Diane L. Persons

Effect of Extracellular Signal-regulated Kinase on p53 Accumulation in Response to Cisplatin

The p53 tumor suppressor protein is a transcription factor that plays a major role in the DNA damage response. After DNA damage, p53 levels increase due primarily to stabilization of the protein. The molecular mechanisms leading to stabilization of p53 after DNA damage have not been completely elucidated. Recently we reported that cisplatin treatment activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and that inhibition of ERK1/2 resulted in enhanced sensitivity to cisplatin. In the present study, we examined the potential role of ERK1/2 activation in regulation of the p53 response to cisplatin. In the ovarian carcinoma cell line A2780, inhibition of ERK1/2 activation with the mitogen-activated protein kinase/ERK kinase 1 (MEK1) inhibitor PD98059 resulted in decreased p53 protein half-life and diminished accumulation of p53 protein during exposure to cisplatin. We also demonstrated that p53 protein co-immunoprecipitated with ERK1/2 protein and was phosphorylated by activated recombinant murine ERK2 in vitro. Furthermore, PD98059 decreased the phosphorylation of p53 at serine 15 during cisplatin exposure, suggesting that ERK1/2 mediates in part phosphorylation of p53 during the cisplatin DNA response. These results strongly suggest that cisplatin-induced ERK activation is an up-stream regulator of the p53 response to DNA damage caused by cisplatin.

Genetic Heterogeneity in HER2 Testing in Breast Cancer Panel Summary and Guidelines

CONTEXT Intratumoral heterogeneity of HER2 gene amplification has been well documented and represents subclonal diversity within the tumor. The reported incidence of intratumor HER2 amplification genetic heterogeneity ranges in the literature from approximately 5% to 30%. The presence of HER2 genetic heterogeneity may increase subjectivity in HER2 interpretation by the pathologist. OBJECTIVES To define HER2 genetic heterogeneity and to provide practice guidelines for examining and reporting breast tumors with genetic heterogeneity for improvement of HER2 testing in breast cancer. DESIGN We convened an expert panel to discuss HER2 gene amplification testing by fluorescence in situ hybridization. Components addressed included a definition of HER2 amplification heterogeneity, practice guidelines for examination of the tissue, and reporting criteria for this analysis. RESULTS Genetic heterogeneity for amplification of HER2 gene status in invasive breast cancer is defined and guidelines established for assessing and reporting HER2 results in these cases. These guidelines are additive to and expand those published in 2007 by the American Society of Clinical Oncology and the College of American Pathologists. CONCLUSION Standardized methods for analysis will improve the accuracy and consistency of interpretation of HER2 gene amplification status in breast cancer.

Cisplatin-induced response of c-jun N-terminal kinase 1 and extracellular signal-regulated protein kinases 1 and 2 in a series of cisplatin-resistant ovarian carcinoma cell lines

The cellular response to cisplatin involves activation of multiple signal transduction pathways, including the mitogen‐activated protein (MAP) kinase pathways. In this study, we compared the cisplatin‐induced activation of two MAP kinases, c‐jun N‐terminal kinase 1 (JNK1) and extracellular signal–regulated protein kinases 1 and 2 (ERK1/2), in the cisplatin‐sensitive ovarian carcinoma cell line A2780 and its derivative cisplatin‐resistant cell lines CP70 and C200. Dose‐dependent and time‐dependent activation of JNK1 and ERK1/2 occurred in each of the three cell lines in response to cisplatin treatment. The requirement of higher concentrations of cisplatin for induction of maximum activation of JNK1 and ERK1/2 was correlated with increased levels of cisplatin resistance. In addition, inhibition of cisplatin‐induced ERK activation, using the MAP/ERK kinase 1 synthetic inhibitor PD98059, resulted in enhanced sensitivity to cisplatin in all three cell lines. These results suggest that cisplatin‐induced ERK1/2 activity is not responsible for the acquired cisplatin resistance in CP70 and C200 cells but rather provides a general cytoprotective effect in both cisplatin‐sensitive and cisplatin‐resistant cell lines. In conclusion, different patterns of cisplatin‐induced JNK1 and ERK1/2 activation are observed in cell lines with different levels of cisplatin sensitivity, and inhibition of cisplatin‐induced ERK1/2 activation enhances sensitivity to cisplatin in both cisplatin‐sensitive and cisplatin‐resistant cell lines. Mol. Carcinog. 29:219–228, 2000.

Interphase Cytogenetics of Esophageal Adenocarcinoma and Precursor Lesions

Limited information is currently available on chromosomal abnormalities in esophageal adenocarcinoma and associated premalignant lesions. In this study, numeric changes affecting chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 17, 18, X, and Y were analyzed by using fluorescence in situ hybridization (FISH) with chromosome-specific centromere DNA probes in 12 esophageal adenocarcinomas. In addition, TP53 overexpression, measured by immunohistochemistry, and amplification of HER-2/neu and C-MYC, detected by FISH, were analyzed within the same tumors. The most common numeric abnormalities detected were gains of chromosomes 12 (8 cases), 6 (7 cases), 7 (7 cases), and 11 (6 cases). The total number of abnormal chromosomes varied from 0 to 10, with an average of 4.6 per case. Overexpression of TP53 was present in 9 of 12 cases. No correlation was noted between the degree of aneusomy and TP53 overexpression. In contrast, HER-2/neu amplification was present in two cases, both with large numbers of aneusomic chromosomes. Amplification of C-MYC was detected in only one case that had a moderate number of numeric abnormalities. In a subset of cases in which premalignant lesions were examined, aneusomy was found to be an early change, frequently present in both Barretts esophagus and dysplastic regions. In contrast, gene amplification and TP53 overexpression were restricted to more advanced areas of dysplasia and malignancy. Screening larger cohorts of patients with Barretts esophagus or dysplasia for numeric abnormalities of chromosomes 6, 7, 11, and 12 may determine whether any of these abnormalities are predictive markers of progression to malignancy.

Regulation of p53 target gene expression by cisplatin-induced extracellular signal-regulated kinase

Abstract. The extracellular signal-regulated kinase (ERK) pathway is among several signal transduction pathways that are activated in response to exposure to the DNA damage-inducing chemotherapeutic agent cisplatin. We have previously reported that inhibition of cisplatin-induced ERK activity enhances sensitivity to cisplatin. Furthermore, we have demonstrated that cisplatin-induced ERK activation is required for optimal p53 protein accumulation following cisplatin-induced DNA damage. In the present study, we expanded our investigations to examine the effect of cisplatin-induced ERK activation on the expression of p53-targeted genes that have been shown to be important in the cellular response to DNA damage including Bax, Bcl-2, Bcl-xl, Cyclin G, Gadd45, p21WAF1, and Mdm2. In the ovarian carcinoma cell line A2780, cisplatin was shown to induce expression of p21WAF1, Gadd45 and Mdm2, but cisplatin had no effect on expression of Bax, Bcl-2, Bcl-xl, or Cyclin G. Inhibition of cisplatin-induced ERK activity by PD98059 resulted in decreased levels of p21WAF1, Gadd45 and Mdm2. These results provide evidence that ERK activity during the cisplatin DNA damage response, regulates in part, these cell cycle control (p21WAF1, Gadd45), DNA repair (Gadd45) and p53-regulatory (Mdm2) proteins.

Detection of Copy Number Alterations in Metastatic Melanoma by a DNA Fluorescence In situ Hybridization Probe Panel and Array Comparative Genomic Hybridization: A Southwest Oncology Group Study (S9431)

Purpose: Gene copy number alteration (CNA) is common in malignant melanoma and is associated with tumor development and progression. The concordance between molecular cytogenetic techniques used to determine CNA has not been evaluated on a large set of loci in malignant melanoma. Experimental Design: A panel of 16 locus-specific fluorescence in situ hybridization (FISH) probes located on eight chromosomes was used to identify CNA in touch preparations of frozen tissue samples from 19 patients with metastatic melanoma (SWOG-9431). A subset (n = 11) was analyzed using bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) of DNA isolated directly from touch-preparation slides. Results: By FISH, most samples showed loss near or at WISP3/6p21, CCND3/6q22, and CDKN2A/9p21 (>75% of samples tested). More than one third of CDKN2A/9p21 losses were biallelic. Gains of NEDD9/6p24, MET/7q31, and MYC/8q24 were common (57%, 47%, and 41%, respectively) and CNA events involving 9p21/7p12.3 and MET were frequently coincident, suggesting gain of the whole chromosome 7. Changes were confirmed by aCGH, which also uncovered many discreet regions of change, larger than a single BAC. Overlapping segments observed in >45% of samples included many of the loci analyzed in the FISH study, in addition to other WNT pathway members, and genes associated with TP53 pathways and DNA damage response, repair, and stability. Conclusions: This study outlines a set of CNAs at the gene and regional level, using FISH and aCGH, which may provide a benchmark for future studies and may be important in selection of individual therapy for patients with metastatic malignant melanoma.

HER-2 fluorescence in situ hybridization: results from the survey program of the College of American Pathologists.

CONTEXT Fluorescence in situ hybridization (FISH) is a common method used to determine HER-2 status in breast cancer. Limited information is available concerning reproducibility of FISH in determining HER-2 gene amplification. OBJECTIVE To present proficiency testing results of FISH for HER-2 conducted by the Cytogenetics Resource Committee of the College of American Pathologists/American College of Medical Genetics. DESIGN During the past 5 years, unstained sections from 9 invasive breast carcinomas were used for HER-2 FISH proficiency testing, allowing for comparison of FISH results among a large number of laboratories. Additional data were collected using an educational (ungraded) challenge and supplemental questions in the surveys. RESULTS The number of laboratories participating in HER-2 FISH proficiency testing has increased steadily during the past 5 years (from 35 in 2000 to 139 in 2004). Reproducibility of test results among laboratories was excellent for breast tumors with low copy number (no HER-2 amplification) and for breast tumors with high copy number (HER-2 amplification). However, there was considerable variation in interpretation of results for a tumor with low-level HER-2 amplification that was tested on 2 separate occasions. Responses to supplemental questions indicated that there was a need for consensus on the use of a separate equivocal/borderline interpretative category and the need for standardization of cutoff values used to define interpretative categories. CONCLUSIONS The College of American Pathologists proficiency survey programs provide useful information concerning the reproducibility of clinical testing for HER-2 by FISH and reflect clinical interpretation of HER-2 FISH analyses from laboratories across the country.

Histiocytic/dendritic cell sarcoma arising from follicular lymphoma involving the bone: a case report and review of literature

Histiocytic/dendritic cell sarcomas arising from follicular lymphoma are very rare and poorly understood lesions. We describe a case, which is unique in that it presented with a hipbone lesion simultaneously with axillary lymphadenopathy. Biopsy of the axillary lymph node showed a low-grade follicular lymphoma. The hipbone lesion was comprised two cell populations, one representing diffuse large B cell lymphoma and the other a histiocytic/dendritic sarcoma. The cells of all three lesions contained an IGH/BCL2 rearrangement, suggesting that both diffuse large B cell lymphoma and histiocytic/dendritic sarcoma differentiation developed from the same low grade precursor (follicular lymphoma). This case illustrates that sarcomatous transdifferentiation of follicular lymphoma can be an unpredictable local phenomenon and that it can occur extra nodally in the bone marrow. It may also occur concurrently with the progression of follicular lymphoma to a diffuse large B cell lymphoma.

Association of apoptosis with the inhibition of extracellular signal–regulated protein kinase activity in the tumor necrosis factor α-resistant ovarian carcinoma cell line UCI 101

Tumor necrosis factor‐α (TNFα) can function as both an autocrine and a paracrine growth factor and may therefore play a role in ovarian tumor progression. TNFα initiates multiple cellular responses, many of which are mediated through the mitogen‐activated protein kinase pathways, which transduce signals from the TNFα receptors through the cytoplasm to the nucleus, resulting in regulation of gene expression. We examined the role of c‐jun N‐terminal kinase 1 (JNK1) and extracellular signal–regulated protein kinase (ERK) 1 and 2 in the cellular growth response to TNFα in the ovarian carcinoma cell line UCI 101. JNK1 activity was increased to a maximum level ninefold above the basal level after 10–20 min of treatment with 10 ng/mL TNFα. A maximum threefold induction of ERK1/2 activity was observed after 1 min of treatment. At concentrations up to 100 ng/mL, TNFα had neither a stimulatory nor an inhibitory effect on growth of UCI 101 cells. However, inhibition of TNFα‐induced ERK1/2 activity by the MAP/ERK kinase 1 inhibitor PD 98059 resulted in 60% inhibition of cell growth in TNFα‐treated UCI 101 cells. This decrease in cell growth was accompanied by apoptosis, as demonstrated by the presence of a 180‐bp DNA ladder. Thus, the inhibition of TNFα‐induced ERK1/2 activity was associated with induction of apoptosis in the TNFα‐resistant cell line UCI 101. Inhibition of TNFα‐induced ERK1/2 activity was accompanied by a subsequent transient increase in TNFα‐induced JNK1 activity. The significance of this increase with respect to apoptosis induction remains to be determined. These findings demonstrated that ERK1/2 activity can modulate cellular sensitivity to TNFα and suggested that the balance between the levels of ERK1/2 and JNK1 activation may be critical in the cellular growth response to TNFα. Mol. Carcinog. 25:14–20, 1999.

Cytogenetic analysis of two sacral chordomas

Cytogenetic analysis of two sacral chordomas revealed two distinct abnormal clones in one of the cases: 44,XY,t(1;3)(q42;q11), -2,der(7)t(2;7)(q23;q32), -21 and 46,X,t(Y;8)(q12;q22), t(1;14)(p34;q32),t(5;10)(q13;p11). All cells analyzed from the second case were cytogenetically normal. To the best of our knowledge, chordomas have not previously been subjected to cytogenetic analysis.