Darren L. Riehle

Pathobiology of pituitary adenomas and carcinomas.

OBJECTIVE:To examine relationships between pituitary tumors and lesion size, invasiveness, resectability, deoxyribonucleic acid ploidy, cell cycle profile, mitotic activity, and immunoreactivity for MIB-1, proliferating cell nuclear antigen (PCNA), p27Kip1, and p53. PATIENTS AND METHODS:One hundred fifty-three adenomas of most pathological subtypes, including 20 medically treated and prolactin and growth hormone-containing tumors, as well as 10 premetastatic tumors and 13 pituitary carcinomas, were studied. RESULTS:Significant (P < 0.05) differences were noted between functional versus nonfunctional adenomas (percent aneuploidy, percent S phase, p27Kip1 labeling indices [LI], male sex, tumor size, and frequency of visual disturbance); Cushing’s versus silent adrenocorticotropin adenomas (percent hypertetraploidy, p53 LI, tumor size, visual disturbance, and resectability); untreated versus medically treated prolactin cell adenomas (MIB-1 LI, p53 LI, and resectability); untreated versus medically treated growth hormone-containing adenomas (percent diploidy, percent S phase, MIB-1 LI, p53 LI, and p27 LI); untreated prolactin cell adenomas versus premetastatic tumors (percent hypertetraploidy, PCNA LI, p53 LI, invasiveness, and resectability); untreated growth hormone-containing adenomas versus premetastatic tumors (percent diploidy, percent S phase, PCNA LI, p53 LI, invasiveness, and resectability); Cushing’s adenomas versus premetastatic tumors (percent diploidy, percent hypertetraploidy, percent S phase, MIB-1 LI, p53 LI, tumor size, invasiveness, visual disturbance, and resectability); Nelson’s adenomas versus premetastatic tumors (p53 LI, tumor size, invasiveness, and resectability); silent adenomas as a whole versus nonfunctional adenomas (percent nondiploid, percent S phase, invasiveness, and respectability); silent adrenocorticotropin adenomas I and II versus silent adenoma Subtype III (invasiveness); silent adrenocorticotropin adenoma Subtypes I and II versus premetastatic tumors (MIB-1 LI and invasiveness); silent adenoma Subtype III versus premetastatic tumors (PCNA and p53 LI); and premetastatic tumors versus metastatic pituitary carcinomas (MIB-1 LI). CONCLUSION:Only trends toward differences were noted between Cushing’s versus Nelson’s adenomas and between prolactinomas of reproductive female patients versus those of menopausal female patients and male patients. Too few “atypical adenomas” were encountered to permit their comparison with premetastatic tumors, but our results suggest that most pituitary carcinomas arise by malignant transformation from adenomas.

Expression of p16 and Retinoblastoma Determines Response to CDK4/6 Inhibition in Ovarian Cancer

Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients. Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052). Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

HER2 gene amplification and EGFR expression in a large cohort of surgically staged patients with nonendometrioid (type II) endometrial cancer

Type II endometrial cancers (uterine serous papillary and clear cell histologies) represent rare but highly aggressive variants of endometrial cancer (EC). HER2 and EGFR may be differentially expressed in type II EC. Here, we evaluate the clinical role of HER2 and EGFR in a large cohort of surgically staged patients with type II (nonendometrioid) EC and compare the findings with those seen in a representative cohort of type I (endometrioid) EC. In this study HER2 gene amplification was studied by fluorescence in situ hybridisation (FISH) and EGFR expression by immunohistochemistry. Tissue microarrays were constructed from 279 patients with EC (145 patients with type I and 134 patients with type II EC). All patients were completely surgically staged and long-term clinical follow up was available for 258 patients. The rate of HER2 gene amplification was significantly higher in type II EC compared with type I EC (17 vs 1%, P<0.001). HER2 gene amplification was detected in 17 and 16% of the cases with uterine serous papillary and clear cell type histology, respectively. In contrast, EGFR expression was significantly lower in type II compared with type I EC (34 vs 46%, P=0.041). EGFR expression but not HER2 gene amplification was significantly associated with poor overall survival in patients with type II EC, (EGFR, median survival 20 vs 33 months, P=0.028; HER2, median survival 18 vs 29 months, P=0.113) and EGFR expression retained prognostic independence when adjusting for histology, stage, grade, and age (EGFR, P=0.0197; HER2, P=0.7855). We conclude that assessment of HER2 gene amplification and/or EGFR expression may help to select type II EC patients who could benefit from therapeutic strategies targeting both HER2 and EGFR.

High expression levels of survivin protein independently predict a poor outcome for patients who undergo surgery for clear cell renal cell carcinoma.

In a previous study of gene array data, the authors identified survivin as a candidate marker of aggressiveness in clear cell renal cell carcinoma (ccRCC). What remained in question was whether survivin expression at the protein level is an independent predictor of disease progression and cancer‐specific survival.

Perineural invasion and MIB-1 positivity in addition to Gleason score are significant preoperative predictors of progression after radical retropubic prostatectomy for prostate cancer

We assessed the use of clinical stage, serum prostate specific antigen, DNA ploidy, proliferation, and traditional histologic findings from the biopsy to predict prostate cancer progression after radical retropubic prostatectomy. Between 1995 and 1998, 454 consecutive patients with cancer on biopsy were treated by radical retropubic prostatectomy. Preoperative serum prostate specific antigen, clinical stage, Gleason score, percentage of cores and surface area positive for cancer, perineural invasion, and DNA ploidy and MIB-1 immunostain quantitation by image analysis were evaluated in a multivariate Cox proportional hazards regression model to predict cancer progression. Cancer progression was defined as a postoperative serum prostate specific antigen level of ≥0.4 ng/mL, local recurrence, or systemic progression. Mean follow-up was 3.4 years (range 17 days to 5.8 years). Cancer progression was observed in 73 patients with a mean time to progression of 2.1 years (range 33 days to 5.1 years). Gleason score (p <0.001), MIB-1 cancer proliferation (p = 0.008), and perineural invasion (p = 0.008) were significantly associated with progression. Patients with cancer Gleason scores of 7 and >7 had a 2.5-fold and nearly 4-fold increased risk, respectively, of cancer progression compared with patients with cancer Gleason scores of ≤6. Patients with perineural invasion at biopsy were twice as likely to progress compared with patients without perineural invasion. Each 1-unit increase in MIB-1 on the natural logarithmic scale increased the risk of cancer progression by 64%. Cancer progression models that include serum prostate specific antigen and clinical stage may require revision to incorporate perineural invasion and MIB-1 proliferative activity in addition to Gleason score.

Claudin-3 and Claudin-4 expression in serous papillary, clear cell, and endometrioid endometrial cancer

OBJECTIVE Tight junction (TJ) proteins claudin-3 and claudin-4 may be differentially expressed in uterine serous papillary carcinoma (USPC), a rare form of endometrial cancer characterized by a particularly poor prognosis. Our aim was to determine the expression pattern and prognostic relevance of claudin-3 and claudin-4 in a large cohort of endometrial cancer patients of diverse histological type and stage. METHODS Claudin-3 and claudin-4 expression was studied in a cohort of 287 patients with endometrial cancer including 137 cases of USPC or clear-cell histology using immunohistochemistry. Patients were completely surgically staged. Outcome data is available on all 287 patients. RESULTS The rate of claudin-3 and claudin-4 expression was significantly higher in USPC and clear-cell endometrial cancer compared to endometrioid endometrial cancer (claudin-3: 78% and 61% versus 38%, p<.0001; claudin-4: 56% and 44% versus 9%, p<.0001). Furthermore, expression of both TJ proteins was significantly associated with poor clinical outcome (claudin-3, DFS RR 1.70, p=.0087, OS RR 1.62, p=.0247; claudin-4, DFS RR 2.66, p<0.0001, and OS RR 2.50, p<0.0001). However, both markers did not maintain prognostic independence in multivariate analyses, as their expression was tightly associated with more advanced disease stages (p<.0001 for both), and higher nuclear grade (p<.0001 for both). CONCLUSION These clinical observations confirm the hypothesis based on preclinical evidence that increased expression of claudin-3 and claudin-4 may contribute to the aggressive phenotype of endometrial cancer of serous papillary or clear-cell histology and suggest their potential utility as diagnostic biomarkers and possible targets for therapeutic intervention.

Identifying and quantifying apoptosis: Navigating technical pitfalls

Apoptosis or programmed cell death is often altered in malignancies and is frequently determined by the terminal transferase–mediated nick end labeling technique (TUNEL). However, commercially available protocols can produce high background and false-positive staining, which renders the distinction between apoptosis and necrosis difficult. In an attempt to develop a rapid and reproducible method for detecting and quantifying apoptosis, we coupled optimization of the Apoptag Plus Peroxidase In Situ Apoptosis Detection kit with quantitative histomorphometric computer imaging software using the Bacus Laboratories Incorporated Slide Scanner (BLISS). Multiple (200–350) unique 40 × images were scanned using the BLISS system and downloaded into the WebSlide Browser program, creating a permanent, scanned record of the area assessed. The stored images were counted, with the final analysis simultaneously taking into account cells that were immunohistochemically positive and the histology of the surrounding cells to reduce the possibility of false positive and negative staining. In addition, cells with equivocal staining can be simultaneously reviewed by other technologists with networked WebSlide Browser access to the same images. Our data show that the advantages offered by the BLISS imaging software greatly reduce the potential drawbacks of using the TUNEL method as a sole means of quantification.

Adenomatoid tumor of the adrenal gland: A clinicopathologic study of five cases and review of the literature

We report the clinicopathologic, immunophenotypic, DNA ploidy, and MIB-1 proliferative findings of five adenomatoid tumors of the adrenal gland. All patients were male, and tumors were incidental radiologic, surgical, or autopsy findings. Mean patient age at diagnosis was 41 years (range 31–64 years). The tumors ranged from 1.2 to 3.5 cm (mean 2.8 cm; median 3.2 cm) in greatest dimension, and all originated within the adrenal gland. The tumors were composed of anastomosing variably sized tubules lined by epithelioid as well as flattened cells. Signet-ring-like cells were present in all cases. The previously described histologic patterns of adenomatoid tumor, adenoid, angiomatoid, cystic, and solid, were observed, and each tumor contained multiple histologic patterns. In three of five cases, there was extra-adrenal extension of tumor into periadrenal adipose tissue. All adenomatoid tumors infiltrated the adrenal cortex, and in four cases the adrenal medulla was involved. All tumors exhibited strong immunoreactivity for calretinin, cytokeratins AE1/AE3, and CAM 5.2, cytokeratin 7, and vimentin. Tumors showed weak and focal immunoreactivity for cytokeratin 5/cytokeratin 6 and were negative for CD15, CD31, CD34, cytokeratin 20, MOC31, and polyclonal carcinoembryonic antigen. Ploidy analysis using Feulgen-stained sections and image analysis showed that three tumors were diploid and two were tetraploid. Tumors exhibited low MIB-1 proliferative activity, ranging from 0.2% to 2.7% (mean 1.6%). In three cases with clinical follow-up, no recurrence or metastases occurred. Adrenal gland adenomatoid tumors are morphologically and immunophenotypically identical to adenomatoid tumors of the genital tract and appear benign.

Effect of Dopamine Agonists on Lactotroph Adenomas of the Human Pituitary.

Dopamine (DA) agonists cause reduction of blood prolactin level and tumor shrinkage in most patients with lactotroph adenoma. Our aim was to investigate the cellular mechanism of tumor shrinkage by determining mitotic, MIB-1, p27, and apoptotic indices, as well as microvessel density (MVD), surface microvessel density (SMD), ploidy, and other nuclear parameters. Surgically removed lactotroph adenomas were selected from 29 patients, of whom 19 were treated with oral bromocriptine (BEC), long-acting injectable BEC (BEC-LAR), or quinagolide and 10 were untreated. In treated adenomas mitotic and MIB-1 indices were lower, whereas the apoptotic indices were not significantly higher compared to untreated adenomas. The decrease in MIB-1 labeling reached significance in adenomas exposed to quinagolide (p<0.05). Aside from the BEC-LAR treated group, wherein p27 expression was significantly reduced (p<0.05), p27 expression did not differ significantly between the treated and untreated groups. MVD density was significantly lower in the treated adenomas, whereas the decrease in SMD did not attain significance. The DNA ploidy and most other nuclear parameters did not differ significantly in the two groups. In conclusion, reduction of mitotic and MIB-1 indices indicates that suppression of cell proliferation contributes to tumor shrinkage, whereas p27 protein expression and apoptosis play no major role in the adenoma involution. Further studies are required to explain the effect of DA agonists on MVD and SMD.

Myocardial fibrosis in patients with symptomatic obstructive hypertrophic cardiomyopathy: correlation with echocardiographic measurements, sarcomeric genotypes, and pro-left ventricular hypertrophy polymorphisms involving the renin-angiotensin-aldosterone system.

INTRODUCTION Hypertrophic cardiomyopathy (HCM) is a heterogeneous disorder of the cardiac sarcomere, resulting in myocyte hypertrophy and disarray, interstitial fibrosis, and cardiac dysfunction. Our aim was to determine whether the amount of fibrosis in HCM correlates with echocardiographic measures of diastolic dysfunction, presence of HCM-susceptibility mutations, or polymorphisms in the renin-angiotensin-aldosterone system (RAAS). METHODS Surgical specimens from patients with obstructive HCM undergoing septal myectomy at the Mayo Clinic (2001-2004) were examined and compared with autopsy-derived tissues from age- and sex-matched normal controls. Digital image analysis was used to quantitate the fibrosis in representative microscopic sections. Genotyping was performed for myofilament-HCM using polymerase chain reaction, high-performance liquid chromatography, and direct DNA sequencing. RAAS polymorphism status was similarly established. RESULTS The study included 59 HCM cases and 44 controls. Patients with HCM exhibited more fibrosis (mean 17%, range 3-45%) than controls (mean 8%, range 3-17%) (P<.0001). A significant relationship existed between amount of fibrosis and maximum wall thickness (P=.02), left ventricular ejection fraction (P=.02), and peak early/late diastolic mitral annulus velocity (E/A ratio) (P=.002). Although there was no association between amount of fibrosis and myofilament-HCM genotype status or polymorphisms in the RAAS cascade, there was a trend toward more fibrosis in patients with > or =1 C-encoding allele in CYP11B2-encoded aldosterone synthase. CONCLUSIONS Patients with HCM undergoing septal myectomy had significantly more myocardial interstitial fibrosis than controls. The amount of fibrosis in HCM patients correlated with degree of septal hypertrophy and left ventricular systolic and diastolic function. Notably, neither mutations in cardiac myofilament proteins or polymorphisms in RAAS exhibited strong associations with severity of myocardial fibrosis.