IGF1R Protein Expression Is Not Associated with Differential Benefit to Concurrent Trastuzumab in Early-Stage HER2+ Breast Cancer from the North Central Cancer Treatment Group (Alliance) Adjuvant Trastuzumab Trial N9831.

Background: Preclinical evidence indicates that increased insulin-like growth factor receptor-1 (IGF1R) signaling interferes with the action of trastuzumab suggesting a possible mechanism of trastuzumab resistance. Thus, we evaluated IGF1R prevalence, relationship with demographic data, and association with disease-free survival (DFS) of patients randomized to chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) in the prospective phase III HER2+ adjuvant N9831 trial.Experimental Design: IGF1R protein expression was determined in tissue microarray sections (three cores per block; N = 1,197) or in whole tissue sections (WS; N = 537) using IHC (rabbit polyclonal antibody against IGF1R ß-subunit). A tumor was considered positive (IGF1R+) if any core or WS had ≥1+ membrane staining in >0% invasive cells. Median follow-up was 8.5 years.Results: Of 1,734 patients, 708 (41%) had IGF1R+ breast tumors. IGF1R+ was associated with younger age (median 48 vs. 51, P = 0.007), estrogen receptor/progesterone receptor positivity (78% vs. 35%, P < 0.001), nodal positivity (89% vs. 83%, P < 0.001), well/intermediate grade (34% vs. 24%, P < 0.001), tumors ≥2 cm (72% vs. 67%, P = 0.02) but not associated with race or tumor histology. IGF1R did not affect DFS within arms. Between Arms A and C, patients with IGF1R+ and IGF1R- tumors had DFS HRs of 0.48 (P ≤ 0.001) and 0.68 (P = 0.009), respectively (Pinteraction = 0.17). Between Arms A and B, patients with IGF1R+ and IGF1R- tumors had DFS HRs of 0.83 (P = 0.25) and 0.69 (P = 0.01), respectively (Pinteraction = 0.42).Conclusions: In contrast to preclinical studies that suggest a decrease in trastuzumab sensitivity in IGF1R+ tumors, our adjuvant data show benefit of adding trastuzumab for patients with either IGF1R+ and IGF1R- breast tumors. Clin Cancer Res; 23(15); 4203-11. ©2016 AACR.

Impact of c-MYC protein expression on outcome of patients with early-stage HER2+ breast cancer treated with adjuvant trastuzumab NCCTG (alliance) N9831.

PURPOSE: This study investigated the association between tumor MYC protein expression and disease-free survival (DFS) of patients randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) in the N9831 (Alliance) adjuvant HER2(+) trastuzumab breast cancer trial. EXPERIMENTAL DESIGN: This analysis included 1,736 patients randomized to Arms A, B, and C on N9831. Nuclear MYC protein expression was determined in tissue microarray sections containing three biopsies per patient or whole tissue sections using standard immunohistochemistry (clone 9E10). A tumor was considered positive for MYC protein overexpression (MYC(+)) if the nuclear 3+ staining percentage was more than 30%. RESULTS: Five hundred and seventy-four (33%) tumors were MYC(+). MYC(+) was associated with hormone receptor positivity (χ(2), P = 0.006), tumors 2 cm or more (χ(2), P = 0.02), and a higher rate of nodal positivity (χ(2), P < 0.001). HRs for DFS (median follow-up: 6.1 years) for Arm C versus A were 0.52 (P = 0.006) and 0.65 (P = 0.006) for patients with MYC(+) and MYC(-) tumors, respectively (P(interaction) = 0.40). For Arm B versus A, HRs for patients with MYC(+) and MYC(-) tumors were 0.79 (P = 0.21) and 0.74 (P = 0.04), respectively (P(interaction) = 0.71). For Arm C versus B, HRs for patients with MYC(+) and MYC(-) tumors were 0.56 (P = 0.02) and 0.89 (P = 0.49), respectively (P(interaction) = 0.17). CONCLUSIONS: Our data do not support an impact of tumor MYC protein expression on differential benefit from adjuvant trastuzumab.

Tissue composition of mammographically dense and non-dense breast tissue.

Mammographic density is a strong risk factor for breast cancer but its underlying biology in healthy women is not well-defined. Using a novel collection of core biopsies from mammographically dense versus non-dense regions of the breasts of healthy women, we examined histologic and molecular differences between these two tissue types. Eligible participants were 40 + years, had a screening mammogram and no prior breast cancer or current endocrine therapy. Mammograms were used to identify dense and non-dense regions and ultrasound-guided core biopsies were performed to obtain tissue from these regions. Quantitative assessment of epithelium, stroma, and fat was performed on dense and non-dense cores. Molecular markers including Ki-67, estrogen receptor (ER) and progesterone receptor (PR) were also assessed for participants who had >0% epithelial area in both dense and non-dense tissue. Signed rank test was used to assess within woman differences in epithelium, stroma and fat between dense and non-dense tissue. Differences in molecular markers (Ki-67, ER, and PR) were analyzed using generalized linear models, adjusting for total epithelial area. Fifty-nine women, mean age 51 years (range: 40-82), were eligible for analyses. Dense tissue was comprised of greater mean areas of epithelium and stroma (1.1 and 9.2 mm(2) more, respectively) but less fat (6.0 mm(2) less) than non-dense tissue. There were no statistically significant differences in relative expression of Ki-67 (P = 0.82), ER (P = 0.09), or PR (P = 0.96) between dense and non-dense tissue. Consistent with prior reports, we found that mammographically dense areas of the breast differ histologically from non-dense areas, reflected in greater proportions of epithelium and stroma and lesser proportions of fat in the dense compared to non-dense breast tissue. Studies of both epithelial and stromal components are important in understanding the association between mammographic density and breast cancer risk.

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.

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.

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.

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.

Conventional assessment of needle biopsy specimens is more useful than digital image analysis of proliferation and DNA ploidy in prediction of positive surgical margins at radical prostatectomy.

OBJECTIVES: The preoperative prediction of the likelihood of positive surgical margins (+SMs) at radical retropubic prostatectomy (RRP) may be useful for counseling and determining the surgical approach. The aim of this study was to assess the additional value of digital image analysis (DIA) of ploidy and proliferation on needle biopsies, in addition to the known preoperative predictors of +SMs at RRP. METHODS: We identified 454 patients treated by RRP at our institution from 1995 to 1998 for prostate cancer verified by transrectal ultrasound-guided biopsy, with a specimen adequate for DIA. Patients receiving preoperative hormonal therapy were excluded. The clinical features, transrectal ultrasound-guided biopsy findings, and DIA evaluation of MIB-I immunostaining and DNA ploidy were assessed in a multivariate logistic regression model to predict for +SMs at RRP. RESULTS: The mean +/- SD age at treatment was 64.5 +/- 6.5 years, the percentage of positive cores was 40.4% +/- 24.3%, the median prostate-specific antigen level was 6.3 ng/mL (range 0.6 to 112.0), median biopsy Gleason score was 6 (range 4 to 9), and median percentage of diploid nuclei was 67% (range 0% to 100%). Of the 454 patients, 185 (40.7%) had +SMs; this finding was time dependent (1995 to 1996, 45% and 1997 to 1998, 31%; P = 0.004). Univariately, preoperative prostate-specific antigen, biopsy Gleason score, extent of cancer on biopsy, MIB-1 expression, percentage of diploid or nondiploid nuclei, and year of surgery were predictive for +SMs. On multivariate analysis, the preoperative prostate-specific antigen level, biopsy Gleason score, percentage of positive cores, and year of surgery remained significant. CONCLUSIONS: The results of our study have shown that the likelihood of +SMs at RRP is best predicted on the basis of conventional prognostic factors. The DIA features of needle biopsies did not provide additional predictive power.

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

BACKGROUND: 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. METHODS: Between 1990 and 1994, 312 patients underwent nephrectomy for ccRCC at Mayo Clinic Rochester and had paraffin tissue available. The authors performed immunohistochemistry with antisurvivin antibody, quantitated the expression by using an image-analysis system, and analyzed the association of survivin expression with disease progression and cancer-specific survival. RESULTS: Within the cohort, 97 patients (31.1%) had high levels of survivin expression. Patients who had high survivin expression levels were at significantly increased risk of death from RCC compared with patients who had low expression levels (risk ratio [RR], 5.3; 95% confidence interval [95% CI], 3.5-7.9). The 5-year cancer-specific survival rate was 43.0% for patients with high survivin expression and 87.2% for patients with low survivin expression. In multivariate analysis, survivin expression remained associated with death from RCC even after adjusting for the Eastern Cooperative Oncology Group performance status; 2002 Tumor, Lymph Node, Metastases (TNM) stage groupings and nuclear grade (RR, 2.4; 95%CI, 1.5-3.8); and the Mayo Clinic composite TNM stage groupings, tumor size, nuclear grade, and tumor necrosis (SSIGN) score (RR, 1.8; 95%CI, 1.1-2.9). Among 273 patients who had localized ccRCC, survivin expression was associated significantly with cancer progression (RR, 3.9; 95%CI, 2.4-6.2). CONCLUSIONS: Survivin expression is an independent predictor of ccRCC progression and death from RCC. Thus, survivin has the potential to offer additional prognostic information and to provide a novel target for the development of new adjuvant therapies.

Role of bcl-2 in endometrioid corpus cancer: an experimental study.

BACKGROUND: Bcl-2 expression appears to be under hormonal control in normal endometrium and to correlate with hormone receptor status in endometrial cancer. The aim of this study was to assess bcl-2 expression in endometrial cancer. MATERIALS AND METHODS: Hysterectomy specimens from 125 patients with endometrial cancer were stained for bcl-2. Estrogen receptor (ER) and progesterone receptor (PR) levels were quantified with a dextran-coated charcoal assay. RESULTS: Bcl-2 expression correlated significantly with endometrioid histology and high levels of ER and PR (p < 0.05). For the entire population, bcl-2 expression was not significantly associated with the presence of extrauterine disease. However, when only patients with grade 1 or 2 endometrioid histology were considered, tumors with bcl-2 expression were significantly (p < 0.05) more likely to present with extrauterine disease than those not expressing bcl-2. CONCLUSION: Bcl-2-mediated inhibition of apoptosis may be important in the acquisition of molecular alterations and development of metastases in a subset of hormone-dependent endometrial cancers.

HER-2/neu overexpression and hormone dependency in endometrial cancer: analysis of cohort and review of literature.

BACKGROUND: Overexpression of HER-2/neu (HER) is associated with unfavorable prognoses in both endometrial and breast cancer. MATERIALS AND METHODS: To determine whether an association exists between HER expression and markers of hormone dependency in endometrial cancers, we subjected hysterectomy specimens from 125 patients to immunohistochemical staining for HER. HER was visually interpreted as negative/weakly positive (HER-) versus strongly positive (HER+). Estrogen receptor (ER) and progesterone receptor (PR) levels were quantitated on fresh tissue using a dextran-coated charcoal assay. RESULTS: HER+ was observed in 12% of endometrioid tumors and 22% of nonendometrioid tumors (p = 0.07). Mean ER and PR levels were 255 fmol/mg and 457 fmol/mg in endometrioid tumors, compared with 74 and 104 in nonendometrioid tumors (p < 0.01). Hyperplasia associated with the tumor was related to high levels of both ER and PR (p < 0.05), but not with HER expression. Age was significantly related to high levels of ER (p = 0.007). Both recurrence and death rates were significantly associated with low levels of ER and PR (p < 0.01). Mean ER and PR levels were 270 and 466 fmol/mg, respectively, in HER-tumors, compared with 95 (p = 0.14) and 138 fmol/mg (p = 0.02) in HER+ tumors. CONCLUSION: HER overexpression may be an important step in hormone-independent growth and proliferation in a subgroup of endometrial cancers.

B-Lymphocytes in plaque and adventitia of coronary arteries in two patients with rheumatoid arthritis and coronary atherosclerosis: preliminary observations.

OBJECTIVES: To describe the subtypes of lymphocytes in coronary arteries of two patients with rheumatoid arthritis (RA) and coronary artery disease (CAD). MATERIALS AND METHODS: The Mayo Clinic database was searched for patients with RA and CAD who underwent an autopsy in 2001. Medical records were reviewed, and coronary arteries were examined microscopically. The percentage of B- and T-lymphocytes was determined using histomorphometry on representative sections stained with CD20 and CD3 antibodies, respectively. RESULTS: Two men were diagnosed with RA at ages 52 and 70 years and died at ages 60 and 82 years. One sustained an acute myocardial infarction 2 years prior to the diagnosis of RA and had stable CAD until an arrhythmic death. The other developed congestive heart failure secondary to ischemic heart disease 5 years after RA was diagnosed. Both patients had severe three-vessel CAD with both stable fibrocalcific plaques and acute lesions. B- and T-lymphocytes were identified in the plaque and adventitia of all coronary arteries. The mean percentage of B-lymphocytes was 37% to 52% in the plaque and 78% to 85% in the adventitia, while that of T-lymphocytes was 38% to 51% and 28%, for plaque and adventitia, respectively. CONCLUSIONS: In typical CAD, lymphocytic infiltrates are almost exclusively T-cells. In contrast, the two patients with RA and CAD showed prominent infiltrates of B-lymphocytes within plaques and adventitia. Thus, the leukocytic response in atherosclerotic plaques may be altered in patients with autoimmune disorders. This warrants further study.

Prognostic value of proliferation, apoptosis, defective DNA mismatch repair, and p53 overexpression in patients with resected Dukes' B2 or C colon cancer: a North Central Cancer Treatment Group Study.

PURPOSE: Molecular studies of colon cancer have provided insights into pathogenesis, yet it is unclear how important these markers are in predicting prognosis. This study investigated the prognostic significance of TUNEL, bcl-2, p53, proliferation marker Ki-67 and DNA mismatch repair (MMR) status in patients with Dukes' stage B2 and C colorectal adenocarcinomas. PATIENTS AND METHODS: Tumor tissue from 366 patients (75% Dukes' C, 25% Dukes' B2) from four randomized North Central Cancer Treatment Group phase III surgical adjuvant trials were used. Eighty-one percent of patients received adjuvant treatment, which was primarily fluorouracil (FU) based (90%). Tumor location was predominantly (87%) the colon. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), Ki-67, p53, bcl-2, and MMR were assayed using immunohistochemistry. Stage, grade, MMR, Ki-67, and previously determined flow cytometry markers (ploidy and S phase) were explored for associations with each other and with overall survival (OS) and disease-free survival (DFS). RESULTS: Univariately, stage B2, low grade, diploid, Ki-67 more than 27%, normal p53, and FU-based adjuvant treatment were significantly associated with improved OS and DFS (P <.05). After adjusting for stage, grade, and ploidy in multivariate analysis, Ki-67 remained significantly related to both OS and DFS (P <.01). Active FU-based adjuvant treatment was significant only for OS in this multivariate model. Neither bcl-2 nor TUNEL were significant. CONCLUSION: This retrospective study indicates that Ki-67 and ploidy may have stronger prognostic impact on OS and DFS than other parameters investigated after adjusting for stage and tumor grade. Prospective studies to elucidate the mechanism and prognostic significance of these findings are necessary.

Multivariate models to predict clinically important outcomes at prostatectomy for patients with organ-confined disease and needle biopsy Gleason scores of 6 or less.

The objective of this study was to determine the clinical and biopsy features associated with outcomes at radical retropubic prostatectomy (RRP) in patients with clinically organ-confined prostate cancers and biopsy Gleason scores (GS) of 6 or less. We reviewed 274 biopsies with GS 6 or less cancers from patients with clinically organ-confined disease between 1995 and 1998 to determine statistically significant predictors for the following outcomes at RRP: tumor volume, small (<0.5 cc), confined (pT2) tumors with RRP GS of 6 or less (potentially "insignificant" tumors), and extraprostatic extension (EPE). Clinical and pathologic features evaluated included age, serum prostate specific antigen (PSA), clinical stage, percent biopsy cores and surface area positive for cancer (tumor extent), perineural invasion, MIB-I proliferation, and DNA ploidy by digital image analysis (DIA). Multivariate analyses showed that biopsy tumor extent (median percent surface area positive 3.3%; P < 0.001 and median biopsy cores positive 28.6%; P = 0.001) and PSA (median 5.5 ng/mL; P = 0.009) predicted tumor volume (median 1.4 cc). Biopsy tumor extent (P = 0.002), PSA (P = 0.002), and percent S-phase nuclei (P = 0.050) predicted potentially "insignificant" tumors at RRP (n = 76, 28%). Percent surface area positive for cancer (P = 0.003) predicted EPE (n = 22, 8%). DNA ploidy (n = 211, 79% diploid) and MIB-I proliferation (median 1.4%) did not add information to predict these RRP outcomes. Biopsy tumor extent and serum PSA were significantly associated with tumor volume. Biopsy tumor extent, serum PSA, and percent S-phase nuclei by DIA were predictive of potentially insignificant tumors. Patients with clinically confined disease, <5% biopsy surface area positive for cancer, <20% biopsy cores positive for cancer, and GS 6 or less, had a 48% chance of having a potentially insignificant tumor at diagnosis if the serum PSA was <10 ng/mL. Percent surface area predicted EPE at RRP. DNA ploidy and MIB-I proliferation by DIA did not provide additional information.

Molecular and histopathologic predictors of distant failure in endometrial cancer.

A case-cohort study was designed to correlate various histopathologic and molecular variables with distant failure in endometrial cancer by analyzing phenotypic and molecular indices in hysterectomy specimens. From an overall population of 283 patients with endometrial cancer, we selected a cohort including all 49 patients who experienced any recurrence and 76 randomly chosen patients without recurrence. Expression of nuclear proliferating cell nuclear antigen (PCNA), MIB-1 (a marker of cell proliferation), and p53 was determined with digital image analysis, and cell membrane HER-2/neu and bcl-2 were quantitated visually. Ploidy and DNA indices were determined with flow cytometry. Overall, 6 immunohistochemical and 11 flow cytometric cases were eliminated because of technical inadequacies. Distant failures were defined as primary recurrences that developed outside the pelvis or vagina. Median follow-up was 91 months. Distant failures occurred in 13% of the patients. Cervical stromal invasion, positive adnexae, myometrial invasion >50%, positive lymph nodes, positive peritoneal cytology, lymphovascular invasion, grade 3 histology, nonendometrioid subtype, p53 >33%, strong HER-2/neu membranous staining, aneuploidy, S-phase fraction > or =9%, proliferative index > or =14%, and DNA index > or =1.5 significantly (P<0.05) predicted distant failures. However, a logistic regression model identified only p53 (OR=43.73; P<0.005), lymphovascular invasion (OR=11.59; P<0.001), and cervical stromal invasion (OR=11.29; P=0.001) as cogent predictors of distant failures. Only 3% of patients without any of these three predictors developed distant failures compared with 36% of those with at least one of the three (P<0.01). Thus, locoregional therapy may be insufficient when at least one of these predictors is present.

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.

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 40x 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.

pT1 clear cell renal cell carcinoma: a study of the association between MIB-1 proliferative activity and pathologic features and cancer specific survival.

BACKGROUND: The majority of patients with pT1 clear cell renal cell carcinoma (RCC) are cured with nephrectomy. However, a few patients will die of RCC. In several studies, MIB-1 proliferative activity was identified as an independent predictor of survival in patients with RCC. The objective of the current study was to examine MIB-1 proliferative activity in a large series of patients with pT1 clear cell RCC who were treated uniformly with radical nephrectomy, and to examine the association between proliferative activity and cancer specific survival in a multivariate model incorporating tumor size, nuclear grade, and tumor necrosis. METHODS: Patients with solitary pT1 clear cell RCC who underwent radical nephrectomy between 1970-1997 were eligible for the current study. For each of the 40 patients who died of RCC, a stratified random sample of at least 3 year-matched patients who still were alive or had died of other causes at the time of last follow-up was selected. Patient age at nephrectomy, patient gender, tumor size, nuclear grade, and tumor necrosis were evaluated, and the MIB-1 proliferative activity was assessed using digital image analysis. Univariate and multivariate Cox proportional hazards models were fit to assess the features associated with cancer specific survival. The associations between MIB-1 proliferative activity and pathologic features were assessed using the Wilcoxon rank sum test. RESULTS: The mean MIB-1 value for those patients who died of clear cell RCC was 6.5% compared with 3.6% for those patients who died of other causes or were still alive at the time of last follow-up. Patients whose tumor had an MIB-1 proliferative activity > o r = 5.0% were more than twice as likely to die of RCC than patients whose tumors had a MIB-1 activity < 5% (P = 0.02). However, after adjusting for tumor size, nuclear grade, and necrosis, MIB-1 proliferative activity was not found to be associated significantly with cancer specific survival. There was a significant association between MIB-1 proliferative activity and tumor size, nuclear grade, and necrosis. CONCLUSIONS: After adjusting for tumor size, nuclear grade, and necrosis, MIB-1 proliferative activity was not found to be an independent predictor of outcome in patients with pT1 clear cell RCC who were treated with radical nephrectomy. There was a significant association between MIB-1 and other well established pathologic prognostic features of pT1 clear cell RCC.

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 > or = 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 < or = 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.

Comparison of quantitative histomorphometry and DNA ploidy in tissue sections of prostate carcinoma.

OBJECTIVE: To examine the ability of quantitative histomorphometry to predict DNA ploidy of prostate carcinoma in biopsy tissue sections assigned after quantitation by nuclear digital image analysis. STUDY DESIGN: Thirty-five diploid, 35 tetraploid and 35 aneuploid prostatic carcinomas in biopsies, assessed by the CAS 200 image analyzer (Bacus Laboratories, Lombard, Illinois, U.S.A.), were reevaluated by the Bacus Laboratories Incorporated Slide Scanner, a microscope that quantifies histologic images. Thirty-one histomorphometric features from cancer cells were captured at 40 x magnification, averaged across tilesfor each case and incorporated into a multivariate discriminant model to determine which features predicted ploidy interpretation by nuclear image analysis using the CAS 200. RESULTS: On average, 60 and 15 minutes were required to perform nuclear image analysis and histomorphometry, respectively. The multivariate discriminant model identified configurable run length, difference variance, contrast, inverse difference moment, sum entropy and diagonal variance as histomorphometry features capable of distinguishing diploid from nondiploid tumors (P < .05). Cross-validation studies showed the model correctly classified 74.3% of the diploid and 57.1% of the nondiploid cases. CONCLUSION: Quantitative histomorphometry can predict the ploidy of prostate carcinoma in biopsy tissue sections. Quantitative histomorphometry has potential as a method of rapidly assessing DNA ploidy otherwise earmarked for nuclear image analysis, resulting in savings of time and expense.