Omar Hameed

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.

Immunohistochemical Stains for p63 and α-methylacyl-coa Racemase, Versus a Cocktail Comprising Both, in the Diagnosis of Prostatic Carcinoma: A Comparison of the Immunohistochemical Staining of 430 Foci in Radical Prostatectomy and Needle Biopsy Tissues

The diagnosis of prostatic carcinoma and especially minimal prostatic carcinoma can sometimes be challenging on needle core biopsy and occasionally immunohistochemistry is an aid in the diagnosis. Immunostains, such as those directed against the basal cell marker p63 and, more recently, employing antibodies reactive with α-methylacyl-CoA racemase (AMACR), can be useful in this situation. The aim of this investigation was to assess the diagnostic utility of a p63/AMACR antibody cocktail and compare the staining pattern it produces with that using the individual antibodies alone. A retrospective review of 31 consecutive radical prostatectomy specimens and 150 prostate needle biopsy samples was performed to select histologic sections showing foci of prostatic carcinoma and/or minimal prostatic carcinoma, high-grade prostatic intraepithelial neoplasia (HGPIN), as well as common benign mimickers of prostatic carcinoma, to include atrophy and basal cell hyperplasia, especially with prominent nucleoli. Serial histologic sections from the corresponding paraffin blocks were stained with hematoxylin and eosin and by immunostains for p63, AMACR, and a prediluted antibody cocktail comprising both. The diagnostic utility of the cocktail was assessed, and the staining characteristics it produced were compared with those using the individual immunostains. In 430 foci, the cocktail produced a p63 staining profile identical to that using the single p63 antibody. Distinction of the nuclear p63 signal from the cytoplasmic AMACR localization was readily accomplished. There was an excellent agreement (kappa = 0.91; P < 0.0001) between the AMACR staining profile using the cocktail and the single AMACR antibody alone. The cocktail was very useful in highlighting prostatic carcinoma associated with HGPIN, flat and cribriform HGPIN, and distorted foci of minimal prostatic carcinoma. These data indicate that use of a p63/AMACR cocktail is essentially equivalent to use of each antibody separately for immunohistochemical confirmation of a diagnosis of prostatic carcinoma in needle biopsy. This cocktail would be of diagnostic utility when only limited tissue is available for immunohistochemical evaluation of small, diagnostically difficult foci in prostate needle biopsy tissue.

Differential expression of immunohistochemical markers in bladder smooth muscle and myofibroblasts, and the potential utility of desmin, smoothelin, and vimentin in staging of bladder carcinoma

Distinguishing bladder muscularis propria from muscularis mucosae can be problematic especially in transurethral resection specimens performed for bladder carcinoma. Moreover, bladder carcinoma can be associated with a proliferative/desmoplastic myofibroblastic response that can resemble smooth muscle and potentially lead to overdiagnosis of muscularis propria invasion. The aim of this study was to investigate the potential role of immunohistochemistry in staging bladder carcinoma by evaluating the expression of different markers in myofibroblasts and nonvascular smooth muscle cells in 15 cases of invasive bladder carcinoma. Reactive myofibroblasts were consistently positive for vimentin and smooth muscle actin, consistently negative for caldesmon, desmin, and smoothelin, and had variable expression of actin and CD10. Nonvascular smooth muscle cells of the bladder were consistently positive for smooth muscle actin, actin, desmin, and caldesmon, and consistently negative for CD10. In contrast to smooth muscle cells of the muscularis propria, which displayed strong smoothelin expression in all 15 cases, the smooth muscle cells of the muscularis mucosae displayed moderate smoothelin expression in only 1 (9%) of 11 cases (P=10−7). Surprisingly, although strongly highlighting endothelial and endomysial cells, the smooth muscle cells of the muscularis propria weakly expressed vimentin in only 1 (7%) of 15 cases, whereas smooth muscle cells of the muscularis mucosae had moderate or strong expression in 9 (82%) of 11 cases (P=0.00016). The sensitivity and specificity of desmin or caldesmon expression for smooth muscle cells were 100%. The sensitivity and specificity of strong smoothelin expression for muscularis propria were 100%, whereas those of absent vimentin expression were 93 and 82%, respectively. Although morphology remains the gold standard, the findings suggest that immunohistochemistry, using a panel composed of desmin, smoothelin, and vimentin, may be potentially useful for staging of bladder carcinoma. Confirmatory larger-scale studies, especially on transurethral resection specimens, are warranted.

Breast cancer subtypes predispose the site of distant metastases.

OBJECTIVES The distant organs to which breast cancer preferentially metastasizes are of significant clinical importance. METHODS We explored the relationship between the clinicopathologic factors and the common sites of distant metastasis in 531 consecutive patients with advanced breast cancer. RESULTS Breast cancer subtype as a variable was significantly associated with all five common sites of relapse by multivariate analysis. The luminal tumors were remarkable for their significant bone-seeking phenotype and were less frequently observed in lung, brain, and pleural metastases and less likely to be associated with multiorgan relapse. The HER2 subtype demonstrated a significant liver-homing characteristic. African Americans were significantly less likely to have brain-only metastasis in patients with brain relapse. CONCLUSIONS These findings further articulate that breast cancer subtypes differ not only in tumor characteristics but also in their metastatic behavior, thus raising the possibility that this knowledge could potentially be used in determining the appropriate strategy for follow-up of patients with newly diagnosed breast cancer.

Hepatoid carcinoma of the pancreas : A case report and literature review of a heterogeneous group of tumors

Hepatoid carcinomas are tumors that display, at least focally, cytologic and/or architectural features of hepatocellular carcinoma. They have been described in several organs, most notably in the stomach and ovary. We report a case of hepatoid carcinoma of the pancreas that developed in a 41-year-old woman in association with a pancreatic endocrine carcinoma. The fine needle aspiration material was characterized by the presence of monotonous, small-to-medium sized tumor cells with round nuclei and finely granular chromatin, intermixed with more atypical tumor cells displaying larger nuclei with coarse clumped chromatin, prominent nucleoli, and moderate amounts of foamy cytoplasm. The excised specimen displayed a poorly differentiated pancreatic endocrine carcinoma associated with well-defined islands of larger tumor cells growing in a perisinusoidal pattern which, based on their immunohistochemical profile and the demonstration of bile, proved to represent a hepatoid component. This case and prior examples in the literature suggest that hepatoid carcinomas of the pancreas appear to be a heterogeneous group of tumors (pure or associated with another histologic component) that are often associated with early liver metastasis and a short survival, although those arising as a component of endocrine tumors seem to fare slightly better. Hepatoid carcinoma of the pancreas should be included in the differential diagnosis of pancreatic tumors composed of large eosinophilic cells.

Diagnostic utility of PAX8, TTF-1 and napsin A for discriminating metastatic carcinoma from primary adenocarcinoma of the lung.

Abstract TTF-1 and napsin A are useful biomarkers for differentiating primary lung adenocarcinoma from metastatic tumors. Studies have shown, however, that TTF-1 and napsin A also can be expressed in extrapulmonary carcinomas, and that a small fraction of primary lung adenocarcinomas do not co-express these two markers. We attempted to determine whether a tissue-specific transcriptional factor, PAX8, can help determine primary sites of lung carcinomas. Immunohistochemical stains for PAX8, TTF-1 and napsin A were performed on 103 cases of metastatic lung carcinomas from a variety of origins and 120 cases of primary lung adenocarcinomas. Our data demonstrated that all 103 metastatic carcinomas were negative for napsin A, while 14 (13.6%; four thyroid, two endometrium, three colon, one prostate, one salivary adenoid cystic, two renal cell carcinomas, and one ovary) showed weak to strong TTF-1 nuclear staining in 5–60% of the tumor cells. All primary lung adenocarcinomas were negative for PAX8, whereas 46 (44.7%) metastatic carcinomas from the kidney (29/33), ovary (6/8), endometrium (5/5), endocervix (1/1), thyroid (4/5) and urinary tract (1/3) were positive for PAX8. Our data demonstrate that of combined use of PAX8, TTF-1 and napsin A is reliable to separate reliably lung primary from metastatic tumors.

The impact of implementation of the Bethesda System for Reporting Thyroid Cytopathology on the quality of reporting, “risk” of malignancy, surgical rate, and rate of frozen sections requested for thyroid lesions

The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) has been anticipated to improve communication between pathologists and clinicians and thereby patient outcomes. In the current study, the impact of TBSRTC on various quality and outcome measures was assessed.

Estrogen and progesterone receptor expression is not always specific for mammary and gynecologic carcinomas: a tissue microarray and pooled literature review study.

Given their frequent expression in breast and gynecologic carcinomas, the National Comprehensive Cancer Network has recommended that the immunohistochemical expression of estrogen (ER) and/or progesterone (PR) receptors in a carcinoma of unknown primary can be used to support a breast or gynecologic origin. Several reports in the literature, however, have described such expression in a variable proportion of nonmammary and nongynecologic carcinomas. The aim of this study was to systematically evaluate the immunohistochemical expression of ER and PR on tissue microarray sections representing 348 nonbreast or gynecologic and nongynecologic tumors of lung, esophageal, gastric, pancreatic, colonic, renal, or bladder origin. We also performed a pooled analysis of the published literature in this regard. Except for 1 (2.5%) out of 40 pancreatic adenocarcinomas that expressed PR, there was no ER or PR expression in any of the other tumors evaluated by immunohistochemistry. A pooled literature review demonstrated that most of the evaluated tumors express ER and/or PR in up to 3% of cases, whereas some lung and bladder carcinomas showed such expression in around 10% of cases. This literature review also demonstrated that the frequency of ER and PR expression was dependent on the antibody clone used. Given that ER and PR expression can occasionally be seen in carcinomas of nonmammary/nongynecologic origin, we conclude that the diagnosis of metastatic breast, ovarian or endometrial carcinoma in a carcinoma of unknown primary should not be based solely on such expression.

PIM Kinase Inhibitor AZD1208 for Treatment of MYC-Driven Prostate Cancer

BACKGROUND PIM1 kinase is coexpressed with c-MYC in human prostate cancers (PCs) and dramatically enhances c-MYC-induced tumorigenicity. Here we examine the effects of a novel oral PIM inhibitor, AZD1208, on prostate tumorigenesis and recurrence. METHODS A mouse c-MYC/Pim1-transduced tissue recombination PC model, Myc-CaP allografts, and human PC xenografts were treated with AZD1208 (n = 5-11 per group). Androgen-sensitive and castrate-resistant prostate cancer (CRPC) models were studied as well as the effects of hypoxia and radiation. RNA sequencing was used to analyze drug-induced gene expression changes. Results were analyzed with χ(2) test. Students t test and nonparametric Mann-Whitney rank sum U Test. All statistical tests were two-sided. RESULTS AZD1208 inhibited tumorigenesis in tissue recombinants, Myc-CaP, and human PC xenograft models. PIM inhibition decreased c-MYC/Pim1 graft growth by 54.3 ± 39% (P < .001), decreased cellular proliferation by 46 ± 14% (P = .016), and increased apoptosis by 326 ± 170% (P = .039). AZD1208 suppressed multiple protumorigenic pathways, including the MYC gene program. However, it also downregulated the p53 pathway. Hypoxia and radiation induced PIM1 in prostate cancer cells, and AZD1208 functioned as a radiation sensitizer. Recurrent tumors postcastration responded transiently to either AZD1208 or radiation treatment, and combination treatment resulted in more sustained inhibition of tumor growth. Cell lines established from recurrent, AZD1208-resistant tumors again revealed downregulation of the p53 pathway. Irradiated AZD1208-treated tumors robustly upregulated p53, providing a possible mechanistic explanation for the effectiveness of combination therapy. Finally, an AZD1208-resistant gene signature was found to be associated with biochemical recurrence in PC patients. CONCLUSIONS PIM inhibition is a potential treatment for MYC-driven prostate cancers including CRPC, and its effectiveness may be enhanced by activators of the p53 pathway, such as radiation.

Stratified epithelium in prostatic adenocarcinoma: a mimic of high-grade prostatic intraepithelial neoplasia

Typically glands of prostatic adenocarcinoma have a single cell lining, although stratification can be seen in invasive carcinomas with a cribriform architecture, including ductal carcinoma. The presence and diagnostic significance of stratified cells within non-cribriform carcinomatous prostatic glands has not been well addressed. The histomorphological features and immunohistochemical profile of cases of non-cribriform prostatic adenocarcinoma with stratified malignant glandular epithelium were analyzed. These cases were identified from needle biopsy cases from the consultation files of one of the authors and from a review of 150 consecutive in-house needle biopsy cases of prostatic adenocarcinoma. Immunohistochemistry was performed utilizing antibodies reactive against high molecular weight cytokeratin (34βE12), p63 and α-methylacyl-coenzyme-A racemase (AMACR). A total of 8 cases were identified, including 2 from the 150 consecutive in-house cases (1.3%). In 4 cases, the focus with glands having stratified epithelium was the sole carcinomatous component in the biopsy, while such a component represented 5–30% of the invasive carcinoma seen elsewhere in the remaining cases. The main attribute in all these foci was the presence of glandular profiles lined by several layers of epithelial cells with cytological and architectural features resembling flat or tufted high-grade prostatic intraepithelial neoplasia, but lacking basal cells as confirmed by negative 34βE12 and/or p63 immunostains in all cases. The AMACR staining profile of the stratified foci was variable, with 4 foci showing positivity, and 3 foci being negative, including two cases that displayed AMACR positivity in adjacent non-stratified prostatic adenocarcinoma. Prostatic adenocarcinoma with stratified malignant glandular epithelium can be identified in prostate needle biopsy samples harboring non-cribriform prostatic adenocarcinoma and resembles glands with high-grade prostatic intraepithelial neoplasia. These ‘PIN-like’ carcinomas can present in pure form. Recognition of this pattern of prostatic adenocarcinoma is necessary to correctly diagnose such cases as invasive carcinoma.