Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer.

The transcriptional coactivator Amplified in Breast Cancer 1 (AIB1) plays a major role in the progression of hormone and HER2-dependent breast cancers but its role in triple negative breast cancer (TNBC) is undefined. Here, we report that established TNBC cell lines, as well as cells from a TNBC patient-derived xenograft (PDX) that survive chemotherapy treatment in vitro express lower levels of AIB1 protein. The surviving cell population has an impaired tube-formation phenotype when cultured onto basement membrane, a property shared with TNBC cells that survive shRNA-mediated depletion of AIB1 (AIB1LOW cells). DNA analysis by exome sequencing revealed that AIB1LOW cells represent a distinct subpopulation. Consistent with their in vitro phenotype AIB1LOW cells implanted orthotopically generated slower growing tumors with less capacity for pulmonary metastases. Gene expression analysis of cultured cells and tumors revealed that AIB1LOW cells display a distinct expression signature of genes in pro-inflammatory pathways, cell adhesion, proteolysis and tissue remodeling. Interestingly, the presence of this AIB1LOW expression signature in breast cancer specimens is associated with shorter disease free survival of chemotherapy treated patients. We concluded that TNBC cell lines contain heterogeneous populations with differential dependence on AIB1 and that the gene expression pattern of AIB1LOW cells may represent a signature indicative of poor response to chemotherapy in TNBC patients.

Exposures in early life: associations with DNA promoter methylation in breast tumors.

There is evidence that epigenetic changes occur early in breast carcinogenesis. We hypothesized that early-life exposures associated with breast cancer would be associated with epigenetic alterations in breast tumors. In particular, we examined DNA methylation patterns in breast tumors in association with several early-life exposures in a population-based case-control study. Promoter methylation of E-cadherin, p16 and RAR-ß2 genes was assessed in archived tumor blocks from 803 cases with real-time methylation-specific PCR. Unconditional logistic regression was used for case-case comparisons of those with and without promoter methylation. We found no differences in the prevalence of DNA methylation of the individual genes by age at menarche, age at first live birth and weight at age 20. In case-case comparisons of premenopausal breast cancer, lower birth weight was associated with increased likelihood of E-cadherin promoter methylation (OR = 2.79, 95% CI, 1.15-6.82, for ⩽2.5 v. 2.6-2.9 kg); higher adult height with RAR-ß2 methylation (OR = 3.34, 95% CI, 1.19-9.39, for ⩾1.65 v. <1.60 m); and not having been breastfed with p16 methylation (OR = 2.75, 95% CI, 1.14-6.62). Among postmenopausal breast cancers, birth order was associated with increased likelihood of p16 promoter methylation. Being other than first in the birth order was inversely associated with likelihood of ⩾1 of the three genes being methylated for premenopausal breast cancers, but positively associated with methylation in postmenopausal women. These results suggest that there may be alterations in methylation associated with early-life exposures that persist into adulthood and affect breast cancer risk.

Familial and racial determinants of tumour suppressor genes promoter hypermethylation in breast tissues from healthy women.

To determine the hypermethylation status of the promoter regions of tumour suppressor genes in breast tissues from healthy women and identify the determinants of these epigenetic changes. Questionnaires and breast tissues were collected from healthy women without a history of cancer and undergoing reduction mammoplasty (N= 141). Methylation for p16(INK4), BRCA1, ERalpha and RAR-beta promoter regions from breast tissues were determined by methylation specific PCR. Associations were examined with chi-square and Fisher's exact test as well as logistic regression. All statistical tests were two-sided. p16(INK4), BRCA1, ERalpha and RAR-beta hypermethylation were identified in 31%, 17%, 9% and 0% of the women, respectively. Women with BRCA1 hypermethylation had an eight-fold increase in the risk of ERalpha hypermethylation (P= 0.007). p16(INK4) hypermethylation was present in 28% of African-Americans, but 65% in European-Americans (P= 0.02). There was an increased likelihood of p16(INK4) or BRCA1 hypermethylation for women with family history of cancer (OR 2.3; 95%CI: 1.05-4.85 and OR 5.0; 95%CI: 1.55-15.81, respectively). ERalpha hypermethylation was associated with family history of breast cancer (OR 6.6; 95%CI: 1.58-27.71). After stratification by race, p16(INK4) in European-Americans and BRCA1 hypermethylation in African-Americans were associated with family history of cancer (OR 3.8; 95%CI: 1.21-12.03 and OR 6.5; 95%CI: 1.33-31.32, respectively). Gene promoter hypermethylation was commonly found in healthy breast tissues from women without cancer, indicating that these events are frequent and early lesions. Race and family history of cancer increase the likelihood of these early events.

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.

Molecular pathology of leukemia and lymphoma.

Molecular dissection of physiologic and pathologic genetic phenomena in hematologic malignancy has provided the pathologist with a broad menu of new assays. By integrating the data gleaned from these techniques we can formulate more rational and biologically based diagnoses, which should lead to the ultimate goal of targeted therapy for these specific entities. We summarize some of the more relevant molecular genetic assays and present an overview of those genetic mechanisms usually evaluated in the current practice of hematopathology. The usefulness of such assays extends beyond refining diagnoses in that they also provide relevant prognostic information. Moreover, since most are based on the polymerase chain reaction and reverse transcriptase polymerase chain reaction, we are more sensitively able to monitor for residual disease after attempts at curative therapy, and our definition of remission has been dramatically altered. However, molecular genetic tests are not without limitations, and we must remain cognizant of their cost effectiveness and be aware of current deficiencies in standardization. The challenge will be to meaningfully and economically harness and integrate the information we obtain from these and future technologies into appropriate clinical practice.

Posttherapy surveillance of B-cell precursor acute lymphoblastic leukemia. Value of polymerase chain reaction and limitations of flow cytometry.

Flow cytometric immunophenotypic analysis is critical in diagnosis and classification of acute leukemia and has been used after therapy to monitor for minimal residual disease. However, the presence of normal B-cell precursors, hematogones, particularly in the context of treated pediatric B-cell precursor acute lymphoblastic leukemia (BP-ALL), may confound such evaluation. In this study, the value of more specific genotypic markers (polymerase chain reaction evaluation of 2 antigen receptor genes) was assessed to resolve this issue. Flow cytometric analysis of enriched mononuclear cells revealed 1% to 20% precursor B cells (PBCs), based on expression of 1 or more pan-B cell antigens in addition to CD10, CD34, and terminal deoxynucleotidyl transferase in all 14 patients studied. Inasmuch as this mimicked the immunophenotype of the original leukemic clone, PBCs, in isolation, were considered suspicious for minimal residual disease. However, 11 of the 14 posttherapy specimens (79%) revealed no monoclonally rearranged antigen receptor genes, and 7 of these 11 patients had trackable genotypic markers at presentation. Accordingly, by PCR these 7 patients had complete molecular remission, supported by clinical follow up of 16 to 73 months. Among the remaining 4 patients with PCR-negative disease, 3 continue in remission, confirming the interpretation of false-positive flow cytometric analysis. In conclusion, flow cytometric monitoring of posttherapy bone marrow specimens from patients with BP-ALL may be misleading, if considered in isolation, in falsely suggesting the presence of minimal residual disease. Rather, PCR for antigen receptor gene rearrangements is a valuable and specific tool, helpful in differentiating hematogones from minimal residual disease in patients with treated BP-ALL whose bone marrow harbors increased PBCs.

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.

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.

Prognostic significance of tumor necrosis factors and their receptors in nonsmall cell lung carcinoma.

BACKGROUND: In vitro studies have shown an antiproliferative effect of tumor necrosis factor (TNF) against various nonsmall cell lung carcinoma (NSCLC) cell lines. However, clinical trials of combined interleukin-2 and TNF-alpha in patients with advanced NSCLC have demonstrated both conflicting and disappointing results. METHODS: Immunohistochemical (IHC) staining was performed on formalin fixed, paraffin embedded tissues from 39 bronchogenic adenocarcinomas and 32 squamous cell carcinomas using polyclonal antibodies against TNF-alpha, TNF-beta, TNF-R1, and TNF-R2 proteins. IHC positivity was correlated with tumor stage, grade, and patient survival. RESULTS: Significant coexpression of TNF-alpha, TNF-beta, TNF-R1, and TNF-R2 was observed in NSCLC (significance range, P < 0.001-0.02). Although immunoreactivity for TNFs remained high in all tumor stages, a loss of TNF-R expression was found in advanced NSCLC (P < 0.006 for TNF-R1 and P < 0.003 for TNF-R2), suggesting down-regulation of TNF-Rs in the process of tumor progression. When all stages were considered together, immunoreactivity for TNF-beta(P < 0.001), TNF-R1, and TNF-R2 (both P < 0.001) significantly correlated with favorable outcome in univariate analysis. However, when stages were studied separately, an association between immunopositivity for TNF-Rs and favorable prognosis was found only in NSCLC without distant metastasis (P < 0.04 and P < 0.005 for TNF-R1 and TNF-R2 in Stage I [according to the American Joint Committee on Cancer staging system] disease, and P < 0.03 and P < 0.02 for TNF-R1 and TNF-R2 in Stage III disease). On multivariate analysis, increased expression of TNF-R1 (P < 0.003) and TNF-R2 (P < 0.001) as well as tumor stage (P < 0.001) independently predicted favorable outcome in patients with NSCLC. CONCLUSIONS: Although NSCLC exhibits strong coexpression of TNF-alpha, TNF-beta, TNF-R1, and TNF-R2, there is a loss/down-regulation of TNF receptors in high stage tumors. TNF-R1 and TNF-R2 positivity independently predicts favorable outcome in NSCLC, particularly in tumors with no clinically distant metastasis. The current study supports a role for TNFs and their receptors in the evolution and progression of NSCLC.

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.

Coexistence of granular cell tumor and ipsilateral infiltrating ductal carcinoma of the breast.

Granular cell tumor is a benign neoplasm that is rarely seen in the breast and can mimic carcinoma, both clinically and by gross pathologic examination. The coexistence of a granular cell tumor with a primary mammary carcinoma can potentially pose diagnostic and therapeutic problems. In this report, we document a granular cell tumor of the breast coexisting with an ipsilateral infiltrating ductal carcinoma in a 74-year-old woman and discuss its clinical significance.

Telomerase activity in human benign prostate tissue and prostatic adenocarcinomas.

Telomerase adds a hexanucleotide telomeric sequence to the chromosomal ends during replication and is postulated to play a role in cellular senescence and immortalization. Thirty-four human prostate tissues (18 malignant; 16 benign) were analyzed for telomerase activity by a sensitive nonradioactive polymerase chain reaction (PCR)-based method using the TRAP-eze telomerase detection kit (Oncor, Inc., Gaithersburg, MD). Telomerase activity in the homogenized tissue extracts was correlated with tumor grade, pathologic stage, and DNA ploidy. Specimens that exhibited the 36 bp internal control band and a ladder of products with 6-base increments starting with 50 nucleotides were considered positive. Fourteen (78%) of 18 prostatic adenocarcinomas (PACs) and only 2 (13%) of 16 benign prostate tissues exhibited telomerase activity. Our results indicate that, in contrast to most benign prostate tissues, telomerase activity can be detected in the majority of PACs and appears to be independent of tumor grade, stage, or DNA ploidy. Telomerase expression is occasionally detected in benign prostatic tissues bordering PACs and may result from either the presence of undetected tumor foci in these stored specimens or the proliferative response of the benign elements to adjacent cancer.