Next-generation sequencing of adrenocortical carcinoma reveals new routes to targeted therapies.

AIMS: Adrenocortical carcinoma (ACC) carries a poor prognosis and current systemic cytotoxic therapies result in only modest improvement in overall survival. In this retrospective study, we performed a comprehensive genomic profiling of 29 consecutive ACC samples to identify potential targets of therapy not currently searched for in routine clinical practice. METHODS: DNA from 29 ACC was sequenced to high, uniform coverage (Illumina HiSeq) and analysed for genomic alterations (GAs). RESULTS: At least one GA was found in 22 (76%) ACC (mean 2.6 alterations per ACC). The most frequent GAs were in TP53 (34%), NF1 (14%), CDKN2A (14%), MEN1 (14%), CTNNB1 (10%) and ATM (10%). APC, CCND2, CDK4, DAXX, DNMT3A, KDM5C, LRP1B, MSH2 and RB1 were each altered in two cases (7%) and EGFR, ERBB4, KRAS, MDM2, NRAS, PDGFRB, PIK3CA, PTEN and PTCH1 were each altered in a single case (3%). In 17 (59%) of ACC, at least one GA was associated with an available therapeutic or a mechanism-based clinical trial. CONCLUSIONS: Next-generation sequencing can discover targets of therapy for relapsed and metastatic ACC and shows promise to improve outcomes for this aggressive form of cancer.

Next-generation sequencing reveals frequent consistent genomic alterations in small cell undifferentiated lung cancer.

AIMS: Small cell lung cancer (SCLC) carries a poor prognosis, and the systemic therapies currently used as treatments are only modestly effective, as demonstrated by a low 5-year survival at only ∼5%. In this retrospective collected from March 2013 to study, we performed comprehensive genomic profiling of 98 small cell undifferentiated lung cancer (SCLC) samples to identify potential targets of therapy not currently searched for in routine clinical practice. METHODS: DNA from 98 SCLC was sequenced to high, uniform coverage (Illumina HiSeq 2500) and analysed for all classes of genomic alterations. RESULTS: A total of 386 alterations were identified for an average of 3.9 alterations per tumour (range 1­10). Fifty-two (53%) of cases harboured at least 1 actionable alteration with the potential to personalise therapy including base substitutions, amplifications or homozygous deletions in RICTOR (10%), KIT (7%), PIK3CA (6%), EGFR (5%), PTEN (5%), KRAS (5%), MCL1 (4%), FGFR1 (4%), BRCA2, (4%), TSC1 (3%), NF1 (3%), EPHA3 (3%) and CCND1. The most common non-actionable genomic alterations were alterations in TP53 (86% of SCLC cases), RB1 (54%) and MLL2 (17%). CONCLUSIONS: Greater than 50% of the SCLC cases harboured at least one actionable alteration. Given the limited treatment options and poor prognosis of patients with SCLC, comprehensive genomic profiling has the potential to identify new treatment paradigms and meet an unmet clinical need for this disease.

Expression of matrix metalloproteinases 3, 10 and 11 (stromelysins 1, 2 and 3) and matrix metalloproteinase 7 (matrilysin) by cancer cells in non-small cell lung neoplasms. Clinicopathologic studies.

Matrix metalloproteinases (MMP's) 3, 10 and 11 (also known as stromelysins 1, 2 and 3, respectively), and matrix metalloproteinase 7 (also known as matrilysin), produced by stromal fibroblast-like cells in the vicinity of various malignancies, are suspected to have an ability to degrade components of extracellular matrix, thus promoting spread of the tumor. MMP's also have been found in epithelial tumor cells in various cancers. Tissue sections from 95 cases of non-small cell lung cancer (NSCLC) were immunostained with antibodies against MMP 3, MMP 10 and MMP 11 and sections from 99 cases of NSCLC were immunostained with an antibody against MMP 7. Cytoplasmic immunoreactivity in the tumor cells was semiquantitatively scored for intensity and distribution and correlated with tumor type, tumor grade, stage, tumor size, lymph node positivity, metastasis and survival. Overexpression of MMP 10 and MMP 11 correlated with higher grade for NSCLC (p = 0.029 and p = 0.016, respectively), and also in a subset of adenocarcinomas (AC) (p = 0.015 and p = 0.009, respectively). Also, MMP 10 and MMP 11 correlated with lymph node involvement in NSCLC (p = 0.025 and p = 0.027 respectively). No correlation was found for MMP 3. Overexpression of MMP-7 correlated with tumor stage (p = 0.0001) and was associated with adverse clinical outcome (p = 0.0001) in NSCLC and also in separate squamous cell carcinoma (SCC) (p = 0.003) and AC (p = 0.004) tumor groups.

A comparison of palpable and impalpable cryptorchid testes using CD-99 immunohistochemistry.

OBJECTIVE: To compare the clinical and morphological features of impalpable and palpable cryptorchid testes, as there is debate about how much effort is appropriate to bring an impalpable undescended testicle into the scrotum. PATIENTS AND METHODS: We reviewed retrospectively 189 cases of undescended testicles in 168 patients who were explored surgically by one surgeon between August 1997 and September 2000. Operative findings of palpability, testicular size and location were collected. The mean tubular diameter (MTD), tubular fertility index (TFI) and mean number of germ cells per tubule (MGCT) were calculated using immunohistochemistry for CD-99, a Sertoli-cell marker, to classify germ cells more accurately. RESULTS: Sixty-three testes (33%) were impalpable; the median age at the time of surgical exploration was 23 months for both groups. The mean (sd) testicular volume for the impalpable and palpable groups were 0.83 (0.38) and 1.22 (0.54) mL, respectively. Using fitted curves of size vs age, impalpable testes were smaller than palpable testes at all ages, with the difference nearly statistically significant (P < 0.06). The MTD, TFI and MGCT decreased with age in both groups, with no statistically significant differences between the groups. A sub-analysis of abdominal and extra-abdominal testes confirmed no significant differences. CONCLUSION: Impalpable testes are smaller at the time of exploration than palpable cryptorchid testes. However, histological factors predict that impalpable testes have a significant chance of future fertility and therefore orchidopexy is appropriate. CD-99 immunohistochemistry makes objective morphological information easier to obtain.

Decreased expression of catenins (alpha and beta), p120 CTN, and E-cadherin cell adhesion proteins and E-cadherin gene promoter methylation in prostatic adenocarcinomas.

BACKGROUND: Catenin/E-cadherin complex proteins play an important role in cell-cell adhesion with decreased expression correlating with adverse prognostic variables in several human malignancies. METHODS: Archival formalin fixed, paraffin embedded (FFPE) sections from 118 prostatic adenocarcinomas (PACs) were immunostained by an automated method (Ventana Medical Systems, Tuscon, AZ) using monoclonal antibodies to catenins alpha and beta, p120 CTN, and E-cadherin proteins. Immunoreactivity was semiquantitatively graded, and results correlated with traditional prognostic parameters. In a subset of 10 randomly selected cases, E-cadherin gene promoter methylation status was determined on FFPE tissues using sodium bisulfite/hydroquinone DNA modification and polymerase chain reaction (PCR) with methylation specific primers (CpG wiz E-cadherin methylation assay; Intergen Co., Purchase, NY). RESULTS: Decreased expression of alpha-catenin (17%), beta catenin (4%), p120 CTN (45%), and E-cadherin (25%) proteins was noted in PACs with downregulation of each protein correlating with high tumor grade (P = 0.01-0.0001). In addition, p120 CTN and E-cadherin expression levels correlated with pathologic stage (P = 0.05; P = 0.02), aneuploidy (P = 0.001; P = 0.0001), and alpha-catenin with aneuploidy (P = 0.0001). p120 CTN loss also correlated with preoperative serum prostate specific antigen (P = 0.05). Two of 10 cases featured no evidence of E-cadherin gene promoter methylation by PCR and both cases retained expression of E-cadherin protein on immunohistochemistry. Of the 8 cases that showed E-cadherin methylation, 5 (68%) featured loss of expression of the protein on immunohistochemistry (P = 0.11). There was no correlation between E-cadherin methylation and adverse prognostic variables. CONCLUSIONS: Decreased expression of catenin/E-cadherin complex cell adhesion proteins is associated with aggressive phenotype in prostatic adenocarcinoma. E-cadherin gene promote methylation is a common event in prostate carcinoma but does not appear to bear prognostic significance in the subset of cases analyzed.

Increased expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinases 1 and 2 correlate with poor prognostic variables in renal cell carcinoma.

PURPOSE: Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix and are implicated in tissue remodeling and tumor infiltration. Tissue inhibitor of metalloproteinases (TIMPs) inhibit enzymes of the MMP family and preserve stromal integrity, thus inhibiting tumor migration. Although numerous studies on several human carcinomas have demonstrated a role for MMPs in tumor metastasis and patient survival, their prognostic role in patients with renal cell carcinoma (RCC) has not been well defined. More importantly, the recently documented paradoxical functions of TIMPs have not been characterized in these neoplasms. EXPERIMENTAL DESIGN: Five-microm, formalin-fixed, paraffin-embedded tissue sections from 153 RCCs were immunostained using specific antibodies against MMP2, MMP9, (Novocastra, Burlingame, CA) TIMP1, and TIMP2 (NeoMarkers, Fremont, CA) proteins. Immunostaining was semiquantitatively scored based on intensity and distribution, and results were correlated with histological and prognostic variables. RESULTS: The rates of increased expression of MMPs and TIMPs in RCC were as follows: MMP2, 67%; MMP9, 43%; TIMP1, 46%; and TIMP2, 73%. Each of these four markers individually correlated with histological tumor type with a vast majority of papillary and sarcomatoid RCCs expressing these proteins as compared with clear cell tumors (P range, 0.0001-0.003). Significant coexpression of MMPs and TIMPs was observed (P = 0.0001). Increased immunoreactivity for each of these proteins correlated with high tumor grade (P range, 0.0001-0.01). On univariate analysis, expression of each of these markers correlated with shortened survival (P range, 0.004-0.05). On multivariate analysis, including tumor grade, stage, and all four markers, only advanced stage (P = 0.047) and increased TIMP1 expression (P = 0.007) independently predicted shortened survival. CONCLUSION: Increased expression of MMP2, MMP9, TIMP1, and TIMP2 proteins in RCCs correlate with poor prognostic variables including shortened patient survival. The paradoxical poor prognostic implication of TIMP overexpression complements the recently documented dual function of TIMPs and warrants further investigation.

Co-downregulation of cell adhesion proteins alpha- and beta-catenins, p120CTN, E-cadherin, and CD44 in prostatic adenocarcinomas.

Cell adhesion molecule expression has been linked to disease outcome in prostatic adenocarcinomas (PACs). We evaluated the coordinated expression of catenin-related proteins, E-cadherin, N-cadherin, and CD44s in PACs. Archival sections from 112 PACs were immunostained by an automated method (Ventana Medical Systems, Tucson, AZ) using monoclonal antibodies to alpha- and beta-catenins, p120CTN, E-cadherin, N-cadherin, and CD44s proteins. Immunoreactivity was semiquantitatively scored, and results were evaluated for association between these markers. Staining results were also correlated with tumor grade, stage, ploidy, preoperative serum PSA, and postoperative biochemical disease recurrence. Decreased expression of alpha- and beta- catenins, p120CTN, E-cadherin, and CD44s proteins (range, 5% to 49%) was noted in PACs, and downregulation of each of these proteins correlated with high tumor grade (P =.02 to.0001). Although loss of E-cadherin and p120CTN each correlated with stage (E-cadherin, P =.02; p120CTN, P =.02) and ploidy (E-cadherin, P =.0001; p120CTN, P =.004), downregulation of CD44s correlated with ploidy (P =.002), serum PSA (P =.005), and postoperative disease recurrence (P =.02). N-cadherin was positive in only 5% of PACs and did not correlate with any prognostic parameters. alpha-Catenin downregulation correlated with decreased expression of E-cadherin (P =.0001). Additionally, decreased expression of each of these 2 proteins respectively correlated with loss of beta-catenin (P =.0001 and.004), p120CTN (P =.005 and.001), and CD44s (P =.008 and.01). beta-Catenin expression levels correlated with p120CTN (P =.01). A trend for co-downregulation of CD44s and p120CTN and of CD44s and beta-catenin was observed. In conclusion, the significant association between decreased expression of various members of the CAM family of proteins supports their collective role in mediating cell-cell adhesion. Altered expression of these proteins may be of prognostic value in patients with prostate cancer.

Decreased CD44 standard form expression correlates with prognostic variables in ovarian carcinomas.

Expression of CD44 standard form (CD44s) was evaluated by automated immunohistochemical analysis using the anti-CD44 A3D8 clone in 101 ovarian epithelial neoplasms including 82 primary tumors (64 carcinomas and 18 tumors of low malignant potential [LMP]), 9 lymph node metastases, 8 malignant ascites, and 2 peritoneal implants. Immunostaining was scored semiquantitatively. Tumors were graded according to the FIGO (International Federation of Gynecology and Obstetrics) classification system. Tumor stage and patient survival were determined from the patient records. While 9 of 18 LMP tumors expressed CD44s, only 15 of 64 carcinomas expressed it. In the carcinomas, univariate analysis revealed that decreased CD44s expression correlated with high tumor grade, advanced stage, and shortened survival. Loss of CD44s expression also was noted in the tumor cells in 8 of 9 lymph node metastases, 7 of 8 malignant ascites, and 1 of 2 implants. Multivariate analysis revealed that only tumor stage independently correlated with patient survival. Loss of CD44s expression determined by immunohistochemical analysis is more common in ovarian carcinomas than in LMP tumors; correlates with prognostic variables including tumor grade, stage, and survival; and may have an important role in the dissemination of ovarian cancer.

Her-2/neu oncogene amplification determined by fluorescence in situ hybridization.

The proto-oncogene HER-2/neu (C-erbB-2) has been localized to chromosome 17q and encodes a transmembrane tyrosine kinase growth factor receptor. The name for the HER-2 protein is derived from human epidermal growth factor receptor (EGFR) because it features substantial homology with the EGFR (1,2). HER-2/neu gene amplification has been associated with the development of breast cancer in animal models (1). The HER-2/neu protein is a component of a four-member family of closely related growth factor receptors including EGFR or HER-1 (erb-B1), HER-2 (erb-B2), HER-3 (erb-B3), and HER-4 (erb-B4) (3). In addition to its association with disease outcome in gastrointestinal, pulmonary, genitourinary, and other neoplasms, amplification of the HER-2/neu gene or overexpression of the HER-2/neu protein has been identified in from 10 to 34% of breast cancers (4-5). The techniques used to evaluate HER-2/neu status in breast cancer have included gene-based assays such as Southern and slot blotting, polymerase chain reaction methods, and, more recently, in situ hybridization featuring both fluorescent and nonfluorescent techniques (4-5).

Comparison of oncostatin M expression in keratoacanthoma and squamous cell carcinoma.

Oncostatin M (OSM) is a 28-kDa glycoprotein, produced by stimulated macrophages and T lymphocytes, that inhibits the proliferation and induces differentiation of a number of different cell lines derived from solid tumors. To determine whether keratoacanthoma (KA) is unique or a variant of squamous cell carcinoma (SCC), we compared the immunohistochemical expression of OSM in the tumor cells and peri- and intratumoral macrophages of 21 mature KAs, 7 regressing KAs, and 27 SCCs. An inverse correlation was identified between OSM tumor labeling and the density of OSM-labeled tumor-associated macrophages for KAs (r = -.4; P = .09). OSM tumor expression was significantly more frequent and more intense in KAs than in SCCs (95% versus 63%; P < .01). In contrast, the density of OSM-labeled macrophages was significantly higher in SCCs compared with mature KAs (7/3 high power fields versus 4/3 high power fields; P = .02). These OSM-positive macrophages were predominantly located at the advancing, infiltrative margins of both neoplasms. Regressing KAs demonstrated a decreased level of OSM tumor expression compared with mature KAs (53% versus 95%; P = .001), but there was no difference in density of OSM-labeled macrophages. Both the above differences and the overlapping patterns of OSM expression suggest that KAs are a variant of SCC where OSM, possibly as an autocrine factor, may mediate KA's overwhelming but not absolute tendency to involute.

HER-2/neu gene amplification by FISH predicts poor survival in Barrett's esophagus-associated adenocarcinoma.

The HER-2/neu oncogene is localized to chromosome 17q and shares significant homology with the epidermal growth factor receptor. HER-2/neu protein overexpression has been associated with poor prognosis in a variety of tumors, but its significance in Barrett's esophagus-associated adenocarcinoma (BEAd) is unknown. Therefore, the aim of this study was to evaluate the prevalence and prognostic value of HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) in 63 cases of BEAd. Routinely processed tissue sections from resection specimens of 63 patients with BEAd (M/F ratio, 10:1; mean age, 63 years) were assayed for HER-2/neu gene amplification by FISH using the Ventana unique sequence probe (Ventana Medical Systems, Inc, Tuscon, AZ). FISH results were correlated with the pathological features of the tumors and with patient survival. Clinical follow-up data were available for 54 patients (mean follow-up, 31 months [range, 1 to 152 months]). The HER-2/ neu gene was amplified in 12 of 63 (19%) cases. The presence of HER-2/neu gene amplification showed a trend toward a correlation with depth of tumor invasion (P = .07), lymph node metastasis (P = .13), and pathological stage (P = .14), but did not correlate with any of the other pathological features, such as degree of differentiation or tumor size. On both univariate and multivariate analysis, HER-2/neu gene amplification was associated with shortened survival (P = .03). HER-2/neu oncogene amplification, as determined by FISH, correlates with shortened patient survival and independently predicts poor outcome in patients with BEAd.

Prognostic significance of p34cdc2 cyclin-dependent kinase and MIB1 overexpression, and HER-2/neu gene amplification detected by fluorescence in situ hybridization in breast cancer.

The HER-2/neu oncogene, localized to chromosome 17q, shares substantial homology with the epidermal growth factor receptor. HER-2/neu gene amplification and protein overexpression have been associated with poor prognosis in breast cancer. Formalin-fixed paraffin-embedded primary invasive breast cancer tissues from 135 women were tested for HER-2/neu gene amplification by automated fluorescence in situ hybridization (FISH) using a sequence probe. The tumors also were evaluated by immunohistochemistry for proliferation markers Ki 67 (MIB1) and p34cdc2 cyclin-dependent kinase. Patients were followed up for a mean of 61 months. There were 70 node-negative and 65 node-positive cases. Ki 67 and p34cdc2 proliferation marker overexpression, HER-2/neu oncogene amplification, large tumor size, high tumor grade, advanced tumor stage, positive lymph node status, and distant metastasis at the time of diagnosis predicted disease-related death in combined node-negative and node-positive breast cancer. HER-2/neu gene amplification, tumor stage, lymph node metastasis, tumor grade, and distant metastasis at the time of diagnosis independently predicted disease outcome. HER-2/neu amplification detected by FISH also predicted disease-related death independent of lymph node status, tumor grade, and distant metastasis in breast cancer patients who received adjuvant therapy.

The prognostic significance of proliferation-associated nucleolar protein p120 expression in prostate adenocarcinoma: a comparison with cyclins A and B1, Ki-67, proliferating cell nuclear antigen, and p34cdc2.

BACKGROUND: In this study, the authors evaluated the prognostic significance of the expression of nucleolar antigen p120, along with other cell proliferation-associated proteins, in prostate adenocarcinomas (PACs) and compared the results with previously reported data on p34cdc2 cyclin-dependent kinase (p34 cdk). METHODS: Archival sections from 132 PACs were immunostained with monoclonal antibodies against p120, cyclin A, cyclin B1, Ki-67, and proliferating cell nuclear antigen (PCNA). The DNA content of each tumor was determined by the Feulgen method using image analysis. The immunohistochemistry (IHC) results were correlated with tumor grade, stage, margin positivity, metastasis, ploidy, and postsurgical disease recurrence. RESULTS: The overall positivity for the various proteins follows: p120, 36%; cyclin A, 35%; cyclin B1, 43%; Ki-67, 46%; and PCNA, 32%. p120 correlated with grade (P = 0.004), stage (P = 0.01), ploidy (P = 0.02), margin positivity (P = 0.03), and metastasis (P = 0.004). Cyclin B1 correlated with ploidy (P = 0.04) and grade (P = 0.05), Ki-67 with grade (P = 0.02) and margins (P = 0.03), and PCNA with grade (P = 0.01). Significant coexpression among these proteins was noted, as was a significant association between the expression of these markers and that previously reported for p34 cdk. In univariate analysis, p120 (P = 0.01), cyclin A (P = 0.01) and p34 cdk (P = 0.002) correlated with disease recurrence. In multivariate analysis of all these proteins, only p34 cdk independently predicted postsurgical recurrence (P = 0.05). CONCLUSIONS: Nucleolar antigen p120 expression appears to be an additional marker of aggressiveness in PACs. The significant coexpression of the various cell cycle regulatory proteins support their collective role in tumor cell proliferation, with p34 cdk positivity being an independent predictor of postsurgical recurrence.

HER-2/neu oncogene amplification by fluorescence in situ hybridization in epithelial tumors of the ovary.

HER-2/neu gene amplification and protein overexpression have been associated with prognosis in breast, lung and prostate cancers but have not been extensively studied in ovarian carcinoma. For the study, we selected 5-micron-thick, formalin-fixed, paraffin-embedded tissue sections from 74 cases of ovarian epithelial tumors of low malignant potential and ovarian carcinoma. Tumors were graded and staged and evaluated for amplification of the HER-2/neu gene by fluorescence in situ hybridization. HER-2/neu amplifications was present in 3 of 13 serous, mucinous, and endometrioid epithelial tumors of low malignant potential and in 40 of 61 epithelial carcinomas. In the carcinoma group, amplification did not correlate with stage, grade, or tumor type. Mean follow-up was 31 months; 1 patient with a low malignant potential tumor and 32 patients with carcinomas died of disease. On univariate and multivariate analysis, survival correlated with stage of disease but not with HER-2/neu amplification. HER-2/neu amplification by fluorescence in situ hybridization can be performed on tissue sections of ovarian neoplasms; amplification is uncommon in ovarian tumors of low malignant potential, but is present in 66% of ovarian epithelial carcinomas. HER-2/neu amplification did not predict outcome in ovarian epithelial neoplasia but may have an important role in tumor development.

Needle biopsy DNA ploidy status predicts grade shifting in prostate cancer.

DNA ploidy analysis of prostate needle biopsy specimens was performed to determine whether ploidy status could predict tumor grade shifting at radical prostatectomy. The paired needle biopsy and radical prostatectomy specimens from 111 randomly selected men with prostate cancer were obtained from the surgical pathology files of the Albany Medical Center Hospital. The original tumor grades were assigned by a staff of 12 surgical pathologists according to the Gleason system. Tumors with original Gleason scores < or = 6 were classified as low grade, and tumors with scores of > or = 7 were considered high grade. DNA ploidy analysis was performed on the needle biopsy specimens using the CAS 200 image analyzer (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA) on Feulgen stained 5-microm tissue sections. There were 88 diploid and 23 nondiploid cases. Thirty-eight of 111 (34%) of cases had grade shifting from needle biopsy to radical prostatectomy specimens. Of 89 low-grade needle biopsy cases, 28 (31%) were upgraded at radical prostatectomy. Of 22 high-grade needle biopsy cases, 10 (45%) were downgraded to low grade at radical prostatectomy. Of the 28 low-grade needle biopsy specimens that were upgraded at radical prostatectomy, 19 (68%) featured an aneuploid histogram and 9 (32%) were diploid. Nineteen of 28 (68%) of aneuploid low-grade tumors on needle biopsy became high-grade at radical prostatectomy. Nine of 10 (90%) diploid high-grade tumors at needle biopsy became low-grade at radical prostatectomy. Of the 38 cases in which ploidy and grade were incongruous, 28 (74%) had grade shifting. In a multivariate regression analysis, a high-grade Gleason score on radical prostatectomy specimens correlated significantly with needle biopsy ploidy (p = 0.0001) but not with needle biopsy grade (p = 0.15). The sensitivity of the needle biopsy grade in the detection of high-grade tumors on radical prostatectomy was 30%, and the specificity was 86%. The sensitivity of ploidy status in the prediction of high grade at radical prostatectomy was 78%, and the specificity was 96%. With a prostate-specific antigen (PSA) level of >0.4 ng/ml as the indicator of post-radical prostatectomy disease recurrence on a subset of 106 patients, on univariate analysis, disease recurrence was predicted by needle biopsy ploidy (p = 0.001) and radical prostatectomy grade (p = 0.04) but not by needle biopsy grade (p = 0.39). On multivariate analysis, needle biopsy DNA ploidy status independently predicted disease recurrence (p = 0.002), whereas needle biopsy and prostatectomy grade did not. These results indicate that DNA ploidy analysis of needle biopsy specimens of prostate cancer predicts grade shifting, that it is a more sensitive and specific indicator of final tumor grade at radical prostatectomy than is the original needle biopsy grade, and that ploidy status independently predicts postoperative disease recurrence.

Prognostic factors in gastric cancer.

Molecular assays related to cell proliferation correlate with stage and/or survival in a variety of tumors. We immunostained formalin-fixed, paraffin-embedded tissue sections from 61 patients with gastric adenocarcinoma (21 biopsy and 40 gastrectomy specimens) for cyclin A, cyclin B1, p34cdc2, p120, MIB1, and proliferating cell nuclear antigen (PCNA) by automated methods. HER-2/neu gene amplification was analyzed by automated fluorescence in situ hybridization (FISH). Immununostains, FISH results, and pathologic stage were compared with length of survival. Forty-three percent of the cases showed amplification of HER-2/neu. Sixty-two percent of cases showed positive immunostaining for cyclin A, 38% for cyclin B1, 31% for p34cdc2, 49% for p120, 69% for MIB1, and 33% for PCNA. On univariate analysis, pathologic stage (P = .003) and HER-2/neu gene amplification (P < .001) correlated with length of survival. Cyclin A, cyclin B1, p34cdc2, p120, MIB1, and PCNA did not correlate with survival. On multivariate analysis, pathologic stage (P = .015) and HER-2/neu gene amplification (P = .002) independently predicted survival. These correlations were unrelated to tubular or signet ring cell histologic characteristics or to location within the cardia or more distally. Pathologic stage and HER-2/neu gene amplification by FISH were independent prognostic factors in gastric cancer, but the various proliferation markers that we studied did not correlate with survival.

Cell proliferation-associated proteins in endometrial carcinomas, including papillary serous and endometrioid subtypes.

Cyclin dependent kinases (cdks) and cyclins regulate the progression of cells through the cell cycle and can be overexpressed in human cancers. The purpose of this study was to evaluate the immunohistochemical profile of these proliferation-associated proteins and correlate the results with clinicopathologic parameters of endometrial carcinomas. Archival tissue sections from 91 endometrial carcinomas were immunostained using monoclonal antibodies against p34CDC2 cdk, cyclins A and B1, p120, Ki-67, and PCNA. Immunoreactivity was semiquantitatively assessed and the results correlated with pathologic features and survival. Of the 91 endometrial carcinomas, 74 were endometrioid (17 villoglandular, 57 of usual type) and 17 were papillary serous carcinomas. The positivity rates for the different proteins in papillary serous and endometrioid tumors, respectively, were as follows: p34CDC2, 24% and 23%; cyclin A, 71% and 64%; cyclin B1, 24% and 26%; p120, 47% and 9%; Ki-67, 82% and 64%; and PCNA, 47% and 47%. Only p120 correlated with histologic tumor type with significantly higher expression in both papillary serous and villoglandular endometrioid carcinomas compared to nonvilloglandular endometrioid carcinomas (p = 0.0001). p120 positivity also correlated with advanced tumor stage (p = 0.0001). Ki-67, cyclin A, and PCNA correlated with patient survival in endometrioid carcinomas on univariate analysis (p = 0.01, 0.02, and 0.003, respectively), but, on multivariate analysis, only tumor grade (p = 0.02) and depth of invasion (p = 0.04) were independent predictors of outcome. In summary, although most of the cell proliferation-associated proteins studied did not appear to be associated with clinicopathologic features of endometrial carcinoma, there was significantly higher expression of p120 in papillary serous and villoglandular endometrioid carcinomas compared to nonvilloglandular endometrioid carcinomas, suggesting a possible role of p120 in tumor behavior. In addition, Ki-67, cyclin A, and PCNA expression correlated with survival in endometrioid carcinoma, but only in a univariate analysis.

Cell proliferation rate by MIB-1 immunohistochemistry predicts postradiation recurrence in prostatic adenocarcinomas.

We examined whether the cell proliferation index by MIB-1, HER-2/neu gene amplification, Gleason grade, and pretreatment level of serum prostate specific antigen (PSA) correlated with postradiation recurrence (PRR) in patients with prostatic adenocarcinoma. Formalin-fixed, paraffin-embedded tissue sections from 42 pretreated cases of prostatic adenocarcinoma (38 needle biopsy and 4 transurethral resection specimens) were immunostained for MIB-1 (MMI, Ventana Medical Systems, Tucson, Ariz). HER-2/neu gene amplification was analyzed by fluorescence in situ hybridization using the Oncor unique sequence probe (Oncor, Gaithersburg, Md). The cell proliferation index by MIB-1 was determined by labeling index; levels of HER-2/neu were analyzed semiquantitatively. Twenty-three of 42 patients (55%) were considered to have PRR on the basis of consecutive elevations of serum levels of PSA to greater than 1.5 ng/mL after completion of treatment (mean follow-up time, 33.4 months). The cell proliferation index correlated with PRR on both univariate and multivariate analyses. Of the 23 tumors that showed PRR, 18 (78%) revealed a high cell proliferation index, compared with 6 of 19 cases (32%) that showed no PRR. Twelve of 23 cases of prostatic adenocarcinoma (52%) in the recurrent group showed HER-2/neu gene amplification, compared with 5 of 19 (26%) in the nonrecurrent group; these findings reached near significance on univariate analysis. Pretreatment levels of serum PSA also reached significance on multivariate analysis. In this preliminary study, the cell proliferation index by MIB-1 reached independent significance in predicting PRR in patients with prostatic adenocarcinoma, whereas HER-2/neu amplification by fluorescence in situ hybridization reached near significance.

Correlation of p34cdc2 cyclin-dependent kinase overexpression, CD44s downregulation, and HER-2/neu oncogene amplification with recurrence in prostatic adenocarcinomas.

PURPOSE: To test whether p34cdc2 overexpression, CD44s downregulation, and HER-2/neu amplification correlate with disease recurrence after radical prostatectomy, and to evaluate a possible biologic association between p34cdc2 and HER-2/neu expression. MATERIALS AND METHODS: Immunohistochemical (IHC) detection of both p34cdc2 cyclin-dependent kinase (CDK) and CD44s expression and fluorescence in situ hybridization (FISH)-based analysis of HER-2/neu gene status were performed on formalin-fixed, paraffin-embedded sections of 106 prostatic adenocarcinomas (PACs). Findings were correlated with Gleason grade, pathologic stage, DNA ploidy, and postsurgical biochemical disease recurrence. RESULTS: CDK overexpression correlated with tumor grade (P = .001), DNA ploidy (P = .001), pathologic stage (P = .04), and disease recurrence (P = .01). CD44s downregulation correlated with grade (P = .03), ploidy (P = .01), and recurrence (P = .02). HER-2/neu amplification correlated with grade (P = .001), ploidy (P = .001), and recurrence (P = .01). On multivariate analysis, CDK overexpression independently predicted recurrence (P = .001) after prostatectomy. CDK expression correlated with HER-2/neu status with 32 of 65 (49%) tumors that overexpressed CDK and showed concomitant HER-2/neu amplification (P = .04). CONCLUSION: This study showed that p34cdc2, CD44s, and HER-2/neu are variably expressed or amplified in prostatic carcinoma and that such alteration may affect tumor behavior. In addition, CDK overexpression and HER-2/neu amplification may be biologically related.

Control of mouse hepatocyte proliferation and ploidy by p53 and p53ser246 mutation in vivo.

The effect of expression of the p53 gene, in the presence or absence of the p53ser246 mutation (p53*), on ploidization (image cytometry), proliferation (expression of proliferating cell nuclear antigen and radioactive thymidine histoautoradiography), and apoptosis (in situ detection of DNA fragments) is determined in hepatocytes of p53-null and p53*-transgenic mice. The mouse p53ser246 mutation is equivalent to the p53ser249 mutation found in human hepatomas associated with hepatitis B virus infection and aflatoxin exposure. The hepatocytes of heterozygous or homozygous p53-knockout mice (p53+/-; p53-/-), as well as knockout mice expressing one allele of p53ser246 (p53+/-, p53*; p53-/-, p53*), do not undergo normal polyploidization with aging and show an increase in the number of cycling (G1-, S-, and M-phase) cells. In addition, p53ser246-transgenic mice (p53+/+, p53*; p53+/-, p53*; and p53-/-, p53*) have a greatly increased number of hepatocytes in the G1 phase. No differences in rates of apoptotic hepatocytes are found among any of the mouse groups studied, so the increased proliferation results in a hyperplasia manifested by a increased number of small periportal cells. We conclude that loss of p53 removes blocks in the cell cycle, leading to increased proliferation, whereas expression of the p53ser246 mutation stimulates G0 to G1 and/or M to G1 transition of hepatocytes. Increased proliferation of hepatocytes, combined with no concomitant increase in apoptosis, may in part explain the enhanced development of hepatocellular carcinomas in p53-knockout and p53*-transgenic mice exposed to aflatoxin.