Marlena S. Fejzo

Lapatinib, a dual EGFR and HER2 kinase inhibitor, selectively inhibits HER2-amplified human gastric cancer cells and is synergistic with trastuzumab in vitro and in vivo.

Purpose: HER2 amplification occurs in 18% to 27% of gastric and gastroesophageal junction cancers. Lapatinib, a potent ATP-competitive inhibitor simultaneously inhibits both EGFR and HER2. To explore the role of HER family biology in upper gastrointestinal cancers, we evaluated the effect of lapatinib, erlotinib, and trastuzumab in a panel of molecularly characterized human upper gastrointestinal cancer cell lines and xenografts. Experimental Design: EGFR and HER2 protein expression were determined in a panel of 14 human upper gastrointestinal cancer cell lines and HER2 status was assessed by fluorescent in situ hybridization. Dose-response curves were generated to determine sensitivity to lapatinib, erlotinib, and trastuzumab. In HER2-amplified cells, the combination of trastuzumab and lapatinib was evaluated using the median effects principal. The efficacy of lapatinib, trastuzumab, or the combination was examined in HER2-amplified xenograft models. Results: Lapatinib had concentration-dependent antiproliferative activity across the panel with the greatest effects in HER2-amplified cells. There was no association between EGFR protein expression and sensitivity to any of the HER-targeted agents. Cell cycle analysis revealed that lapatinib induced G1 arrest in sensitive lines and phosphorylated AKT and phosphorylated ERK were decreased in response to lapatinib as well. The combination of lapatinib and trastuzumab was highly synergistic in inhibiting cell growth with a combination index of <1. The combination also induced greater decreases in AKT and ERK activation, G0-G1 cell cycle arrest, and increased rates of apoptosis. In vivo studies showed that the combination of lapatinib and trastuzumab had greater antitumor efficacy than either drug alone. Conclusion: Together, these data suggest that lapatinib has activity in HER2-amplified upper gastrointestinal cancer and supports the ongoing clinical investigation of lapatinib in patients with HER2-amplified disease. Clin Cancer Res; 16(5); 1509–19

Frozen Tumor Tissue Microarray Technology for Analysis of Tumor RNA, DNA, and Proteins

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified this technology by using frozen tissues embedded in OCT compound as donor samples and arraying the specimens into a recipient OCT block. Tumor tissue is not fixed before embedding, and sections from the array are evaluated without fixation or postfixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. While paraffin tissue arrays can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, this method allows optimal evaluation by each technique and uniform fixation across the array panel. We show OCT arrays work well for DNA, RNA, and protein analyses, and may have significant advantages over the original technology for the assessment of some genes and proteins by improving both qualitative and quantitative results.

Characterization of Genes Encoding Known and Novel Human Mast Cell Tryptases on Chromosome 16p13.3

Tryptases are serine proteases implicated in asthma and are very highly expressed in human mast cells. They fall into two groups, α and β. Although several related tryptase mRNAs are known, it is unclear which if any are transcripts of separate haploid genes. The studies described here investigated the nature and number of human tryptases and sought possibly novel members of the family. To this end, two human bacterial artificial chromosome (BAC) clones containing tryptase genes were identified and mapped to chromosome 16p13.3, of which ∼2.2 megabases are syntenic with the part of mouse chromosome 17 containing tryptase genes mouse mast cell protease (mMCP)-6 and -7. Sequencing and restriction mapping suggest that the BACs may partially overlap. Sequenced BAC genes correspond to three known β-tryptases (βI, βII, and βIII), an α-like gene, and a pair of novel hybrid genes related partly to α/β-tryptases and partly to orthologs of mMCP-7. βII and βIII, βI and αII, as well as the two mMCP-7-like genes, may be alleles at single loci; in total, there are at least three nonallelic tryptase genes in the isolated BAC clones. DNA blotting and restriction analysis suggest that the BACs include most members of the immediate tryptase family. Thus, chromosome 16p13.3 harbors a cluster of known and previously undescribed members of the tryptase gene family.

Translocation breakpoints upstream of the HMGIC gene in uterine leiomyomata suggest dysregulation of this gene by a mechanism different from that in lipomas

Uterine leiomyomata are the most common pelvic tumors in women and are the indication for more than 200,000 hysterectomies annually in the United States. Rearrangement of chromosome 12 in bands q14‐q15 is characteristic of uterine leiomyomata and other benign mesenchymal tumors, and we identified a yeast artificial chromosome (YAC) spanning chromosome 12 translocation breakpoints in a uterine leiomyoma, a pulmonary chondroid hamartoma, and a lipoma. Recently, we demonstrated that HMGIC, which is an architectural factor mapping within the YAC, is disrupted in lipomas, resulting in novel fusion transcripts. Here, we report on the localization of translocation breakpoints in seven uterine leiomyomata from 10 to > 100 kb upstream of HMGIC by use of fluorescence in situ hybridization. Our findings suggest a different pathobiologic mechanism in uterine leiomyomata from that in lipomas. HMGIC is the first gene identified in chromosomal rearrangements in uterine leiomyomata and has important implications for an understanding of benign mesenchymal proliferation and differentiation. Genes Chromosom Cancer 17:1–6 (1996).

The psychosocial burden of hyperemesis gravidarum

Objective:To describe the psychosocial burden of hyperemesis gravidarum (HG) in a large cohort of affected women, focusing on previously unreported problems.Study Design:Women with HG described their pregnancy history in an open-ended survey administered internationally through an HG website during 2003 to 2005.Result:Of the 808 participants, 626 (77.5%) were American. A large majority (82.8%) reported that HG caused negative psychosocial changes, consisting of (1) socioeconomic changes, for example, job loss or difficulties, (2) attitude changes including fear regarding future pregnancies and (3) psychiatric sequelae, for example, feelings of depression and anxiety, which for some continued postpartum. Women who reported that their health-care provider was uncaring or unaware of the severity of their symptoms were nearly twice as likely to report these psychiatric sequelae (odds ratio 1.86, 95% confidence interval 1.06 to 3.29, P=0.032).Conclusion:Over 80% of a large cohort of women with HG reported that HG caused a negative psychosocial impact.

The hD52 (TPD52) gene is a candidate target gene for events resulting in increased 8q21 copy number in human breast carcinoma.

Chromosome band 8q21 is frequently overrepresented in human cancer, but to date no 8q21 target gene has been proposed. The hD52 (TPD52) gene is of potential significance in breast and other cancers due to its location and expression pattern. Fine mapping of hD52 placed this locus within the peak of the 8q21 amplicon delineated in the SK‐BR‐3 breast carcinoma cell line, and a positive association between hD52 gene dosage and transcript levels was subsequently demonstrated in four breast carcinoma cell lines, including SK‐BR‐3. Increased copy number (ICN) was measured using Southern blot analyses in 3/32 human breast carcinomas at hD52, and the related hD54 gene in 20q13.2–q13.3. Subsequent immunohistochemical analysis of hD52 expression in 19 breast carcinomas with varying hD52 gene dosages demonstrated a significant positive association between hD52 dosage and hD52 expression using a Spearman rank correlation coefficient (rs = 0.573, α = 0.01) and a Wilcoxon rank‐sum test (α = 0.05). On the basis of its map location and expression pattern in breast carcinoma, we therefore propose hD52 as a candidate target gene at chromosome band 8q21. Genes, Chromosomes and Cancer 29:48–57, 2000.

Periostin promotes ovarian cancer angiogenesis and metastasis

OBJECTIVE Perostin (PN) has been found to be overexpressed in a variety of human malignancies including ovarian cancer. In the present study, we investigated PN expression status in a large cohort of ovarian tumors with the focus on biological influence of PN related on ovarian tumor angiogenesis and metastasis. METHODS PN expression was determined by cDNA microarray, PN northern blot and PN IHC tissue array analyses. Exogenous PN expression in ovarian cancer cells OVCAR-3 and OV2008 were achieved through retroviral transfection and confirmed by PN western blot and ELISA. The effects of exogenous PN expression on tumor angiogenesis and metastatic growth were accessed in orthotopic mouse models. The in vitro cell adhesion, migration and invasion assays were performed to investigate the potential mechanisms involved in PNs in vivo effects. RESULTS PN was frequently overexpressed in ovarian tumors. Higher PN levels significantly correlated with clinical late stages (III/IV) and cancer recurrence. PN was produced by engineered PN-overexpressing cells at levels comparable to that of A2780 cells, an ovarian carcinoma cell line with endogenous PN expression. PN overexpression did not change cell growth rates in vitro; however it significantly promoted intraperitoneal tumor metastatic growth in immunodeficient mice, which was associated with increased tumor angiogenesis and decreased tumor cell apoptosis. In vitro purified PN promoted cell adhesion, migration, and invasion of both human umbilical endothelial cells (HUVECs) and/or ovarian cancer cells. CONCLUSIONS Our data indicate PN plays a critical role in both ovarian tumor angiogenesis and metastasis. Thus PN may represent a clinically effective new target for therapy of ovarian cancer.

Tumor protein D52 (TPD52) is overexpressed and a gene amplification target in ovarian cancer.

Recurrent chromosome 8q gain in ovarian carcinoma is likely to reflect the existence of multiple target loci, as the separate gain of chromosome bands 8q21 and 8q24 has been reported in independent studies. Since tumor protein D52 (TPD52) has been identified as a chromosome 8q21 amplification target in breast and prostate carcinoma, we compared TPD52 expression in normal ovarian epithelium (n = 9), benign serous adenomas (n = 11), serous borderline tumors (n = 6) and invasive carcinomas of the major histologic subtypes (n = 57) using immunohistochemistry. These analyses revealed that all normal ovarian epithelium samples and benign serous tumors were predominantly TPD52‐negative, whereas TPD52 was overexpressed in most (44/57; 77%) ovarian carcinomas regardless of histologic subtype. TPD52 subcellular localization was predominantly cytoplasmic, although nuclear localization was also frequently observed in mucinous and clear cell carcinomas. In an independent cohort of stage III serous carcinomas (n = 18), we also directly compared in situ TPD52 expression using immunohistochemistry and TPD52 copy number using interphase FISH analyses. This revealed that TPD52 dosage and TPD52 expression were significantly positively correlated. TPD52 therefore represents a novel molecular marker in ovarian cancer, which is broadly expressed across the different histologic subtypes and whose upregulation frequently reflects increased TPD52 copy number.

High Prevalence of Severe Nausea and Vomiting of Pregnancy and Hyperemesis Gravidarum among Relatives of Affected Individuals

OBJECTIVE The goal of this study was to determine the prevalence of severe nausea and vomiting of pregnancy/hyperemesis gravidarum among relatives of affected individuals. STUDY DESIGN Family history data were obtained on 1224 self-reported cases of hyperemesis gravidarum. Cases completed an online survey administered by the Hyperemesis Education and Research Foundation between 2003 and 2006. RESULTS Approximately 28% of cases reported their mother had severe nausea and vomiting or hyperemesis gravidarum while pregnant with them. Of the 721 sisters with a pregnancy history, 137 (19%) had hyperemesis gravidarum. Among the most severe cases, those requiring total parenteral nutrition or nasogastric feeding tube, the proportion of affected sisters was even higher, 49/198 (25%). Nine percent of cases reported having at least two affected relatives including sister(s), mother, grandmother, daughters, aunt(s), and cousin(s). CONCLUSION There is a high prevalence of severe nausea and vomiting of pregnancy/hyperemesis gravidarum among relatives of hyperemesis gravidarum cases in this study population. Because the incidence of hyperemesis gravidarum is most commonly reported to be 0.5%, this study provides strong but preliminary evidence for a genetic component to extreme nausea and vomiting of pregnancy.

Molecular cytogenetic analysis of consistent abnormalities at 8q12-q22 in breast cancer

Studies using comparative genomic hybridization (CGH) indicate that portions of chromosome arm 8q from 8q12 to 8qter are present at an increased relative copy number in a broad range of solid tumors. In this study we define an ∼ 1 Mb wide region that appears to be frequently abnormal in copy number or structure in breast cancer cell lines and primary tumors. This was accomplished by fluorescence in situ hybridization (FISH) with yeast artificial chromosomes (YACs) mapped to 8q12‐q22. Eleven breast cancer cell lines and ten primary tumors were analyzed. A minimal region of rearrangement was localized to the CEPH‐YAC 928F9 in three breast cancer cell lines with unbalanced translocation breakpoints mapping in this region. Unbalanced translocations also were detected in two primary tumors mapping between CEPH‐YAC clones 890C4 and 936B3, flanking 928F9. An increased copy number in the minimal region was detected in nine cell lines and in multiple primary tumors. This suggests the possibility that a single gene mapping to 928F9 is involved in breast cancer development or progression and may be deregulated by copy number increases in some tumors and by translocation in others. Four expressed sequence tags were mapped to YAC 928F9 and analyzed for rearrangements by Southern analysis and for abnormal expression by Northern analysis. Genes Chromosomes Cancer 22:105–113, 1998.