Susan J. Maygarden

Steroid 5α-Reductase Isozymes I and II in Recurrent Prostate Cancer

Purpose: Prostate cancer recurs during androgen deprivation therapy despite reduced circulating androgens. We showed that recurrent prostate cancer tissue has testosterone levels similar to androgen-stimulated benign prostate, whereas dihydrotestosterone levels were reduced 82% to 1.45 nmol/L, sufficient for androgen receptor activation. The altered testosterone/dihydrotestosterone ratio in recurrent prostate cancer suggests loss of 5α-reducing capability. The aim of this study was to characterize steroid 5α-reductase isozymes I (S5αRI) and II (S5αRII) in prostate tissues. Experimental Design: A tissue microarray was constructed from 22 recurrent prostate cancer specimens and matched pairs of androgen-stimulated benign prostate and androgen-stimulated prostate cancer from 23 radical prostatectomy specimens. Immunoblots were constructed from eight recurrent prostate cancers, eight androgen-stimulated benign prostate, and eight androgen-stimulated prostate cancer specimens. Isozyme expression was examined in microarray sections and immunoblots using S5αRI and S5αRII polyclonal antibodies. Isozyme activities were measured in 12 recurrent prostate cancer, 12 androgen-stimulated benign prostate, and 12 androgen-stimulated prostate cancer specimens. Results: Nuclear immunostaining exhibited higher S5αRI expression than S5αRII in recurrent prostate cancer, androgen-stimulated benign prostate, and androgen-stimulated prostate cancers (P < 0.0001); mean expression was 125, 150, and 115 for S5αRI versus 10, 29, and 37 for S5αRII, respectively. Cytoplasmic immunostaining was moderate and similar for both isozymes in the three tissue types (P > 0.05). Immunoblots confirmed immunohistochemistry; S5αRI was expressed in recurrent prostate cancer specimens and S5αRII was not detected. The activity of S5αRI (114.4 pmol/mg epithelial protein/minute) was 3.7-fold higher than S5αRII (30.7 pmol/mg epithelial protein/minute) in recurrent prostate cancer specimens. Conclusions: Expression levels and isozyme activity shifts from S5αRII toward S5αRI in recurrent prostate cancer. Dual inhibition of S5αRI and S5αRII should reduce dihydrotestosterone biosynthesis and may prevent or delay growth of recurrent prostate cancer.

Metastatic Sublines of an SV40 Large T Antigen Immortalized Human Prostate Epithelial Cell Line

The available human prostate cancer cell lines that are metastatic in athymic nude mice all have complex, highly aneuploid karyotypes. Other prostatic cells immortalized by transforming genes of SV40 or HPV and converted to tumorigenicity by additional genetic manipulation are not reported to be metastatic.

Comparison of the clinical and pathologic staging in patients undergoing radical cystectomy for bladder cancer

PURPOSE Radical cystectomy (RCx) is perhaps the most effective therapeutic approach for patients with muscle-invasive bladder cancer. Unfortunately, clinical staging is imprecise and the degree of understaging remains high. This study retrospectively evaluated patients undergoing RCx with regard to pathologic outcomes and degree of upstaging to better identify features that may lessen clinical understaging. MATERIALS AND METHODS 141 consecutive patients with urothelial bladder carcinoma who were candidates for RCx with curative intent were retrospectively evaluated. Preoperative clinical and pathological (i.e. TURBT) features were compared to pathological outcomes in the cystectomy specimen. Patients were also evaluated as to whether cystectomy was performed as their primary (n = 91) versus secondary (n = 50) treatment for recurrent/progressive disease. Date of cystectomy (<or= 5 years vs. > 5 years prior to study) was also analyzed. RESULTS Of the 141 patients, 54% were upstaged on operative pathology. The greatest degree of upstaging occurred in those with invasive disease preoperatively (cT2-T3). Twenty-six percent of all patients had node-positive disease, and 75% of cT3 patients were node-positive. Seven of 101 (7%) patients with clinical T2 disease were unresectable at the time of surgery. In the primary (vs. secondary) RCx group, more patients were upstaged (63% vs. 40%), non-organ confined (62% vs. 38%), and LN positive (31% vs. 20%). In the more modern cohort, the degree of upstaging was not improved. CONCLUSIONS Pathologic findings after RCx often do not correlate with preoperative staging. Over half of patients undergoing cystectomy are upstaged on their operative pathology. An improved understanding of the relative frequency of upstaging in cystectomy patients may have important implications in the decision-making and selection for neoadjuvant and adjuvant therapies for these high-risk populations.

Downregulation of the β4 integrin subunit in prostatic carcinoma and prostatic intraepithelial neoplasia

Abstract Integrins are adhesion receptors thought to be important in the process of cancer cell invasion and metastasis. Unlike other integrins, which attach a cell to extracellular matrix molecules, the α 6 β 4 integrin participates in the formation of hemidesmosomes, attaching epithelial cells to the basement membrane. Investigations of the α 6 β 4 integrin in human prostatic carcinoma have yielded conflicting results and have been primarily qualitative rather than quantitative. Expression of the β 4 integrin subunit was determined using rat monoclonal antibody 439-9B and image analysis in regions of benign prostatic epithelium (BPE), high-grade prostatic intraepithelial neoplasia (PIN), and prostatic carcinoma (CaP) in 38 patients treated by radical prostatectomy for clinically localized CaP. The β 4 integrin subunit was significantly downregulated in CaP compared with BPE; PIN stained intermediate in intensity between BPE and CaP. Thirty-four of 35 patients showed downregulation of the β 4 integrin subunit, and all 15 patients with PIN had downregulation of β 4 in PIN as compared with BPE. Degree of downregulation of the β 4 integrin subunit did not add prognostic significance to the information present at initial biopsy (age, clinical stage, clinical grade, and serum prostate-specific antigen level). There was no correlation between intensity of staining of CaP, absolute change in staining, or percent loss of β 4 integrin subunit staining with age, pathological stage, or Gleasons score. Downregulation of the β 4 integrin in CaP and PIN compared with BPE maybe correlated with neoplastic transformation of the prostate and loss of hemidesmosomes or basal epithelial cells.

Establishment of Short-Term Primary Human Prostate Xenografts for the Study of Prostate Biology and Cancer

Human tissue xenograft models are currently the only tool for conducting in vivo analyses of intact human tissue. The goal of the present study was to develop reliable methods for successful generation of short-term primary tissue xenografts from benign and tumor-derived human prostate tissue. Primary human prostate xenografts were established in athymic nu/nu mice from eight of eight benign and five of five prostate cancer tissues, collected from a total of 10 patients who underwent radical prostatectomy for the treatment of prostate cancer. An average of 13 xenografts was established per specimen. Two tissue specimens were cryopreserved for >1 month before successful generation of prostate xenografts. After 1 month in vivo, xenograft tissues were harvested and examined regarding: gross evidence of vascularization; tissue morphology; proliferation; apoptosis; and expression of androgen receptor, prostate-specific antigen, and high molecular weight cytokeratins specific for basal cells in the prostate. Direct comparison of the original tissue specimen and the 1-month xenografts revealed similar histology; similar apoptotic and proliferative fractions in most cases; and comparable expression levels and expression patterns of androgen receptor, prostate-specific antigen, and high molecular weight cytokeratins. These data demonstrate that primary human prostate xenografts, benign and malignant, can be established routinely from human prostate tissue surgical specimens, and that the xenografts maintain tissue architecture and expression of key prostatic markers. The development of this methodology, including the technique for cryopreservation of human tissue, will allow multiple (successive) analyses of human prostate tissue to be conducted throughout time using a tissue sample derived from a single patient; and simultaneous analysis of human prostate tissues derived from a cohort of patients.

Bronchial margins in lung cancer resection specimens: utility of frozen section and gross evaluation

Pathology reports for all lobectomy and pneumonectomy specimens at UNC Hospitals between 1991 and 2000 (n=405) were reviewed for correlation between frozen section and final bronchial margin, gross distance between tumor and margin and tumor type. Frozen section was performed in 268 cases (66%). A total of 243 were true negatives (90.6 %), 16 (6.0%) were true positives, four (1.5%) were false positives and five (1.9%) were false negatives. The site of tumor in true-positive cases was mucosal (11), submucosal (three), lymphatics (one), peribronchial (one). The site of tumor in false-negative cases was submucosal (two), lymphatics (one), peribronchial (two). In 137 cases, no bronchial frozen section was performed; there was one case (0.7%) with positive margin. There was no correlation between final margin positivity and distance between gross tumor and margin. Tumor distance to margin in positive margin cases varied from grossly involved to 3 cm away. There were 72 cases in which wedge resection was performed before lobectomy in which no gross tumor remained in the lobectomy, and in all cases final bronchial margins were negative. In all, 373 of cases (92%) were nonsmall carcinomas. Of these, 10 (2.7%) had positive margins. Tumors other than nonsmall cell carcinoma accounted for a disproportionate number of positive margins. In all, 3/6 of adenoid cystic/mucoepidermoid carcinoma, 1/7 small cell carcinoma and 1/1 lymphoma cases had positive margins. In conclusion, frozen section evaluation of bronchial margins is helpful in central lung tumors. Mucosal tumor is preferentially identified in frozen section. Gross evaluation of margins is problematic, as intramucosal carcinoma or tumor in lymphatics may not be detected, but 3 cm was a ‘safe’ distance for gross tumor from margin. In lobectomies following wedge resection in which no gross tumor remained, all had negative margins. Salivary gland-type tumors have a high incidence of positive margins, and frozen section is particularly indicated in these tumors.

Human alveolar macrophage-56 and carcinoembryonic antigen monoclonal antibodies in the differential diagnosis between primary ovarian and metastatic gastrointestinal carcinomas

The immunohistochemical expression and localization of monoclonal antibodies to carcinoembryonic antigen (CEA) and human alveolar macrophage (HAM-56) were evaluated in primary ovarian and metastatic gastrointestinal (GI) carcinomas. Immunohistochemistry was performed using an avidin-biotin-peroxidase complex method with capillary gap technology on formalin-fixed, paraffin-embedded tissues from 41 primary ovarian epithelial neoplasms, 17 metastatic gastrointestinal malignancies, and 10 tumors of uncertain primary origin. Overall, immunostaining for HAM-56 was positive in 35 (85%) ovarian epithelial neoplasms compared with only two (12%) gastrointestinal cancers. Carcinoembryonic antigen was positive in 16 (39%) ovarian versus 13 (76%) GI tumors. Of the primary ovarian neoplasms, 22 were positive for HAM-56 only, 13 were positive for both HAM-56 and CEA, three were positive for CEA only (all mucinous neoplasms), and three were negative for both. Of the primary GI neoplasms, 12 were positive for CEA only (including all eight colon cancers), one was positive for both HAM-56 and CEA, one was positive for HAM-56 only, and three were negative for both. Of the 10 neoplasms of unknown origin at initial presentation, six were positive for HAM-56 only, three were positive for CEA only, none was positive for both HAM-56 and CEA, and one was negative for both. Only three of these 10 neoplasms remained of indeterminate origin after pathological review and clinical follow-up. When positive, CEA was usually strong and generalized in GI cancers but weak and focal in ovarian neoplasms. The HAM-56 positivity in ovarian neoplasms was typically focal and largely limited to areas with glandular or papillary differentiation with apical linear accentuation. We conclude that an immunohistochemical panel using both HAM-56 (Enzo Diagnostics, Syosset, NY) and CEA monoclonal antibodies is helpful in differentiating primary ovarian neoplasms from metastatic gastrointestinal malignancies, and in evaluating metastatic adenocarcinoma of unknown primary site.

Comparison of clinical and pathological features in African-American and Caucasian patients with localized prostate cancer

To examine patient characteristics, prostate specific antigen (PSA) levels, and established preoperative and pathological prognostic factors to determine differences between Caucasian and African‐American patients with localised prostate cancer, as it remains controversial whether African‐American men present with more aggressive disease.

Rapid On-Site Pathologic Evaluation Does Not Increase the Efficacy of Endobronchial Ultrasonographic Biopsy for Mediastinal Staging

BACKGROUND Endobronchial ultrasonography with transbronchial needle aspiration (EBUS-TBNA) has been shown to be equivalent to mediastinoscopy in lung cancer staging for mediastinal node involvement. Rapid on-site evaluation (ROSE) to determine the adequacy of nodal sampling has been claimed to be beneficial. METHODS A retrospective evaluation was performed in 170 patients who underwent EBUS-TBNA from July 2008 to May 2011. The patients were classified as having either high or low pretest probability for mediastinal disease based on history and radiographic imaging. ROSE was compared with the final pathology reports based on slides and cell blocks. RESULTS One hundred thirty-one (77%) patients were classified as being in the high pretest cohort based on clinical staging. Of these, 101 (77%) patients had adequate tissue sampling based on ROSE, with 70 (69%) patients having positive mediastinal disease. In the 30 (23%) patients who had inadequate tissue by ROSE, the final analysis of all the prepared slides and cell blocks allowed for a diagnosis in all but 8 patients. The sensitivity and specificity of ROSE in the high pretest probability cohort were 89.5% and 96.4%, respectively, whereas the overall sensitivity and specificity of EBUS-TBNA was 92.1% and 100%, respectively. Despite having inadequate tissue on ROSE in 30 of 131 patients, sufficient tissue was available on final analysis for diagnosis in 22 of 30 patients. CONCLUSIONS ROSE does not impact clinical decision making if a thorough mediastinal staging using EBUS is performed. Despite inadequate tissue sampling assessment by ROSE, a final diagnosis was made in most patients, potentially avoiding an additional surgical procedure to prove mediastinal disease.

Viral Response to Chemotherapy in Endemic Burkitt Lymphoma

Purpose: Some EBV-directed therapies are predicted to be effective only when lytic viral replication occurs. We studied whether cyclophosphamide chemotherapy induces EBV to switch from latent to lytic phases of infection in a series of EBV-associated Burkitt lymphomas. Experimental Design: Children with first presentation of an expanding, solid maxillary or mandibular mass consistent with Burkitt lymphoma underwent fine-needle aspiration just prior to the initiation of cyclophosphamide therapy and again 1 to 5 days later. Aspirated cells were examined for latent and lytic EBV infection using in situ hybridization to EBV-encoded RNA (EBER), immunohistochemical analysis of the lytic EBV proteins BZLF1 and BMRF1, reverse transcription PCR targeting BZLF1 transcripts, and EBV viral load measurement by quantitative PCR. Results: Among 21 lymphomas expressing EBER prior to chemotherapy, 9 of 10 still expressed EBER on day 1 after therapy whereas only 2 of 11 (18%) specimens still expressed EBER at days 3 to 5, implying that chemotherapy was fairly effective at eliminating latently infected cells. Neither of the lytic products, BZLF1 or BMRF1, were significantly upregulated at the posttherapy time points examined. However, EBV genomic copy number increased in 5 of 10 samples 1 day after treatment began, suggesting that viral replication occurs within the first 24 hours. Conclusion: Cyclophosphamide may induce the lytic phase of EBV infection and is fairly effective in diminishing EBER-expressing tumor cells within 5 days. These findings provide the rationale for a trial testing synergistic tumor cell killing using cyclophosphamide with a drug like ganciclovir targeting lytically infected cells. Clin Cancer Res; 16(7); 2055–64. ©2010 AACR.