor Ca affected nitroprusside-induced relaxation. Thus high oral Ca by itself does not impair endothelium-dependent relaxation in young adult high salt-fed SHRSP, but yet it suppresses the enhancing effect of high oral K on such relaxation and does so without altering BP, K balance, or endothelium-independent relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical detection of cell proliferation-related antigens in cytologic smears of human malignant neoplasms using PC10, reactive with proliferating cell nuclear antigen, and Ki-67. A comparative study.

Cytospins of the MCF-7 cell line and 93 consecutive smears of human malignant neoplasms were immunochemically evaluated for the expression of proliferating cell nuclear antigen (PCNA) and Ki-67-related antigen. Results expressed as the labeling index (LI) were compared with histologic sections. The PCNA LI and Ki-67 LI were lower in cytologic smears than in histologic sections, though the differences were not statistically significant. A positive linear relationship was found between these markers in both cytologic and histologic samples. The PCNA LI was generally lower than the Ki-67 LI, but in seven malignant neoplasms, PCNA LI was greater than the corresponding values of the KI-67 LI. We conclude that cell proliferation can be reliably evaluated on cytologic preparations; PCNA may behave as a Ki-67-like reagent in some tumors, and PCNA may sometimes overestimate the cell growth fraction assessed by Ki-67 immunoreactivity.

Expression of proliferating cell nuclear antigen, Ki-67 antigen, estrogen receptor protein, and tumor suppressor p53 gene in cytologic samples of breast cancer: an immunochemical study with clinical, pathobiological, and histologic correlations.

Sixty-six unselected breast cancers were analyzed in cytologic smears and histologic sections for the expression of Ki-67, proliferating cell nuclear antigen (PCNA), estrogen receptor protein (ERP), and p53 protein using a standard immunochemical method. The results, expressed as both positive cases and labelling index (LI), were compared with clinical and pathobiological variables. Ki-67 and PCNA immunostaining was seen in all cases, whereas ERP was detectable in 46/63 cases and p53 protein in 20/66 cases. The expression of these markers was generally lower in cytology than in histology, though the differences were not statistically significant. PCNA-LI and Ki-67-LI were closely correlated (P < 0.001), the mean PCNA:Ki-67 ratio being 0.92 +/- 0.57. Occasional discrepancies, however, were found. PCNA and Ki-67 expression was associated with an increase in histologic grade and a decrease in ERP content of tumors, whereas p53 was statistically associated with no clinical or pathobiological variables. The data suggest that proliferative activity and oncogene overexpression may be reliably evaluated in breast cancer by FNA cytology, though PCNA is not a suitable indicator for cell proliferation. The results do not resolve the issue as to whether immunostaining for p53 protein constitutes a dedifferentiation product of the tumor, or is a fundamental aspect of the malignant progression. Survival studies in a larger series of tumors are thus needed to elucidate this point.