Growth Response and Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells in the Presence of Novel Multiple Myeloma Drug Melflufen.

Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide-drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome.

The Peptide-Drug Conjugate Melflufen Modulates the Unfolded Protein Response of Multiple Myeloma and Amyloidogenic Plasma Cells and Induces Cell Death.

Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by clonal plasma cell secretion of misfolded light chains that assemble as toxic amyloid fibrils, depositing in vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell-directed therapeutics are expected to reduce production of toxic light chain by eliminating amyloidogenic cells in bone marrow, thereby diminishing amyloid fibril deposition and providing the potential for organ recovery. Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly releases alkylating agents inside tumor cells. Melflufen is highly lipophilic, permitting rapid uptake by cells, where it is enzymatically hydrolyzed by aminopeptidases, resulting in intracellular accumulation of the alkylating agents, including melphalan. Previous data demonstrating sensitivity of myeloma cells to melflufen suggest that the drug might be useful in AL amyloidosis. We describe the effects of melflufen on amyloidogenic plasma cells in vitro and ex vivo, demonstrating enhanced cytotoxic effects in comparison to melphalan, as well as novel mechanisms of action through the unfolded protein response (UPR) pathway. These findings provide evidence that melflufen-mediated cytotoxicity extends to amyloidogenic plasma cells, and support the rationale for the evaluation of melflufen in patients with AL amyloidosis.

Aminopeptidase Expression in Multiple Myeloma Associates with Disease Progression and Sensitivity to Melflufen.

Multiple myeloma (MM) is characterized by extensive immunoglobulin production leading to an excessive load on protein homeostasis in tumor cells. Aminopeptidases contribute to proteolysis by catalyzing the hydrolysis of amino acids from proteins or peptides and function downstream of the ubiquitin-proteasome pathway. Notably, aminopeptidases can be utilized in the delivery of antibody and peptide-conjugated drugs, such as melflufen, currently in clinical trials. We analyzed the expression of 39 aminopeptidase genes in MM samples from 122 patients treated at Finnish cancer centers and 892 patients from the CoMMpass database. Based on ranked abundance, LAP3, ERAP2, METAP2, TTP2, and DPP7 were highly expressed in MM. ERAP2, XPNPEP1, DPP3, RNPEP, and CTSV were differentially expressed between relapsed/refractory and newly diagnosed MM samples (p < 0.05). Sensitivity to melflufen was detected ex vivo in 11/15 MM patient samples, and high sensitivity was observed, especially in relapsed/refractory samples. Survival analysis revealed that high expression of XPNPEP1, RNPEP, DPP3, and BLMH (p < 0.05) was associated with shorter overall survival. Hydrolysis analysis demonstrated that melflufen is a substrate for aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP. The sensitivity of MM cell lines to melflufen was reduced by aminopeptidase inhibitors. These results indicate critical roles of aminopeptidases in disease progression and the activity of melflufen in MM.

Prognostic significance of esterase gene expression in multiple myeloma.

BACKGROUND: Esterase enzymes differ in substrate specificity and biological function and may display dysregulated expression in cancer. This study evaluated the biological significance of esterase expression in multiple myeloma (MM). METHODS: For gene expression profiling and evaluation of genomic variants in the Institute for Molecular Medicine Finland (FIMM) cohort, bone marrow aspirates were obtained from patients with newly diagnosed MM (NDMM) or relapsed/refractory MM (RRMM). CD138+ plasma cells were enriched and used for RNA sequencing and analysis, and to evaluate genomic variation. The Multiple Myeloma Research Foundation (MMRF) Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile (CoMMpass) dataset was used for validation of the findings from FIMM. RESULTS: MM patients (NDMM, n = 56; RRMM, n = 78) provided 171 bone marrow aspirates (NDMM, n = 56; RRMM, n = 115). Specific esterases exhibited relatively high or low expression in MM, and expression of specific esterases (UCHL5, SIAE, ESD, PAFAH1B3, PNPLA4 and PON1) was significantly altered on progression from NDMM to RRMM. High expression of OVCA2, PAFAH1B3, SIAE and USP4, and low expression of PCED1B, were identified as poor prognostic markers (P < 0.05). The MMRF CoMMpass dataset provided validation that higher expression of PAFAH1B3 and SIAE, and lower expression of PCED1B, were associated with poor prognosis. CONCLUSIONS: Esterase gene expression levels change as patients progress from NDMM to RRMM. High expression of OVCA2, PAFAH1B3, USP4 and SIAE, and low expression of PCED1B, are poor prognostic markers in MM, suggesting a role for these esterases in myeloma biology.

Melflufen, a peptide-conjugated alkylator, is an efficient anti-neo-plastic drug in breast cancer cell lines.

Melphalan flufenamide (hereinafter referred to as "melflufen") is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins Æ´H2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.

Prognostic but not predictive role of platelet-derived growth factor receptors in patients with recurrent glioblastoma.

Platelet-derived growth factor receptor (PDGFR) signaling has been implicated in the pathogenesis of glioblastomas and represents a target for the tyrosine kinase inhibitor imatinib. To examine the prognostic or predictive role of PDGFRs in recurrent glioblastomas, expression was examined in tumor samples of 101 patients of CSTI571BDE40, a randomized trial comparing hydroxyurea monotherapy and a combination of hydroxyurea and imatinib. Furthermore, PDGFRα phosphorylation was investigated using in situ proximity ligation assay. PDGFRα protein was expressed in 33% of tumors and was associated with male sex, young age, presence of R132H mutated isocitrate dehydrogenase 1 protein and short median survival (142 vs. 187 days, p = 0.028). Tumor PDGFRα phosphorylation was also associated with short survival (p = 0.030). The subset of patients with PDGFRα positive glioblastoma did not have longer survival on treatment with hydroxyurea and imatinib compared with hydroxyurea monotherapy. In conclusion, both PDGFRα protein expression and phosphorylation status had a prognostic role in recurrent glioblastomas but did not define a group that showed benefit from the combination therapy consisting of hydroxyurea and imatinib.

The tyrosine kinase c-Abl promotes proliferation and is expressed in atypical teratoid and malignant rhabdoid tumors.

BACKGROUND: Atypical teratoid/rhaboid tumors (AT/RTs) and extracranial malignant rhabdoid tumors are highly malignant neoplasms with a dismal prognosis. These tumors predominantly affect infants and targeted, adjuvant treatment approaches would be highly desirable. METHODS: In the current study, the authors investigated the expression and functional role of tyrosine kinases in 2 malignant rhabdoid tumor cell lines (A204 and G401) and in a series of 5 AT/RTs and 18 malignant rhabdoid tumors (13 rhabdoid tumors of the kidney and 5 extrarenal rhabdoid tumors). RESULTS: Both cell lines consistently expressed the tyrosine kinase c-Abl, which promoted proliferation as assessed by small interfering RNA knockdown. Blockage of c-Abl using the tyrosine kinase inhibitor imatinib resulted in reduced cellular growth in both cell lines. Furthermore, c-Abl was expressed in all rhabdoid tumors, whereas expression of platelet-derived growth factor receptor subtypes alpha and beta was infrequent and c-Kit expression was absent. CONCLUSIONS: The current data pointed toward a role for c-Abl in the biology of malignant rhabdoid tumors and provided a rationale for the investigation of tyrosine kinase inhibitors that target c-Abl for the treatment of these aggressive tumors.

Amplification and overexpression of KIT, PDGFRA, and VEGFR2 in medulloblastomas and primitive neuroectodermal tumors.

Medulloblastomas (MB) and primitive neuroectodermal tumors (PNET) are the most common malignant brain tumors in children. These two tumor types are histologically similar, but have different genetic backgrounds and clinical outcomes. Other brain tumors, such as gliomas, frequently have coamplification and overexpression of receptor tyrosine kinases KIT, platelet-derived growth factor receptor alpha (PDGFRA), and vascular endothelial growth factor receptor 2 (VEGFR2). We investigated protein expression and gene copy numbers of KIT, PDGFRA, and VEGFR2 in 41 MB and 11 PNET samples by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). KIT and PDGFRA expression was detected in both MBs and PNETs, whereas VEGFR2 expression was weak in these tumors. KIT, PDGFRA, and VEGFR2 amplifications were all present in 4% of MBs/PNETs, and KIT amplification was associated with concurrent PDGFRA and VEGFR2 amplifications (P

Mutation and copy number analysis of LNX1 and Numbl in nervous system tumors.

Alterations at chromosome locus 4q12 are frequently found in gliomas; this locus contains the receptor tyrosine kinase--encoding genes KIT, PDGFRA, and KDR (alias VEGFR2). Notable among the genes at this locus is LNX1, the ligand of Numb protein X. LNX1 encodes a PDZ domain containing protein, which interacts with the cell fate determinant Numbl, a Numb homolog-like gene involved in the maintenance of neural progenitor cells during embryonic neurogenesis. We performed a mutation analysis for LNX1 and Numbl genes. In addition, gene copy numbers of LNX1, Numbl, and KIT in human nervous system tumors were analyzed by chromogenic in situ hybridization. Tissue samples from 90 patients were screened for LNX1 and Numbl mutations, and tissue sections from 56 samples were analyzed for gene amplification status. Our analysis revealed missense mutations in LNX1 exons 3 and 5 and a single-nucleotide polymorphism in Numbl exon 6. In addition, polyglutamine repeat polymorphism was found in Numbl exon 10. Chromogenic in situ hybridization showed gene amplification of LNX1 in 10%, Numbl in 5%, and KIT in 6% of nervous system tumors. Both gene sequence alterations and amplifications of LNX1 and Numbl are present in a subset of human gliomas, and the role of these genes in neurogenesis suggests that they may contribute to development of glial tumors.

KIT overexpression induces proliferation in astrocytes in an imatinib-responsive manner and associates with proliferation index in gliomas.

Activating gene mutations, gene amplifications and overexpressed proteins may be useful as targets for novel therapies. Alterations at chromosome locus 4q12 are associated with gliomas and the region harbors the receptor tyrosine kinase gene KIT, which is frequently amplified in gliomas, and also overexpressed in a subset of gliomas. KIT and its ligand stem cell factor are widely expressed in embryonic and adult mouse brain, and they play a role in many signal transduction pathways involved in cellular proliferation, differentiation and cancer cell metastasis. However, the function of KIT in gliomagenesis or disease progression remains unresolved as well as its role in neural and brain tumor development. In this study, we utilized lentivirus-mediated gene transfer to deliver the KIT gene into mouse astrocytes. The growth properties of KIT overexpressing cells were analyzed using several in vitro functional assays. The effect of receptor tyrosine kinase inhibitor imatinib on astrocyte growth was also investigated. Our results indicate that overexpression of KIT in mouse astrocytes promotes cell proliferation, and the increased proliferation is partly inhibited by imatinib treatment. Furthermore, KIT overexpression induces phenotypic changes in the cells suggesting that KIT may play a role in astrocyte growth regulation.

Copy number alterations of the polycomb gene BMI1 in gliomas.

Gliomas are heterogeneous tumours that grow in an uninhibited fashion, and these brain tumour cells share numerous characteristics with neural stem cells. The BMI1 gene encodes a component of the polycomb protein complex regulating epigenetically gene activity via histone modification. It functions for instance during the development of the central nervous system and maturation of neural cells. BMI-1 protein expression is deregulated in several forms of cancer and gene amplification has been identified in mantle cell lymphomas. Since BMI1 is located at chromosome 10p, a region implicated frequently in brain tumourigenesis, we investigated the genetic status and the corresponding expression patterns of BMI1 in a series of 100 low- and high-grade primary and recurrent gliomas. Chromogenic in situ hybridisation (CISH) with probes directed against BMI1 at 10p13 and the centromere of chromosome 10 was used in the analyses. Of all gliomas, 59% demonstrated aberrant copy numbers of BMI1. Deletions of the BMI1 locus were found in most types of tumours, and in a univariate survival analysis these cases had poor prognosis. Increased copy numbers of the BMI1 locus (3-5 copies) were found in all histological types, especially in high-grade astrocytomas. No difference in prognosis between cases with normal copy numbers and cases with increased copy numbers could be observed. This data suggests that BMI1 gene is aberrant at the chromosomal level in a subset of gliomas, and possibly contributes to brain tumour pathogenesis.

Platelet-derived growth factor receptor expression and amplification in choroid plexus carcinomas.

Platelet-derived growth factor (PDGF) receptor signaling has been implicated in the development of glial tumors, but not yet been examined in choroid plexus carcinomas, pediatric tumors with dismal prognosis for which novel treatment options would be desirable. Therefore, protein expression of PDGF receptors alpha and beta as well as amplification status of the respective genes, PDGFRA and PDGFRB, were examined in a series of 22 patients harboring choroid plexus carcinoma using immunohistochemistry and chromogenic in situ hybridization (CISH). The majority of choroid plexus carcinomas expressed PDGF receptors with 6 cases (27%) displaying high staining scores for PDGF receptor alpha and 13 cases (59%) showing high staining scores for PDGF receptor beta. Correspondingly, copy-number gains of PDGFRA were observed in 8 cases out of 12 cases available for CISH and 1 case displayed amplification (six or more signals per nucleus). The proportion of choroid plexus carcinomas with amplification of PDGFRB was even higher (5/12 cases). PDGFRB amplification status and PDGF receptor beta protein expression scores were significantly correlated (P=0.01, Spearman). Expression status of PDGF receptor alpha or PDGF receptor beta was not significantly associated with progression-free survival. To conclude, expression and amplification of PDGF receptors, particularly PDGF receptor beta, are frequent in choroid plexus carcinomas, providing a first rationale for the development of treatments targeting PDGF receptor signaling in these rare malignant pediatric tumors.

Patterns of PIK3CA alterations in familial colorectal and endometrial carcinoma.

While the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is known to be activated in multiple sporadic cancers, the role of this pathway in familial tumors is mostly unknown. We searched for alterations in the catalytic domain of PI3K (PIK3CA), PTEN and KRAS, all of which may contribute to PI3K/AKT pathway activation, in a total of 160-familial colorectal (CRC) and endometrial carcinomas (EC), stratified by the presence vs. absence of germline mutations in DNA mismatch repair (MMR) genes. PIK3CA alterations (consisting of point mutations or low-level amplification, which were mutually exclusive with 1 exception) occurred in 10/70 (14%) of CRCs and 19/90 (21%) of ECs. Within ECs, amplification was significantly associated with the subgroup lacking germline mutations in MMR genes (familial site-specific endometrial cancer) (p = 0.015). Decreased or lost PTEN expression was characteristic of endometrial tumourigenesis (51/81, 63%, in EC compared with 24/62, 39%, in CRC, p = 0.004) and KRAS mutations of colorectal tumourigenesis (19/70, 27% in CRC vs. 9/89, 10%, in EC, p = 0.006) regardless of the MMR gene mutation status. PIK3CA alterations frequently coexisted with PTEN or KRAS changes. Combined with published studies on sporadic tumors, our data broaden the understanding of the role for PI3K pathway genes in human tumorigenesis.

Amplification of KIT, PDGFRA, VEGFR2, and EGFR in gliomas.

Receptor tyrosine kinase aberrations are implicated in the genesis of gliomas. We investigated expression and amplification of KIT, PDGFRA, VEGFR2, and EGFR in 87 gliomas consisting of astrocytomas, anaplastic astrocytomas, oligodendrogliomas, or oligoastrocytomas in tumor samples collected at the time of the diagnosis and in samples of the same tumors at tumor recurrence. Gene amplifications were investigated using either chromogenic in situ hybridization or fluorescence in situ hybridization, and protein expression using immunohistochemistry. In samples collected at glioma diagnosis, KIT and PDGFRA amplifications were more frequent in anaplastic astrocytomas than in astrocytomas, oligodendrogliomas, and oligoastrocytomas [28% versus 5% (P = 0.012) and 33% versus 2% (P = 0.0008), respectively]. VEGFR2 amplifications occurred in 6% to 17% of the gliomas at diagnosis, and EGFR amplifications in 0% to 12%. Amplified KIT was more frequently present in recurrent gliomas than in newly diagnosed gliomas (P = 0.0066). KIT amplification was associated with KIT protein expression and with presence of PDGFRA and EGFR amplifications both at the time of the first glioma diagnosis and at tumor recurrence, and with VEGFR2 amplification at tumor recurrence. Three (4%) primary gliomas and 10 (14%) recurrent gliomas that were evaluable for coamplification of KIT, PDGFRA, and VEGFR2 showed amplification of at least two of these genes; the amplicon contained amplified KIT in all 13 cases. In conclusion, besides glioblastoma, amplified KIT, PDGFRA, and VEGFR may also occur in lower-grade gliomas and in their recurrent tumors. It is currently not known whether specific tyrosine kinase inhibitors are effective in the treatment of such gliomas.

Molecular genetic analysis of the REST/NRSF gene in nervous system tumors.

The gene for RE1-silencing transcription factor (REST) alias neuron-restrictive silencer factor NRSF, acts as a transcriptional repressor in the neuronal differentiation pathways in non-neuronal cells, and plays an important role in neuronal development. Inactivating mutations or overexpression of REST have previously been reported in various types of cancer, but no data is available for the role of REST alterations in gliomas. REST gene was screened for mutations in 161 nervous system tumors consisting of astrocytomas, glioblastomas, oligodendrogliomas, oligoastrocytomas, medulloblastomas, meningiomas and schwannomas. REST exons 1-3 were analyzed using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. The gene copy numbers of REST were investigated by chromogenic (CISH) and fluorescence in situ hybridization (FISH) techniques. Non-synonymous SNPs (P797L, P815S) were found in eight different brain tumor samples. No truncating or activating novel mutations of REST were discovered. Since REST is located at 4q12, a chromosome region implicated in brain tumorigenesis, we conducted gene copy number analyses in medulloblastomas and gliomas. The majority of gliomas (67%) demonstrated low-level amplifications of REST, and only one oligodendroglioma showed high-level amplification of the gene. In medulloblastomas, 38% of samples were determined as aneuploidic, no high-level amplifications were found. Our data suggests that REST is neither activated nor inactivated via mutations in gliomas, while high-level amplification may rarely occur.

Platelet-derived growth factor receptor family mutations in gastrointestinal stromal tumours.

OBJECTIVE: Activating mutations of either KIT or platelet-derived growth factor receptor alpha (PDGFRA) genes are present in the majority of gastrointestinal stromal tumours (GISTs). The type of gene mutation is associated with the aggressiveness of the disease, response to imatinib therapy, and the tumour site in the gastrointestinal tract. However, a subgroup of GISTs does not harbour these mutations. MATERIAL AND METHODS: Thirty-three GISTs were studied for mutations in exons encoding the juxtamembrane and the activation loop domains of KIT, PDGFRA, PDGFRB, CSF1R, and FLT3 genes using denaturing high-performance liquid chromatography and gene sequencing. RESULTS: Twenty-two (67%) GISTs had mutation in KIT and 3 (9%) in PDGFRA. The three PDGFRA mutations were all detected in exon 18 of the gene. Three of the 5 GISTs that had weak to moderate KIT expression had a PDGFRA mutation as compared to none of the 26 cases with strong KIT immunopositivity (p=0.022). No mutations were found in PDGFRB, CSF1R or FLT3 in the 8 cases that did not harbour KIT or PDGFRA mutations. CONCLUSIONS: KIT and PDGFRA are the most commonly mutated type III receptor tyrosine kinase genes in GIST. GISTs with PDGFRA mutations often have reduced expression of the KIT protein in immunohistochemistry, suggesting that immunohistochemistry may be potentially useful in identification of such GISTs.

Gastrointestinal stromal tumors with KIT exon 11 deletions are associated with poor prognosis.

BACKGROUND & AIMS: Gain-of-function mutations in the KIT receptor tyrosine kinase gene and rare mutations in the platelet-derived growth factor receptor alpha (PDGFRA) gene are important events in gastrointestinal stromal tumor (GIST) development. Different mutations are reportedly associated with distinctive phenotypes and possibly clinical behavior. We investigated the correlation among mutation type, phenotype, and clinical course in a preimatinib, population-based series of GIST with long-term follow-up. METHODS: Genomic DNA from 177 GIST patients was analyzed for KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18 mutations using denaturating high-performance liquid chromatography and bidirectional sequencing. RESULTS: KIT exon 11 mutations were detected in 101 of 177 GIST (61 deletions, 23 missense mutations, and 17 duplications); wild-type (WT) KIT and PDGFRA were detected in 63; KIT exon 9 and exon 17 mutations in 6 and 1, respectively; and PDGFRA exons 12 and 18 mutations in 3 each. GIST >5 cm vs GIST

Allelic imbalance of HER2 variant in sporadic breast and ovarian cancer.

Both breast and ovarian cancers are associated with HER2 receptor activation, which usually results from receptor overexpression and/or gene amplification. The HER-2 gene harbors a polymorphism at codon 655 (GTC/valine to ATC/isoleucine) in the transmembrane domain region, which has been associated with an elevated risk of breast cancer. The objective of this study was to determine whether the polymorphism is under a selection pressure during breast and ovarian carcinogenesis. The Ile/Val genotype was present in 41% (9/22) of the normal DNA of breast cancer patients. An allelic imbalance in the tumor tissue was found in three breast tumors, with overrepresentation of the Val allele. HER-2 was amplified and overexpressed in these tumors. Half of the eight ovarian tumor patients carried heterozygous Ile/Val genotypes. In contrast to breast tumors, all these ovarian cancer specimens showed the presence of the Ile allele. In our selected set of tumors, the Val allele was overrepresented in the subset of HER2-positive breast cancers and the Ile allele in serous ovarian cancer. Further analyses of tumors with known gene amplifications and overexpression may reveal novel associations between germline polymorphisms and development of sporadic tumors.

Gene amplification, mutation, and protein expression of EGFR and mutations of ERBB2 in serous ovarian carcinoma.

EGFR and erbB-2 are targets for specific cancer therapy. The purpose of this study was to examine the frequency and clinicopathological correlations of gene amplification, protein expression, and mutations of EGFR and ERBB2 in serous carcinoma, the most common and aggressive type of ovarian cancer. Tissue microarray constructed of 398 carcinomas was examined by chromogenic in situ hybridization (CISH) and by immunohistochemistry. Cases with amplification of EGFR by CISH were further analyzed by fluorescence in situ hybridization. One hundred ninety-eight samples were analyzed for mutations in exons 18, 19, or 21 of EGFR and in exon 20 of ERBB2 using denaturating high-performance liquid chromatography and direct sequencing. Amplification of EGFR was present in 12% (41/333), low-level gain in 43% (144/333), and protein overexpression in 17% (66/379) of the tumors. Both increased copy number and overexpression of EGFR were associated with high tumor grade, greater patient age, large residual tumor size, high proliferation index, aberrant p53, and poor patient outcome. Furthermore, increased copy number of EGFR was associated with increased copy number of ERBB2. No mutations were identified in EGFR, whereas one tumor had an insertion mutation in exon 20 of ERBB2. Both amplification and protein overexpression of EGFR occur in serous ovarian carcinoma, but EGFR copy number has a stronger prognostic value. This makes EGFR amplification a potentially useful criterion for selecting patients in clinical trials testing the effect of EGFR inhibitors in serous ovarian carcinoma.