Sarcomatoid renal cell carcinoma of papillary origin. A case report and cytogenic evaluation.

Sarcomatoid renal cell carcinoma (SRCC) is an aggressive tumor variant thought to arise predominantly from dedifferentiation of clear cell carcinoma. A few reports of SRCC associated with non-clear cell tumors led to the presumption that SRCC may arise from any renal cell carcinoma, although direct evidence of this is lacking. Cytogenetic studies on 3 previously documented SRCCs associated with papillary renal cancers showed either 3p deletions or absence of trisomy 7, 17 in the sarcomatoid tumors, suggesting origin from a coexistent clear cell tumor. The present case represents the first conclusive evidence of direct progression of non-clear cell carcinoma to SRCC with both tumor components containing multiple copies of chromosomes 7 and 17. Many genetic anomalies, including p53 mutations, frequently recognized in SRCC were not recognized in this case, highlighting the importance of cytogenetic evaluation of all SRCC. The patient is well and without evidence of tumor progression 1 year after surgery, and the sinister outlook of SRCC in association with clear cell carcinoma may not apply in SRCC of non-clear cell origin.

Distinction between intraductal carcinoma of the prostate (IDC-P), high-grade dysplasia (PIN), and invasive prostatic adenocarcinoma, using molecular markers of cancer progression.

BACKGROUND: Prostate ducts and acini whose lumens are filled with malignant cells represent a well-recognized histological pattern recently termed intraductal carcinoma of the prostate (IDC-P). These tumors are often associated with rapid disease progression, and most recur after radical surgery. Controversy exists as to whether IDC-P should be recognized as a separate entity, an extension of high-grade dysplasia (PIN) or invasive carcinoma as described by the Gleason grading system. This study investigates the use of molecular markers in defining the position of IDC-P in the evolutionary hierarchy of prostate cancer progression. METHODS: IDC-P, high-grade dysplasia, and invasive cancers from a cohort of 20 selected radical prostatectomy specimens were screened for loss of heterozygosity (LOH), using 12 polymorphic microsatellite markers frequently lost in prostate cancer. RESULTS: LOH was absent in Gleason grade 3 cancer, infrequent in high-grade dysplasia (9%) and Gleason grade 4 cancer (29%), but common in IDC-P (60%). In IDC-P, and to a lesser extent Gleason grade 4 cancers, multiple sites of allelic loss in individual cases were usual. CONCLUSIONS: Allelic instability provides further evidence that IDC-P is not a simple extension of dysplasia, nor does it represent invasion of Gleason grade 3 cancers into the ductal/acinar system. IDC-P and Gleason grade 4 cancer represent late but possibly separate events in prostate cancer evolution.

Identification of the glycosaminoglycan keratan sulfate in the prostatic secretory cell.

BACKGROUND: Prostate secretory granules (PSG) represent the basic secretory unit of the prostate gland, containing many of its exocrine proteases. Recent analysis of intraluminal corpora amylacea, a proposed by-product of PSG secretion, detected sulfated glycosaminoglycans (GAG) possibly keratan sulfate (KS), indicating a secretory mechanism for GAG in the human prostate surface epithelial cell. METHODS: Immunostains using anti-KS and anti-prostate-specific antigen (PSA) were evaluated on 10 sequential radical prostatectomy specimens, three of which had received neoadjuvant antiandrogen therapy. Extracts of normal secretory tissue as well as a sample composed almost entirely of prostatic stroma were subjected to Western blot analysis, using the same antibody panel. RESULTS: Keratan sulfate secretion from the normal prostate epithelial cell has been confirmed and correlates, as does PSA, with the presence of cytoplasmic PSG. No such correlation exists in most adenocarcinomas or in benign epithelium after androgen ablation. Western blot analyses confirmed tissue immunostains and demonstrated a secretory proteoglycan of 70-95 kDa. CONCLUSIONS: Recognition of PSG heralds a novel secretory mechanism within the human prostate gland that is linked to the secretion of KS. The role of KS in normal prostate secretion remains unknown, although it appears downregulated in neoplastic and androgen-ablated cells.

Variant chromophobe renal cell carcinoma.

Separation of renal cell tumors into different prognostic groups is an imperative function of the diagnostic pathologist. Recently, chromophobe renal carcinoma has been described as a tumor that is morphologically distinct from conventional "clear cell" carcinoma and that has a low metastatic potential. Identification is based on routine light microscopic features and is confirmed by special stains, immunohistochemistry, and electron microscopy. We present a variant of chromophobe renal carcinoma that did not show the typical cytomorphologic features on light microscopy after formaldehyde fixation. After fixation in Solufix (a commercial fixative), these features were recognized and the diagnosis was confirmed. The tumor also showed an unusual form of calcification and psammoma body formation not previously recognized in chromophobe tumors. Molecular biological assessment was inconclusive, but excluded a chromosome 3p deletion usually found in conventional renal carcinoma. The use of a different primary fixative may provide a cost-effective screening tool to detect variant renal tumors and may have important prognostic implications.

Luminal contents of benign and malignant prostatic glands: correspondence to altered secretory mechanisms.

Recent changes in tissue fixation strategy, using glutaraldehyde, have clarified the secretory mechanisms of the normal prostate identifying cytoplasmic prostatic secretory granules, structures not preserved by formalin fixation. This normal secretory mechanism was absent in most adenocarcinomas, depicting an important metabolic change in transformed prostate cells. The current study further investigates differences between benign and malignant prostate secretion and relates them to the production of corpora amylacea by benign glands and crystalloids or mucin by cancer. In all normal prostate cells examined (6 cases), prostate secretory granules (PSG) were approximately 1-microm, brightly eosinophilic granules filling the cytoplasm of secretory cells and released in packets by a specialized apocrine cell structure. After apocrine decapitation and luminal dispersal, some of the cytoplasmic and PSG remnants condensed to form eosinophilic bodies (EB) with a glycoprotein rim and central protein core. EB were observed adsorbing and layering onto the surface of prostatic corpora amylacea representing their chief mode of enlargement. Biochemical analysis and x-ray diffraction studies confirmed sulfated glycosaminoglycans of similar structure as the main constituent of both PSG and corpora amylacea. Peripheral zone amphiphilic "dark cell" carcinoma (9 cases) contained almost no PSG, and showed neither apical decapitation nor EB formation, but mucin secretion was frequently detected. Crystalloids that share the same staining characteristics and sulfur content as PSG and corpora amylacea were identified in 3 selected "clear cell" carcinomas, all of which showed at least focal PSG secretion. The recognition of these differing secretory mechanisms and their deviation from normal further defines the histological criteria and spectrum of prostate malignancy.

Androgen receptor expression of proliferating basal and luminal cells in adult murine ventral prostate.

Maintenance of the size and differentiated function of the adult prostate is dependent on testicular androgens. In this study, simultaneous androgen receptor (AR) immunohistochemistry and [(3)H]thymidine labelling was used to characterise the proliferating epithelial cells of the murine ventral prostate. Proliferation in the adult prostate was more prevalent in the basal cell population with 1.8&percent; AR-negative cells labelled with [(3)H]thymidine as compared with 0.7% AR-expressing luminal cells. Three weeks following castration of mice, the atrophied prostate contained rudimentary glands composed of both luminal and basal cells with the proportion of AR-expressing basal cells reduced from 50 to 25%. Administration of testosterone enanthate to castrated mice induced a recapitulation of the prostate gland that was preceded by up-regulation of AR expression in basal cells to normal adult levels (50% AR-positive cells) by 12 h following testosterone injection. Proliferation of AR-positive luminal cells peaked at 48 h (22.8%) while proliferation of AR-negative basal cells peaked at 96 h (6.1%) following testosterone administration. These results suggest that distinct populations of luminal and basal cells are resistant to castration-induced involution of the prostate but remain responsive to direct or indirect testosterone effects and recapitulate the gland following administration of testosterone.

Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle. Association with eralpha and erbeta status.

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)

Lymphoepithelioma-like carcinoma of the renal pelvis.

We report the first documented case of undifferentiated carcinoma of the renal pelvis with a prominent lymphoid stroma (lymphoepithelioma-like carcinoma [LELC]). LELCs are morphologically identical to nasopharyngeal carcinoma and are rarely seen in the urinary tract, with only isolated cases involving the urinary bladder and ureter. The tumor was composed entirely of large pale staining malignant epithelial cells with ill-defined borders arranged in syncytial sheets separated by mainly reactive lymphocytes, occasional plasma cells and histiocytes. Tumor cells were immunoreactive to cytokeratin and were negative for leukocyte common antigen. Awareness of LELC is important, as it should be distinguished from lymphoma or inflammatory lesions including, xanthogranulomatous pyelonephritis.

Characterization of cytoplasmic secretory granules (PSG), in prostatic epithelium and their transformation-induced loss in dysplasia and adenocarcinoma.

Cytoplasmic clarity is a histological feature of normal prostatic secretory cells, but in this study, tissue fixation in strong (>2.5%) glutaraldehyde dramatically altered cytological staining. Secretory cytoplasm appeared red and granular on routine stains because of myriad intensely staining eosinophilic granules (PSG). Immunostaining for prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) showed their exclusive localization to the PSG. Electron microscopy confirmed these findings and also showed that after fixation in many agents, including formaldehyde, PSG appeared empty, accounting for the artefactual "clear cell" appearance on light microscopy. PSG were most densely concentrated apically in a bud-shaped luminal compartment in which cytokeratin was selectively absent. Normal exocrine secretion was visualized as detachment of apocrine buds or their in situ disintegration. Distinctively in dysplasia and almost all carcinomas, PSG were rare to absent, and proteases were free in the cytoplasm, often concentrated beneath the apical membrane. The apocrine compartment was absent, with no observed secretory mechanism. Tumor cells had dark amphiphilic cytoplasm after all fixatives. This provided a reliable method of distinguishing malignant from benign glands in tissues fixed in strong glutaraldehyde. Clear cell carcinomas, whose cytoplasm mimicked routinely fixed normal secretory cells, surprisingly had almost no PSG. Instead, their "granules" were lipid-filled vacuoles reflecting a secretory pathway not seen in normal cells, dysplasia, or the common "dark cell" carcinomas. These observations may define two distinctive biological pathways of prostate cancer evolution and may facilitate diagnostic decisions on needle biopsy samples.

Prostate specific origin of dipeptidylpeptidase IV (CD-26) in human seminal plasma.

PURPOSE: A number of peptidases which can metabolize certain bioactive peptides and growth factors have been identified in seminal plasma. Our goal in this study was to determine molecular properties and the tissue source(s) for one of these peptidases, dipeptidylpeptidase IV (DPP IV), in human seminal plasma. MATERIALS AND METHODS: We measured the activities of DPP IV with the dipeptide glycylprolyl-p-nitroanalide and its molecular forms using immunoblotting of seminal plasmas of men who were vasectomized or with different sperm concentrations, and in prostatic and seminal vesicle secretions of men undergoing prostatic surgery. RESULTS: DPP IV in seminal plasma of vasectomized men was a membrane associated dimer comprised of subunits of approximately 110 kDa. Its activity did not differ in seminal plasmas of vasectomized, azoospermic, oligozoospermic and normozoospermic men indicating no correlation with the concentration of sperm originally present in the semen. The DPP IV antigen (CD -26) and enzymic activity were present in prostatic secretion, but absent from that of the seminal vesicles. These data indicate that the prostate gland is the primary source of DPP IV activity in seminal plasma. There was little variation in its activities in repeat seminal plasma samples from the same individual, and there was no change in its activity with age to 50 years. CONCLUSIONS: DPP IV in seminal plasma was derived from the prostate gland and it may be useful as a bioindicator of prostate function and/or disease with age in men.

Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates.

Genomic subtractive hybridization of closely related Pasteurella multocida isolates has generated clones useful in distinguishing hemorrhagic septicemia-causing type B strains from other P. multocida serotypes. Oligonucleotide primers designed during the sequencing of these clones have proved valuable in the development of PCR assays for rapid species- and type-specific detection of P. multocida and of type B:2 in particular. This study demonstrated that the primer pair designed from the sequence of the clone 6b (KTT72 and KTSP61) specifically amplified a DNA fragment from types B:2, B:5, and B:2,5 P. multocida and that the primers KMT1T7 and KMT1SP6 produced an amplification product unique to all P. multocida isolates analyzed. It was also shown that PCR amplification performed directly on bacterial colonies or cultures represents an extremely rapid, sensitive method of P. multocida identification.

Estrogen receptor-negative epithelial cells in mouse mammary gland development and growth.

The mouse mammary gland undergoes rapid proliferation during puberty, then cyclical proliferation and involution during adulthood within a 5-day estrous cycle. Although proliferation of mammary epithelial cells is directed by elevated serum levels of estrogen acting via the estrogen receptor (ER), the ER status of the proliferating cells remains unknown. We examined the ER expression of proliferating epithelial cell types during pubertal development and normal adult growth using simultaneous immunohistochemistry for ER and tritiated thymidine (3H-Tdr) autoradiography. These studies demonstrate that during pubertal growth (4-6 weeks) ER-negative cells comprise more than 50% of the epithelial cell populations in the terminal end buds (TEBs) and ducts. Furthermore, the majority of proliferating cells in both TEBs and ducts are ER-negative. These findings indicate that proliferation of cells within both the TEBs and the mammary ducts contribute to pubertal growth of the mammary gland and that the greater proportion of dividing cells are ER-negative. Similar patterns of cell growth were observed in the normal estrous cycle when the majority of dividing cells were ER-negative during both pro-estrous and estrous. Intensive labelling of cells with 3H-Tdr was used to identify long-lived mammary epithelial cells which retained 3H-Tdr 2 weeks following labelling (i.e., following 3 estrous cycles). Of the small number of mammary epithelial cells retaining 3H-Tdr label, most were ER-positive luminal cells and only a few were ER-negative basal cells. This study indicates that pubertal growth of the mammary gland comprises division of ER-negative cap cells and of both ER-negative and ER-positive cells in the body of the TEBs and elongating mammary ducts. Similarly, estrogen-driven proliferation of ER-negative and ER-positive luminal cells and ER-negative basal cells maintains the differentiated mammary gland in the adult mouse.

Assessment of vascularity in breast carcinoma by computer-assisted video analysis (CAVA) and its association with axillary lymph node status.

Case-control methodology was used to evaluate the significance of vascularity in small breast carcinomas with regard to the presence or absence of axillary lymph node metastases. Vascularity was assessed in 32 axillary node positive primary breast tumours (LN+ve) less than 2 cm in size and compared with 56 control axillary node negative primary tumours (LN-ve), which were matched for histological type and grade and tumour size. This study design employed computer-assisted video analysis (CAVA) to assess the total blood vessel perimeter (BVP), total blood vessel area (BVA), and total blood vessel density (BVD) throughout a tissue section that encompassed an entire cross section of the tumour and its immediate periphery. The BVA and BVD in these tumours were not significantly different between LN+ve and LN-ve groups. The LN-ve carcinomas had, on average, a significantly (P<0.05) higher total BVP (3355 microm/mm2) than LN+ve tumours (2771 microm/mm2). 'Hot spot' areas were also independently assessed by two pathologists and the same areas measured by CAVA. A strong correlation (P<0.001) between the two methods of assessment of BVD of the neovascular 'hot spots' was found; however, no association with axillary lymph node metastasis was found using either method of assessment. In conclusion, vascularity assessed by either blood vessel density or blood vessel size in primary invasive breast cancers less than 2 cm in diameter showed no association with axillary lymph node metastasis; in fact a negative association was found with total BVP of whole tumour sections and BVD in 'hot spots' using CAVA. Further, this study has established a computer-assisted method of quantifying vascularity in solid neoplasms and is a positive step towards a standardised approach to this diverse and methodologically variable area.

Protein kinase CK2 activities in human prostatic and seminal-vesicle secretions and seminal plasma.

Human prostatic secretion and seminal plasma contain certain protein kinase activities. Protein kinases play important roles in regulating a vast variety of cellular functions. The objective of this study was to determine whether one of these protein kinase activities in human prostatic secretion and seminal plasma is due to CK2, a messenger-independent, serine/threonine protein kinase that has considerable potential as a regulatory enzyme. By employing an anti-CK2 antibody and a CK2-specific peptide substrate, we have established that CK2 is present in these secretions. Approximately 70% of the CK2 activity present in seminal plasma of normozoospermic men (n = 49) is correlated to the number of sperm originally present in the semen. Further, both the prostate gland and the seminal vesicles are sources of CK2 activity in the seminal plasma of vasectomized men (n = 38). Although there was considerable variation between individuals in CK2 activity, the variation in repeat semen samples of the same vasectomized men (n = 6) was within 21%. There was no correlation of CK2 activity in seminal plasma with age for vasectomized (27-48 years, n = 38), oligozoospermic (28-43 years, n = 24), or normozoospermic men (26-48 years, n = 49). These data suggest that the majority of CK2 activity in the seminal plasma of normozoospermic men originates from sperm but that the prostate and seminal vesicles are accessory sex-gland sources of this enzyme.

Urinary continence after radical retropubic prostatectomy. Analysis and synthesis of contributing factors: a unified concept.

OBJECTIVE: To assess the effects of three types of apical dissection on urinary continence after radical retropubic prostatectomy and to evaluate possible contributing factors, e.g. preservation of the bladder neck and preprostatic sphincter, age, anastomotic strictures, previous transurethral resection and nerve-sparing surgery. PATIENTS AND METHODS: Having undergone one of three types of apical dissection, 280 patients were evaluated: in Group 1 (sphincter-damaging) 134 patients underwent the original technique of ligating and transecting the venous complex; in Group 2 (sphincter-repairing), 76 patients had the venous complex with part of striated sphincter incorporated within anastomotic suture(s); and in Group 3 (sphincter-preserving), 70 patients had the venous complex alone ligated using the 'bunching' technique of Myers. The outcome was analysed for the number becoming continent and the time to continence. RESULTS: Continence was achieved in 93% overall, with 90%, 93% and 99% achieving continence in Groups 1, 2 and 3, respectively. The mean time to continence was 68 days overall, taking 100, 52 and 30 days for the respective groups. Twenty patients (7%) did not achieve full continence; 15 had minor incontinence and five severe, with none of the latter being in Group 3. The group (preservation of external sphincter), age and freedom from development of anastomotic strictures were the most important factors both in regaining continence and decreasing the time to continence. CONCLUSIONS: Preservation of as much as possible of the normal anatomy of the sphincter mechanisms and their nerve supplies results in an excellent return to continence after radical retropubic prostatectomy.

Analysis of haemorrhagic septicaemia-causing isolates of Pasteurella multocida by ribotyping and field alternation gel electrophoresis (FAGE).

Ribotyping and field alternation gel electrophoresis (FAGE) were used to examine 19 Pasteurella multocida isolates, and to assess the ability of these techniques to differentiate P. multocida strains that cause haemorrhagic septicaemia (HS). Reproducible patterns were obtained from both methods, with FAGE demonstrating greater discriminatory power than ribotyping. FAGE analysis was particularly useful in distinguishing North American cultures originating from the 1922 Yellowstone National Park Buffalo 'B' strain, demonstrating the ability to detect genetic alterations induced by repeated subculture. A remarkable homogeneity was observed among Asian HS strains following ribotyping and FAGE analysis, with a clear distinction observed between virulent and avirulent HS isolates. This study has illustrated the value of genomic fingerprinting methods in distinguishing strains of similar serotype, and the capability of these methods to produce detailed characterisation of P. multocida isolates.

REP-PCR analysis of Pasteurella multocida isolates that cause haemorrhagic septicaemia.

Amplification of multiple P multocida genomic DNA fragments by outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence, generated complex profiles in a PCR-based fingerprinting method known as REP-PCR. Polymorphisms within REP-PCR profiles were used to characterise 38 isolates of P multocida. The high degree of homogeneity observed among haemorrhagic septicaemia (HS) strains of serotype B and E provided evidence of a disease-associated REP profile that may serve as a novel method for the identification of HS strains regardless of serotype. REP-PCR profiles of other P multocida serotypes were highly variable, illustrating the potential of this technique for the molecular fingerprinting of fowl cholera or atrophic rhinitis isolates. A specific amplified REP fragment was isolated and used to probe membrane-bound digested P multocida genomic DNA. Hybridisation patterns not only distinguished HS-causing isolates from non-HS P multocida, but also demonstrated a degree of relatedness between HS and HS-like strains.

Detection of p53 gene mutation by rapid PCR-SSCP and its association with poor survival in breast cancer.

We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non-isotopic single-strand conformation polymorphism (SSCP) method we screened for mutations in exons 4-10 of the p53 gene in 375 primary breast cancers from patients with a median follow-up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S-phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node-negative and hormone receptor-positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S-phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction-SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information.

Detection of a population of long-lived cells in mammary epithelium of the mouse.

The fate of dividing mouse mammary epithelial cells was followed by use of tritiated thymidine (3H-Tdr) autoradiography. Loss of label consistent with halving kinetics was observed at various times after injection; however, heavily labelled cells were frequently observed at two weeks and later, when none was expected. The grain count over these heavily labelled cells was often comparable with that 1 h after 3H-Tdr injection. Extensive serial sectioning revealed that the heavily labelled cells were often single cells surrounded by many unlabelled cells or that their label was in stark contrast (in excess of 20 reduced silver grains) to the surrounding group of cells whose label was just above background (a maximum of 3 grains). In addition, by injecting mice at different stages of oestrus, we demonstrated that these long-lived cells, although influenced by oestrus, replicated independently of the oestrogen peak. Our data support a model for mouse mammary epithelium that has a single 'stem' cell positioned within a group of its progeny to form a discrete proliferative unit. This model requires many such stem cells within the mammary epithelium and is consistent with similar models proposed for other tissues.

Cloning of a unique sequence specific to isolates of type B:2 Pasteurella multocida.

Two Carter type B Pasteurella multocida isolates, Izatnagar 52 and 25, isolated from cases of haemorrhagic septicaemia (HS), were used in a modified subtractive hybridisation technique with the specific aim of cloning unique DNA sequences related to the pathogenesis of HS. Biochemical and protein analyses have shown these isolates to be similar, but reports indicate that they have differences in pathogenicity. The subtracted inserts were screened against genomic DNA from a wide range of P multocida isolates, with two distinct fragments demonstrating specific hybridisation with Carter type B isolates that cause HS. No identity was observed with either Carter type E isolates or non-HS type B strains. The clones were sequenced and a search of the GenBank database revealed significant identity of the clone A3b (296 nt) to P haemolytica lipoprotein, whereas there was no significant identity with 6b (956 nt). Both these fragments had a high level of identity (72.8 to 76.9 per cent) to the H influenzae Rd genome.