FOXL2 Mutation Status Research Articles

Detection of FOXL2 C134W Mutation Status by a Novel BaseScope In Situ Hybridization Assay is Highly Sensitive and Specific for Adult Granulosa Cell Tumors

Adult granulosa cell tumors (AGCTs) are a molecularly distinct group of malignant ovarian sex cord-stromal tumors (SCSTs) characterized by a nearly ubiquitous c.402C>G/p.C134W mutation in FOXL2 (hereafter referred to as “C134W”). In some cases, AGCT exhibits marked morphologic overlap with other SCSTs and has an identical immunophenotype, and molecular testing may be necessary to help confirm the diagnosis. However, molecular testing is time consuming, relatively expensive, and unavailable in many pathology laboratories. We describe the development and validation of an in situ hybridization (ISH) custom BaseScope assay for the detection of the FOXL2 C134W mutation. We evaluated 106 ovarian SCSTs, including 78 AGCTs, 9 juvenile granulosa cell tumors, 18 fibromas (cellular and conventional), and 1 SCST, not otherwise specified, as well as 53 epithelial ovarian tumors (42 endometrioid carcinomas and 11 carcinosarcomas) and 1 STK11 adnexal tumor for the presence or absence of FOXL2 wild-type and FOXL2 C134W RNA expression via BaseScope-ISH. Fifty-one tumors had previously undergone DNA sequencing of the FOXL2 gene. Across the entire cohort, the FOXL2 C134W probe staining was positive in 77 of 78 (98.7%) AGCTs. Two of 81 (2.5%) non-AGCTs also showed positive staining, both of which were epithelial ovarian tumors. The assay worked in tissue from blocks >20 years old. There was 100% concordance between the FOXL2 sequencing and BaseScope-ISH results. Overall, assessment of FOXL2 mutation status by custom BaseScope-ISH demonstrated 98.7% sensitivity and 97.5% specificity for the diagnosis of AGCT. BaseScope-ISH for FOXL2 C134W represents a reasonable alternative to sequencing, is quicker and less expensive, and is more easily incorporated than molecular testing into many pathology laboratories. It also has the advantage of requiring less tissue, and the neoplastic cells can be directly visualized on stained sections.

Large Ovarian Follicle Cyst: Benign Mimic of Cystic Adult Granulosa Cell Tumor.

While most ovarian follicle cysts are <8 cm in greatest dimension, much larger follicle cysts (up to 18.5 cm) have been reported. To our knowledge, the FOXL2 mutation status of such cases has not been documented in the literature. Here, we report the features of a 14 cm ovarian cyst with no FOXL2 mutation detected by targeted next-generation sequencing. While adult granulosa cell tumor was the chief entity in our differential diagnosis, the absence of convincing nuclear grooves, lack of architectural variability, presence of a theca layer, and absence of FOXL2 mutation were consistent with a diagnosis of ovarian follicle cyst.

Juvenile Granulosa Cell Tumors of the Ovary

To explore the clinical and pathologic features of ovarian juvenile granulosa cell tumors (JGCTs). Clinical data, histopathologic observations, immunohistochemical results, FOXL2 mutation status, and follow-up information of 7 JGCT cases were studied. The patients most commonly presented with abdominal distension and pain (5 cases), followed by precocious puberty (1 case) and a pelvic mass (1 case). Six patients had stage I disease, and 1 had stage IV disease. The microscopic examinations typically showed lobular growth punctuated by variably sized and shaped follicles. Rare features included a reticular-cystic appearance mimicking a yolk sac tumor (2 cases), a lobular appearance similar to a sclerosing stromal tumor (1 case), strands and cords (1 case), pseudopapillary appearance (2 cases), spindle cell appearance (1 case), microcystic appearance (1 case), hobnail cells (1 case), and rhabdomyoid cells (1 case). No FOXL2 mutation was encountered. After a median follow-up of 53 months, only 1 patient with a strongly diffuse TP53-positive tumor died of the disease, and 2 successfully had babies. JGCT is a rare neoplasm with a wide morphologic spectrum and is easily confused with other tumors. Familiarity with the characteristics, rare atypical appearances, and immunohistochemical results may aid in obtaining a correct diagnosis.

DICER1 and FOXL2 Mutation Status Correlates With Clinicopathologic Features in Ovarian Sertoli-Leydig Cell Tumors.

Sertoli-Leydig cell tumors (SLCTs) are rare ovarian sex cord-stromal neoplasms. The only known recurrent genetic abnormality is DICER1 mutation, with rare mutations reported in FOXL2. We set out to establish a molecular classifier using DICER1 and FOXL2 somatic mutation status and clinicopathologic features in 42 SLCTs. Five tumors (12%) were well differentiated, 31 (74%) moderately differentiated, and 6 (14%) poorly differentiated. Eight (19%) had heterologous elements, and 2 (5%) showed retiform differentiation; all 10 were moderately differentiated. DICER1 RNase IIIb domain mutations were identified in 18/41 (44%; 17 moderately, 1 poorly differentiated), including all cases with retiform or heterologous elements. FOXL2 c.402C>G (p.C134W) mutation was identified in 8/42 (19%) tumors (5 moderately, 3 poorly differentiated). DICER1 and FOXL2 mutations were mutually exclusive. Median age for the cohort was 47 years (range, 15 to 90 y). Patients with DICER1 mutations were younger (median, 24.5 y; range, 15 to 62 y) than patients with FOXL2 mutation (median, 79.5 y; range, 51 to 90 y) (P<0.0001). Nine of 10 tumors with retiform or heterologous elements occurred in premenopausal patients (median, 26.5 y; range, 15 to 57 y). Patients with tumors that were wild type for DICER1 and FOXL2 (15/42, 37%) had an intermediate age (median, 51 y; range, 17 to 74 y). All tumors were FOXL2 positive by immunohistochemistry. Patients with FOXL2 mutation trended toward presenting more often with abnormal bleeding (P=0.13); DICER1-mutant patients trended toward having more androgenic symptoms (P=0.22). Our data suggest at least 3 molecular subtypes of SLCT with distinct clinicopathologic features: DICER1 mutant (younger, more androgenic symptoms, moderately/poorly differentiated, retiform or heterologous elements), FOXL2 mutant (postmenopausal, abnormal bleeding, moderately/poorly differentiated, no retiform or heterologous elements), and DICER1/FOXL2 wild type (intermediate age, no retiform or heterologous elements, including all well-differentiated tumors).

Abstract B46: DICER1 and FOXL2 mutations correlate with clinicopathologic features of ovarian Sertoli-Leydig cell tumors

Abstract Sertoli-Leydig cell tumors are rare mixed ovarian sex cord-stromal neoplasms consisting of a mixture of Sertoli cells, Leydig cells, and occasionally heterologous elements or retiform differentiation. The only known recurrent genetic abnormality is mutations in DICER1, with rare mutations reported in FOXL2. We set out to determine the DICER1 and FOXL2 mutation status in a series of 51 SLCTs with the goal of establishing a molecular classifier using mutation status and clinicopathologic features. Five tumors were well differentiated, 39 moderately differentiated, and 7 poorly differentiated. Nine tumors had heterologous elements (8 epithelial, one mesenchymal), and four showed retiform differentiation; all of these were moderately differentiated. DICER1 mutations were identified in 16/45 of successfully genotyped tumors (36%, 15 moderately differentiated, 1 poorly differentiated), including 7 with heterologous elements and 3 of 4 with retiform differentiation. FOXL2 c.402C&amp;gt;G mutation was identified in 10/51 tumors (20%, 8 moderately differentiated, 2 poorly differentiated). DICER1 and FOXL2 mutations were mutually exclusive. Median age for the entire cohort was 44 years (range 15-90). Patients with DICER1 mutations were younger (median 23.5 years, range 15-62) than patients with neither DICER1 nor FOXL2 mutation (median 41.5 years, range 16-74). Tumors with FOXL2 mutation occurred exclusively in postmenopausal patients (median 78 years, range 54-90), whereas 11/13 tumors with either heterologous elements (median 26 years, range 16-62) or retiform differentiation (median 38 years, range 18-57) occurred in premenopausal patients. Our data suggest the existence of at least three molecular subtypes of SLCT: DICER1 mutant (younger, moderately/poorly differentiated, including heterologous elements or retiform differentiation), FOXL2 mutant (postmenopausal, moderately/poorly differentiated, no heterologous elements or retiform differentiation), and DICER1/FOXL2 wild-type (no specific age, no heterologous elements, including all well-differentiated tumors). These findings suggest that well-differentiated and moderately/poorly differentiated SLCTs may be different histomolecular entities with different etiologies. Citation Format: Anthony N. Karnezis, Yemin Wang, Jamie Magrill, Jacqueline Keul, Stefan Kommoss, Basile Tessier-Cloutier, Lily Proctor, Dietmar Schmidt, C Blake Gilks, David G. Huntsman, Friedrich Kommoss. DICER1 and FOXL2 mutations correlate with clinicopathologic features of ovarian Sertoli-Leydig cell tumors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B46.

FOXL2 Mutation Analysis of Ovarian Sex Cord-Stromal Tumors: Genotype-Phenotype Correlation With Diagnostic Considerations.

Correlation of FOXL2 mutation status with morphologic features and reticulin staining patterns was performed in a comprehensive single-institutional cohort of ovarian sex cord-stromal tumors. Fifty-one cases were included, 35 of which were morphologically diagnosed as adult granulosa cell tumor, 4 as Sertoli-Leydig cell tumor, 11 as fibroma/fibrothecoma and 1 as a thecoma. Of the adult granulosa cell tumors, 31 (88.6%) harbored FOXL2 mutation. Abundant pale cytoplasm was seen in 51.6% (16/31) of FOXL2 mutated tumors, compared with 6.7% (1/15) among FOXL2 wild type tumors (P=0.003). Nearly half of FOXL2 negative tumors showed individual pericellular reticulin staining pattern, while none of the FOXL2 positive cases demonstrated this feature (P=0.0001). Nested reticulin pattern was observed in 67.7% of FOXL2 positive tumors, compared with 20% of FOXL2 negative cases (P=0.004). Indeterminate reticulin staining pattern was seen in nearly one third of cases in both groups. Nested reticulin pattern was 87.5% specific and 67.7% sensitive for FOXL2 mutation, while individual reticulin pattern was 100% specific for absence of FOXL2 mutation. No statistical significance was observed between the 2 groups in tumor size, mitotic activity, nuclear atypia, and nuclear grooves. Follow-up was available for 44 patients ranging from 0.3 to 259 months (mean: 67.5 mo). Two patients developed recurrence, both of them harbored FOXL2 mutation. In conclusion, the pathology diagnosis of sex cord-stromal tumors continues to be difficult, and reticulin staining remains a valuable tool as an initial step in the diagnostic work-up. Individual pericellular reticulin pattern essentially rules out adult granulosa cell tumor, while cases with indeterminate or nested patterns can be subjected to FOXL2 mutation testing to aid the diagnosis.

FOXL2 Mutation Status in Granulosa Theca Cell Tumors of the Ovary.

Ovarian sex-cord stromal tumors that have between 10% and 50% granulosa cells in a prominent fibrothecomatous background have been referred to as granulosa theca cell tumors or mixed granulosa theca cell tumors. The classification and prognosis of these tumors is not clear. Most adult granulosa cell tumors of the ovary harbor a mutation in the FOXL2 gene, whereas fibromas and thecomas lack this mutation. The aim of our study was to assess the FOXL2 mutation status of ovarian granulosa theca cell tumors and to correlate the mutation status with morphologic and clinical characteristics. A FOXL2 mutation was detected in 6 of 12 (50%) granulosa theca cell tumors. Tumors with higher cellularity of granulosa cells were more likely to harbor a FOXL2 mutation as were tumors in which the granulosa cells formed large lobules. No conclusions could be drawn regarding the clinical and prognostic significance of the presence of a mutation given the small number of cases and limited clinical follow-up. Our study shows that half of granulosa theca cell tumors harbor the same FOXL2 mutation that characterizes adult granulosa cell tumors but there is no outcome evidence to guide whether mutation status should alter the classification of the tumor or the management of the patient.

Characteristics and outcome of recurrence in molecularly defined adult-type ovarian granulosa cell tumors

ObjectiveAdult-type ovarian granulosa cell tumors (AGCTs) have an unpredictable tendency to relapse. In a carefully validated patient cohort, we evaluated the prognostic factors related to AGCT recurrence. MethodsWe identified all patients diagnosed with AGCT during 1956–2014 in Helsinki University Hospital, with a minimum follow-up of one year (n=240). After a histological review supplemented with FOXL2 (402C-G) mutation status analysis, we analyzed the clinical data for association with relapse. ResultsThe final cohort included 164 (68%) molecularly defined AGCTs (MD-AGCTs). The majority of the women were postmenopausal (63%), and 92% of tumors were stage I. The median follow-up time was 15.5years. Fifty-two (32%) patients developed tumor recurrence, of whom 55% had successive recurrences. Multiple-site recurrences were common, and nearly half of the recurrences were asymptomatic. The median time to the first relapse was 7.4years, and 75% of relapses occurred within ten years after primary diagnosis. The median disease-free survival was 11.3years. Premenopausal status at initial diagnosis, FIGO stage Ic versus Ia, and tumor rupture associated with relapse. However, tumor rupture was the only independent predictive factor. Of the relapsed patients, 48% died of AGCT in a median time of 15.3years. ConclusionTumor rupture is the strongest predictive factor for recurrence, and these patients might benefit from a more aggressive initial treatment approach. AGCT requires active follow up for 10 to 15years after primary diagnosis, since recurrences may develop late, asymptomatically and in multiple anatomical locations.

FOXL2 mutation is absent in uterine tumors resembling ovarian sex cord tumors.

Uterine tumors resembling ovarian sex cord tumors (UTROSCTs) are rare uterine neoplasms characterized by pure or predominant epithelial-like patterns that share morphologic, immunohistochemical, and ultrastructural features with ovarian sex cord tumors. FOXL2 immunoexpression has recently been found in sex cord stromal tumors of the ovary, including granulosa cell tumors, Sertoli-Leydig cell tumors, thecomas, and fibromas, but mutations have been identified mostly in adult granulosa cell tumors. In this study, we investigated FOXL2 mutation status and protein expression in UTROSCTs. Mutational analysis using a TaqMan real-time polymerase chain reaction-based allelic discrimination assay was performed on formalin-fixed, paraffin-embedded tissue from 15 UTROSCTs. FOXL2 mutation was absent in all tumors. FOXL2 immunoexpression was tested in all 15 tumors. Intensity of staining was scored as weak, moderate, or strong. Percentage of tumor cells with nuclear staining was recorded as follows: 0 (negative); 1+ (1% to 25%); 2+ (26% to 50%); 3+ (51% to 75%); and 4+ (76% to 100%). Nuclear expression of FOXL2 was present in 6 of 15 (40%) UTROSCTs. One tumor demonstrated strong 4+ staining. Moderate expression was seen in 3 cases, including 2 and 1 showing 2+ and 1+ staining, respectively. Weak expression was observed in 2 tumors demonstrating 3+ and 1+ staining. Although UTROSCTs show overlapping morphologic, immunohistochemical, and ultrastructural features with sex cord stromal tumors of the ovary, they do not harbor FOXL2 mutation despite focal immunoreactivity in a subset of these tumors.

Adult granulosa cell tumour‐like areas occurring in ovarian epithelial neoplasms: report of a case series with investigation of FOXL2 mutation status

To look for FOXL2 mutation in rare ovarian epithelial lesions showing stromal components with morphological features of adult granulosa cell tumour (AGCT). We report the 402C→G FOXL2 mutation status in five epithelial ovarian lesions in women aged 45-77 years showing stromal proliferations that were morphologically indistinguishable from AGCT. The lesions were mucinous cystadenoma, mixed epithelial cystadenoma, endometriotic cyst, mucinous borderline tumour (intestinal type), and mucinous carcinoma. In one case, the AGCT component formed a discrete nodule, and in the others it was distributed within the septa and cyst walls. FOXL2 mutation was present in two cases and absent in three cases. One mutation-positive case showed an AGCT nodule abutting a mucinous borderline tumour, interpreted as a collision tumour. The other positive case had an AGCT component within the septa of a mucinous carcinoma, and both components are likely to be neoplastic. In the three cases without FOXL2 mutation, the stromal component most likely represents a non-neoplastic AGCT-like proliferation. Areas typical of AGCT are rarely associated with epithelial ovarian lesions. These are heterogeneous and likely to be truly neoplastic in only a subset of cases. FOXL2 mutation testing may be useful in confirming a true neoplastic AGCT component.

A current perspective on the pathological assessment of FOXL2 in adult‐type granulosa cell tumours of the ovary

The diagnosis of adult-type granulosa cell tumours of the ovary (aGCT) is based on histomorphology aided by immunohistochemical staining for sex cord markers. Recently a single, recurrent somatic point mutation (402C→G) in FOXL2 was described in aGCT. We have investigated the impact of FOXL2 mutation testing in a large cohort of aGCT diagnosed previously by conventional histology and immunohistochemistry. Formalin-fixed, paraffin-embedded tissue cores from a cohort of 52 aGCT diagnosed previously by expert gynaecopathologists were analysed immunohistologically. FOXL2 mutation status was determined by Sanger sequencing and high-sensitivity TaqMan allelic discrimination assay. Histomorphology was reassessed by two expert gynaecopathologists. FOXL2 mutation analyses could be performed successfully in 46 cases, 40 of which were positive for the c.402C>G mutation, confirming a diagnosis of aGCT. In the six cases negative for the c.402C>G mutation, one case was confirmed on review as FOXL2 wild-type aGCT, whereas in the remaining five cases diagnoses other than aGCT were made. In cases where a diagnosis of aGCT is a consideration and unequivocal diagnosis is not possible based on morphology and routine immunostains, FOXL2 mutation testing can help to confirm the diagnosis. It is particularly relevant for accurate subclassification within the group of sex cord-stromal tumours.

Adult granulosa cell tumours (GCT): Clinicopathological outcomes including FOXL2 mutational status and expression

ObjectivesThe aim of this research was to use nucleic acids isolated from formalin-fixed paraffin-embedded (FFPE) tissue to investigate the diagnostic potential and prognostic significance of FOXL2 in adult-type GCTs, particularly as a marker of identifying early stage patients that are likely to relapse. MethodsWe performed a retrospective review of GCT patients referred to the Auckland Gynae-Oncology Multidisciplinary Team from 1955 to 2012. Baseline characteristics, clinical course, histopathology and survival data was recorded. Using nucleic acids extracted from FFPE tumour blocks, FOXL2 mutation status and expression was determined by DNA sequencing and RT-qPCR, respectively, and correlated with clinical data. Results57 adult GCT patients were identified, however FFPE tumour blocks were available for only 37 of these patients. Sequencing results confirmed the presence of the FOXL2 mutation in 70% of patients. FOXL2 mutation positive adult tumours showed a trend towards higher FOXL2 expression than wildtype adult tumours, particularly in stage I patients (p=0.051). In addition, patients with homozygous FOXL2 mutations had a significantly higher relapse rate (p=0.04). There was no significant correlation between FOXL2 mutation status or FOXL2 expression and any other clinical variables. ConclusionsFFPE tumour blocks are a valuable resource of molecular information, especially when studying rare tumours such as GCTs. The FOXL2 mutation appears to have some diagnostic potential, however additional work in a larger cohort needs to be completed to confirm the prognostic significance of this gene mutation, and its expression.

FOXL2 molecular testing in ovarian neoplasms: diagnostic approach and procedural guidelines

A single, recurrent somatic point mutation (402C→G) in FOXL2 has been described in almost all adult-type granulosa cell tumors but not other ovarian neoplasms. Histopathological features of adult-type granulosa cell tumors can be mimicked by a variety of other tumors, making diagnosis of adult-type granulosa cell tumor challenging. It has been suggested that molecular testing for FOXL2 mutation might be a useful tool in the diagnosis of adult-type granulosa cell tumors. The aim of this study was to demonstrate how testing for the FOXL2 mutation can be used in a gynecological pathology consultation service and to establish clear procedural guidelines for FOXL2 testing. Immunohistochemistry for FOXL2 was done using an anti-FOXL2 polyclonal antiserum. If immunohistochemistry was positive, FOXL2 mutation status was subsequently analyzed using a TaqMan assay. A dilution experiment was done to assess the sensitivity and minimum tumor cellularity requirements for our TaqMan assay. Twenty problematic cases were assessed, where the differential diagnosis after the initial investigations included adult-type granulosa cell tumors. Differential diagnoses included: thecoma, Sertoli–Leydig cell tumor, juvenile granulosa cell tumor, endometrial stromal sarcoma and others. In all cases, FOXL2 immunohistochemistry was positive and in six samples the FOXL2 mutation was detected, thus confirming a diagnosis of adult-type granulosa cell tumor. The TaqMan assay was able to reliably detect the FOXL2 mutation with input DNA in the range of 2.5–20 ng, and with a minimum of 25% tumor cell nuclei. The analysis of the FOXL2 mutational status in clinical samples is a useful diagnostic tool in situations where the differential diagnosis is between adult-type granulosa cell tumor and other ovarian tumors. The TaqMan assay requires a minimum of 2.5 ng DNA, with optimal assay performance for 5 to 10 ng DNA input. Laser capture or needle-macrodissection should be undertaken to enrich samples with tumor cell content below 25%.

Adult granulosa cell tumors (GCT): 56 years of clinicopathologic outcomes including FOXL2 mutational status.

5536 Background: GCT account for 2-3% of ovarian cancers with a tendency for late relapse. Treatment is primarily surgical. The role of chemotherapy and hormonal therapy is more controversial. The FOXL2 mutation (402C→G) has been identified as a potential driver mutation and may be useful in diagnosis and treatment. Methods: We performed a retrospective review of GCT patients (pts) referred to the Auckland Gynae-Oncology Multidisciplinary Team from 1955 to 2011. Baseline characteristics, clinical course, histopathology and survival data was recorded. FOXL2 mutation status was determined by DNA sequencing, and correlated with clinical data. Results: 56 GCT pts were identified. Median (med) age 48.6 years (y) (22-86). Stage I were 82.1%. 48% of tumours were ≥10cm. Med follow up was 10.0y (0.2-40.4). 25 pts progressed, med time to progression (TTP) was 4.5y (0.1-17.7). Med progression free survival was 14.5y. Med overall survival (OS) was 21.8y but med disease specific survival was not reached. 9/18 pts died of disease. Stage III GCT and size ≥10cm had a higher risk of relapse (RR 3.1 and 2.9) and death (RR 8.2 and 8.6) respectively. 17/46 (37%) Stage I pts progressed. Med TTP was 8.3y (1.3 to 17.7), med OS was 29.0y. Stage I relapse rate was higher in tumours ≥10cm (RR 3.9 p&lt;0.01). 12/17 1st relapses were treated with surgery. 10/17 pts received ≥1 line of chemotherapy and 7 ≥1 hormonal therapy. Clinical benefit rates (CR, PR and SD&gt;6m) for first-line chemotherapy was 25% and 71% for hormones. All 7 Stage III pts progressed with med OS of 6.3yr (0.2-12.3y). Currently the FOXL2 mutation statuses are known for 18 patients. 89% carried the mutation. Homozygous, heterozygous and wild-type mutations had no difference in risk of relapse or death. Further FOXL2 mutation analysis is ongoing. Conclusions: This long term series confirms the protracted natural history of this disease. Early stage GCT, despite progression has a good prognosis with med OS &gt;25y. Stage and tumour size remain the most consistent prognostic factors. Whilst surgery remains the mainstay of therapy, the high response rate to hormonal therapy deserves investigation. Currently the FOXL2 mutation status does not appear prognostic but this needs further research.

FOXL2 mutation and large-scale genomic imbalances in adult granulosa cell tumors of the ovary

Adult granulosa cell tumors (AGCTs) are a rare class of ovarian tumors with recurrent cytogenetic abnormalities including trisomy 12, trisomy 14, monosomy 16/deletion 16q, and monosomy 22. Over 90% contain a missense point mutation (C134W) in the FOXL2 gene at 3q22.3. The relationship between FOXL2 mutation and cytogenetic abnormalities is unclear, although both are presumably early events in tumorigenesis. In addition, FOXL2 C134W mutant allele imbalance has been noted in a minority of AGCTs, but the mechanism for allelic imbalance has not yet been described. We used a microarray platform designed for formalin-fixed, paraffin-embedded (FFPE) tissue specimens, the Affymetrix OncoScan FFPE Express 330K Molecular Inversion Probe (MIP) array, to explore the correlation between genomic imbalances detected by microarray and FOXL2 mutation status detected by pyrosequencing in a series of 21 archived AGCTs. Tumors were characterized by histopathologic features, stage, and alpha-inhibin expression by immunohistochemistry. All tumors were positive for inhibin, and 18/21 tumors contained a FOXL2 mutation. The most common genomic imbalances were a gain of 14q, a loss of 16q, and a loss of 22q. Three tumors showed evidence of FOXL2 mutant allele imbalance by pyrosequencing; microarray revealed a 32.5 Mb deletion encompassing FOXL2 in 1 case and a 70.9 Mb stretch of homozygosity encompassing FOXL2 in the other case. The third case, with a FOXL2 mutant allele imbalance, showed a diminished mutant allele population (32%) despite high estimated tumor content (>90%), suggesting tumor heterogeneity for the mutation. This study provides the first correlation of FOXL2 mutation status and genomic imbalances in AGCTs, and it further elucidates the mechanisms for mutant allele imbalance in cancer.

Correlation of FOXL2 mutation status with genomic imbalances in adult granulosa cell tumors of the ovary

Correlation of FOXL2 mutation status with genomic imbalances in adult granulosa cell tumors of the ovary Katherine Geiersbach , Elke A. Jarboe , Mona S. Jahromi , Christine L. Baker , Christian N. Paxton , Sheryl R. Tripp , Joshua D. Schiffmann c,d Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA; ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT, USA; Department of Oncological Sciences, Huntsman Cancer Institute, Salt Lake City, UT, USA; Department of Pediatrics, University of Utah Health School of Medicine, Salt Lake City, UT, USA

Genome-wide analysis of chromosomal abnormalities in acute lymphoblastic leukemia

Correlation of FOXL2 mutation status with genomic imbalances in adult granulosa cell tumors of the ovary Katherine Geiersbach , Elke A. Jarboe , Mona S. Jahromi , Christine L. Baker , Christian N. Paxton , Sheryl R. Tripp , Joshua D. Schiffmann c,d Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA; ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT, USA; Department of Oncological Sciences, Huntsman Cancer Institute, Salt Lake City, UT, USA; Department of Pediatrics, University of Utah Health School of Medicine, Salt Lake City, UT, USA

FOXL2 Is a Sensitive and Specific Marker for Sex Cord-Stromal Tumors of the Ovary

Sex cord-stromal tumors (SCSTs) of the ovary are relatively uncommon tumors. Diagnosis of SCST rests primarily on the histomorphology of these tumors, and tumors with an atypical or unconventional appearance can pose diagnostic challenges. Previously, we had identified FOXL2 (402C→G) mutation as being characteristic of adult granulosa cell tumors (aGCTs). However, molecular screening for this mutation is not always possible and adds time and cost to the diagnostic process. In this study, we investigated the potential diagnostic use of immunostaining for FOXL2 on formalin-fixed paraffin-embedded tissue sections. Using a commercially available polyclonal antiserum against FOXL2 protein, immunoexpression of FOXL2 was tested in 501 ovarian tumor samples, including 119 SCSTs, using whole tissue sections and tissue microarrays. Staining was correlated with FOXL2 mutation status. In addition, we compared FOXL2 immunoexpression with that of α-inhibin and calretinin, the 2 traditional immunomarkers of SCST, in a subset of 89 SCSTs. FOXL2 immunostaining was present in 95 of 119 (80%) SCSTs, including >95% of aGCTs, juvenile granulosa cell tumors, fibromas, and sclerosing stromal tumors. Only 50% of Sertoli-Leydig cell tumors (N=40) expressed FOXL2. One of 11 steroid cell tumors and 3 of 3 female adnexal tumors of probable Wolffian origin showed FOXL2 immunoreactivity, whereas all other non-SCSTs tested (N=368) were negative for FOXL2 expression. Thus, the sensitivity and specificity of FOXL2 immunoreactivity for SCST are 80% and 99%, respectively. The FOXL2 (402C→G) mutation was confirmed to be both a sensitive and relatively specific indicator of aGCT. Forty-five of 119 SCSTs were mutation positive. These cases were 39 of 42 (93%) aGCTs, 3 of 40 Sertoli-Leydig cell tumors, 2 of 5 thecomas, and 1 of 4 (25%) SCSTs of unclassified type. SCSTs harboring a FOXL2 mutation consistently immunoexpressed FOXL2 (44 of 45, 98%), but FOXL2 immunostaining was also seen in many SCSTs that lacked a mutation (49 of 73, 67%). FOXL2 immunostaining showed higher sensitivity for the diagnosis of SCST, compared with α-inhibin and calretinin, and FOXL2 staining was typically more intense in positive cases compared with either α-inhibin or calretinin. In the SCSTs that were negative for FOXL2 expression, α-inhibin and/or calretinin immunostaining yielded positive results. In conclusion, FOXL2 is a relatively sensitive and highly specific marker for SCST. FOXL2 staining is present in almost all SCSTs with a FOXL2 mutation, and also in a majority of SCSTs without a mutation. FOXL2, together with α-inhibin and calretinin, forms an immunomarker panel that will result in positive staining with 1 or more markers in essentially all cases of SCST.

Mutational analysis of FOXL2 codon 134 in granulosa cell tumour of ovary and other human cancers

A missense somatic mutation in the FOXL2 gene affecting codon 134 has recently been reported in granulosa cell tumour (GCT) and thecoma of the ovary. Such a recurrent nature of the mutation strongly suggests that the FOXL2 mutation may play an important role in the development of ovarian sex cord-stromal tumours. The aim of this study was to characterize the FOXL2 mutation in human tumour tissues. We analysed 1353 tumour tissues from various origins, including ovarian tumours and other common cancers, by single-strand conformation polymorphism analysis. We found the FOXL2 codon 134 missense mutation in 53 of 56 adult GCTs (94.6%) and two of the 16 thecomas (12.5%), but none in other tumours. Histologically, FOXL2 mutation-negative adult GCT showed that GCT cells were admixed with fibrothecomatous cells, and FOXL2 mutation-positive thecomas showed that luteinized theca cells were predominant. However, immunostaining of either inhibin alpha or FOXL2 did not differentiate the FOXL2 mutation status of adult GCTs and thecomas. There was no FOXL1 mutation and no common oncogenic mutation in the adult GCTs and thecomas. Our data indicate that the FOXL2 codon 134 mutation occurs exclusively in GCT and thecoma, and suggest the possibility that the development of most GCTs and a fraction of thecomas may be dependent on this mutation. Our data also suggest that the FOXL2 mutation status, as well as some histological features, may be important in the diagnosis of ovarian sex cord-stromal tumours.