Anne-Marie Mes-Masson

Mutation of FOXL2 in granulosa-cell tumors of the ovary

BACKGROUND Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Founder BRCA1 and BRCA2 mutations in French Canadian breast and ovarian cancer families

We have identified four mutations in each of the breast cancer-susceptibility genes, BRCA1 and BRCA2, in French Canadian breast cancer and breast/ovarian cancer families from Quebec. To identify founder effects, we examined independently ascertained French Canadian cancer families for the distribution of these eight mutations. Mutations were found in 41 of 97 families. Six of eight mutations were observed at least twice. The BRCA1 C4446T mutation was the most common mutation found, followed by the BRCA2 8765delAG mutation. Together, these mutations were found in 28 of 41 families identified to have a mutation. The odds of detection of any of the four BRCA1 mutations was 18.7x greater if one or more cases of ovarian cancer were also present in the family. The odds of detection of any of the four BRCA2 mutations was 5.3x greater if there were at least five cases of breast cancer in the family. Interestingly, the presence of a breast cancer case <36 years of age was strongly predictive of the presence of any of the eight mutations screened. Carriers of the same mutation, from different families, shared similar haplotypes, indicating that the mutant alleles were likely to be identical by descent for a mutation in the founder population. The identification of common BRCA1 and BRCA2 mutations will facilitate carrier detection in French Canadian breast cancer and breast/ovarian cancer families.

CHARACTERIZATION OF FOUR NOVEL EPITHELIAL OVARIAN CANCER CELL LINES

Dear Editor: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. EOCs originate from either the surface epithelium itself or from the crypts or inclusion cysts on the surface epithelium of the ovary (Whitman et al., 1993). EOCs are designated according to their cell type: serous, mucinous, endometrioid, clear cell, Brenner, or undifferentiated (Serov et al., 1973). They are graded according to degree of differentiation: borderline or low malignant potential represent minimal deviation from their benign counterpart while well differentiated tumors are grade 1, moderately differentiated are grade 2, and poorly differentiated are grade 3 carcinomas. At surgery EOCs are also classified according to stage, grade and amount of residual disease based on criteria established by the International Federation of Gynecology and Obstetrics (FIGO). A common behavior of EOC (in one-third of all cases) is seeding of the peritoneal fluid leading to subsequent implantation over peritoneal surfaces with ascites formation (Morrow and Curtin, 1998). EOCs can arise sporadically and in rarer instances in association with familial cancer syndromes (Easton et al., 1993). It is presently unclear whether the underlying molecular events in the development of all EOCs are similar or whether distinct events are associated with particular subtypes. We have previously described an efficient and rapid technique for the establishment of primary cultures from EOC (Lounis et al., 1994). While these primary cultures are ideally suited to certain studies, the major drawback is the inability to maintain these cultures over extended periods for long-term experiments and for tumor assays. Most human ovarian cancer cell lines described were derived from ascites or pleural effusion (DiSaia et al., 1975; Fogh and Trcmpe, 1975; Sinna et al., 1979; Bast et al,, 1981; Hamilton et al., 1983; Langdon et al., 1988; Golombick et al., 1990; Wong et al., 1990; Golombick and Bezwda, 1991; Yamada et al., 1991; Grunt et al., 1993; Provencher et al., 1993; Hirte et al., i994; Buller et al., 1995; Alama et al., 1996), with only a few cell lines derived from primary ovarian solid tumors (Woods et al., 1979; Langdon et al., 1988; Crickard et al., 1989) or metastases (Buick et al., 1985). In addition, most cell lines originate from tumor material obtained following adjuvant therapy such as chemotherapy or radiation therapy, which could introduce confounding genetic events. Following the derivation of primary EOC cultures (Lounis et al., 1994), we were able to establish four independent spontaneously immortalized epithelial ovarian cancer cell lines from patients who were never exposed to chemotherapy or radiation therapy. The cell lines were derived from ovarian malignant tumors (TOV-21G, TOV-81D, and TOV-112D) and from an ovarian malignant ascites (OV-90). The ovarian tumor cell lines are derived from different histopathologies of ovarian tumors: a clear cell carcinoma (TOV-21G), a papillary serous adenocarcinoma (TOV-81D), an endometrioid carcinoma (TOV-112D), and an adenocarcinoma (OV-90). The clinical information is summarized in Table 1. All patients were diagnosed with advanced disease. Three tumors were grade 3 while one, TOV-81D, was classified as grade 1-2. In Canada, the average age at diagnosis is 54 yr (NCIC, 1999) and therefore TOV112D was derived from a patient with early age of onset ovarian cancer (42 yr at the time of diagnosis). This patient survived less then 3 mo despite an optimal cytoreduetive surgical procedure and platinol-based chemotherapy. TOV-81D is particularly interesting since it was derived from a patient with a familial history of breast and ovarian cancer. In this patient the disease was rather indolent with relapse occurring greater than 5 yr later. Since all the cell lines described here were derived from women of French-Canadian descent they were screened for mutations found to occur in this population (Tonin et al., 1998). Through these studies it was possible to identify a germline BRCA2 mutation, a nucleotide 8765delAG mutation in exon 20, in TOV-81D. All cell lines expressed BRCA1 and BRCA2 as detected by reverse transcriptasepolymerase chain reaction (RT-PCR). Three of the cell lines grow as monolayer cultures on a solid surface (TOV-21G, TOV-81D, and OV-90) while the TOV-112D cell line is loosely adherent and the cells have a tendency to compact and form foci. TOV-81D cells have a very flat morphology and are large surface cells with abundant cytoplasm (Fig. 1D). The epithelial morphology is highly similar to the morphology of cell cultures derived from normal ovarian epithelium (Lounis et al., 1994) and resembles the morphology of a previously described ovarian cell line SKOV-3 (Fogh and Trempe, 1975) which was established from the ascites of a patient with an ovarian adenocarcinoma (Fig. 1E). In contrast, the cellular morphology of the TOV-21G (Fig. 1A), TOVl12D (Fig. 1B), and OV-90 (Fig. 1C) differ from SKOV-3, with ceils being generally smaller and more refractile (Fig. 1). In particular, the OV-90 cells maintain a classic morphology, characterized by ruffled membranes (Lounis et al., 1994), which have been observed in primary cultures from ovarian ascites. The expression of epithelial specific keratins was verified (Table 2). The TOV-21G, OV-90, and TOV-112D cell lines show strong immunofluorescence against the keratin specific CAM5.2 antibody while the TOV-81D cell line reacted weakly with the CAM5.2 antibody. Staining was as reported previously for OV and TOV cultures (Lounis et al., 1994) and was consistent with a staining pattern associated with transformed calls. In addition, we have assessed the expression of antigens MH99 (Mattes et al., 1983, 1987) and B72.3 whose expression correlates with epithelial ovarian carcinomas (Thor et al.~ 1986). All cell lines showed strong positive immunofluorescent staining for both MH99 and B72.3, and this staining appeared stronger than the staining pattern observed for SKOV-3 (Table 2). Staining for CAM5.2, MH99, and B72.1 were negative in the NIH3T3 fibroblast control cell line (Table 2). Finally, the expression of the HER2/NEU oncogene, has been associated with poor prognosis in ovarian cancer (Slamon et al., 1989), was determined. All four ovarian cell lines express neu to some extent

Characterization of the intra-prostatic immune cell infiltration in androgen-deprived prostate cancer patients

INTRODUCTION Our goal was to study the hormonal regulation of immune cell infiltration in prostate cancer patients treated by androgen deprivation therapy (ADT) using an optimized computer-assistance quantification approach. METHODS The relative density of immune cell subtypes (CD3(+), CD8(+), CD20(+), CD56(+), CD68(+) and Foxp3(+)) was analyzed by immunohistochemistry in archived prostate specimens from control patients (radical prostatectomy only, n=40) and ADT-treated patients (ADT prior to radical prostatectomy, n=35) using an image analysis software and a whole-slide scanner. RESULTS ADT-treated patients had significantly increased relative density of CD3(+) (p<0.001) and CD8(+) T lymphocytes (p<0.001) as well as CD68(+) macrophages (p<0.001). Elevated abundance of CD56(+) Natural Killer (NK) cells was associated with a lower risk of prostate cancer progression (p=0.044), while a high density of CD68(+) macrophages was related to an increased risk of biochemical recurrence (p=0.011). CONCLUSIONS Our results demonstrate that the infiltration of specific immune cell subtypes is modulated by ADT. Furthermore our data confirm that NK cells have a protective role against tumor progression while macrophages seem to favor the development of advanced prostate cancer.

NF-κB nuclear localization and its prognostic significance in prostate cancer

To detect the subcellular localization of NF‐κB (p65) in human prostate cancer tissues of different histological grades, and to test whether NF‐κB localization alone, or combined with the histological grade, can be used to predict patient outcome.

Regulation of E2Fs and senescence by PML nuclear bodies

The tumor suppressor PML (promyelocytic leukemia protein) regulates cellular senescence and terminal differentiation, two processes that implicate a permanent exit from the cell cycle. Here, we show that the mechanism by which PML induces a permanent cell cycle exit and activates p53 and senescence involves a recruitment of E2F transcription factors bound to their promoters and the retinoblastoma (Rb) proteins to PML nuclear bodies enriched in heterochromatin proteins and protein phosphatase 1α. Blocking the functions of the Rb protein family or adding back E2Fs to PML-expressing cells can rescue their defects in E2F-dependent gene expression and cell proliferation, inhibiting the senescent phenotype. In benign prostatic hyperplasia, a neoplastic disease that displays features of senescence, PML was found to be up-regulated and forming nuclear bodies. In contrast, PML bodies were rarely visualized in prostate cancers. The newly defined PML/Rb/E2F pathway may help to distinguish benign tumors from cancers, and suggest E2F target genes as potential targets to induce senescence in human tumors.

KIF1A, an axonal transporter of synaptic vesicles, is mutated in hereditary sensory and autonomic neuropathy type 2

Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system.

Expression and Nuclear Localization of ErbB3 in Prostate Cancer

Purpose: The ErbB1 and ErbB2 receptors have been implicated in prostate cancer progression, but less is known about the role and biology of other ErbB receptor family members in prostate cancer. The aim of this study was to analyze the expression and localization of ErbB3 in prostate tissues and prostate cancer cell lines. Experimental Design: Immunohistochemistry of ErbB3 was done on prostate cancer tissue sections from 143 patients and on a tissue microarray containing 390 cores of radical prostatectomy-derived specimens representing normal, prostatic intraepithelial neoplasia, and malignant tissues from 81 patients. ErbB3 subcellular localization was studied by Western blot analysis in LNCaP, 22Rv1, PC-3, and DU145 prostate cancer cell lines. Results: Immunohistochemistry analysis of prostate cancer tissues revealed that >90% of prostate cancer tissues displayed cytoplasmic ErbB3 staining. Minimal ErbB3 nuclear staining was observed in normal prostate tissues and benign prostatic hyperplasia tissues; in contrast, ErbB3 was frequently localized in the nucleus of cancerous tissues. This nuclear localization was more frequent (P < 0.001) in hormone-refractory tissues (17 of 17, 100%) compared with hormone-sensitive samples (37 of 92, 40.2%). Additionally, in the tissue microarray, increased nuclear ErbB3 was associated with increasing Gleason grade. Interestingly, Western blot analysis of cytoplasmic and nuclear subcellular fractions showed that ErbB3 nuclear localization was more prevalent in hormone-sensitive prostate cancer cell lines (LNCaP and 22Rv1) compared with hormone-insensitive cell lines (PC-3 and DU145). Conclusions: ErbB3 nuclear localization discriminates normal from malignant prostate tissues and between tumors from hormone-sensitive versus hormone-refractory prostate cancer. ErbB3 nuclear staining seems to be associated with risk of disease progression. The high frequency of ErbB3 nuclear localization in hormone-refractory tissues indicates that ErbB3 warrants further study to understand its association with prostate cancer disease progression.

Overexpression of her-2/neu in human prostate cancer and benign hyperplasia

Overexpression of the neu oncoprotein has been described in several tumor models including breast and prostate cancer. Overexpression of neu has been reported to have prognostic significance in certain tumors but controversy continues regarding the role and frequency of neu overexpression in prostatic cancer. The objectives of the study were twofold. First, to characterize neu expression in prostate cancer in comparison to benign prostatic hyperplasia. Second, to determine whether neu expression correlates with Gleason grade in prostate cancer. Thirty-nine prostate cancers obtained from radical prostatectomy specimens and 10 benign prostatic hyperplasia specimens were included in the study. Specimens were formalin fixed and paraffin-embodied. neu expression was studied by immunohistochemical staining using a monoclonal neu specific AB-3 antibody. All 39 specimens (100%) of prostate cancer showed positive immunostaining of variable degree while 2 (20%) benign prostatic hyperplasia specimens showed positive staining. Thus, neu oncogene is overexpressed in localized prostate cancer compared to benign prostatic hyperplasia. The degree of neu immunostaining did not correlate with Gleason grade and there appeared to be a tendency towards an inverse relationship. The prognostic significance of the varying overexpression is unknown.

Subtype-specific mutation of PPP2R1A in endometrial and ovarian carcinomas

PPP2R1A mutations have recently been described in 3/42 (7%) of clear cell carcinomas of the ovary. PPP2R1A encodes the α‐isoform of the scaffolding subunit of the serine/threonine protein phosphatase 2A (PP2A) holoenzyme. This putative tumour suppressor complex is involved in growth and survival pathways. Through targeted sequencing of PPP2R1A, we identified somatic missense mutations in 40.8% (20/49) of high‐grade serous endometrial tumours, and 5.0% (3/60) of endometrial endometrioid carcinomas. Mutations were also identified in ovarian tumours at lower frequencies: 12.2% (5/41) of endometrioid and 4.1% (2/49) of clear cell carcinomas. No mutations were found in 50 high‐grade and 12 low‐grade serous carcinomas. Amino acid residues affected by these mutations are highly conserved across species and are involved in direct interactions with regulatory B‐subunits of the PP2A holoenzyme. PPP2R1A mutations in endometrial high‐grade serous carcinomas are a frequent and potentially targetable feature of this disease. The finding of frequent PPP2R1A mutations in high‐grade serous carcinoma of the endometrium but not in high‐grade serous carcinoma of the ovary provides clear genetic evidence that these are distinct diseases. Copyright