Clinical and genetic analysis of recurrent adult-type granulosa cell tumor of the ovary: Persistent preservation of heterozygous <i>c</i>.<i>402C>G FOXL2</i> mutation

<div><p>Background</p><p>Adult-type granulosa cell tumors of the ovary (aGCTs) are rare tumors that represent 2–5% of ovarian malignancies. The prognosis of this tumor is favorable, and it is characterized by slow progression. 10–30% of these tumors recur after 4–7 years of the primary surgery and the 5-year survival rate from the first recurrence is 55%, for the incompletely resected patients. At this time, complete resection is the only prognostic factor for better outcome, and establishing a novel strategy for identification and/or treatment of recurrent tumors is crucial. After the discovery of heterozygous <i>c</i>.<i>402C>G FOXL2</i> mutations in 97% of cases of aGCT, much effort has been made to find the role of the mutation on the pathogenesis of aGCT, however, little is known about the role of the mutation in disease progression.</p><p>Methods</p><p>We analyzed the clinical data of 56 aGCT patients to find a marker of recurrence. In particular, we compared the <i>FOXL2</i> status in 5 matched primary and recurrent samples by immunohistochemistry, and TaqMan allelic discrimination assay to address the role of <i>FOXL2</i> in potential mechanisms of recurrence.</p><p>Results</p><p>The clinical data analysis was consistent with complete resection as an indicator of disease eradication, though the sample size was limited. The genetic analysis showed all the samples, including recurrent tumor samples up to 14 years after the primary surgery, expressed heterozygous <i>c</i>.<i>402C>G FOXL2</i> mutation and the FOXL2 protein expression.</p><p>Conclusion</p><p>This report describes the preservation of heterozygous <i>c</i>.<i>402C>G FOXL2</i> mutation in recurrent aGCTs. This finding adds further credence to the concept that the <i>c</i>.<i>402C>G FOXL2</i> mutation is oncogenic and integral to this disease.</p></div

Histological finding and p53 expression of Pt #5.

<p>(a) The primary tumor of the Pt #5 revealed the bland tumor cells with variably nuclear grooves and a predominant diffuse pattern with minor trabecular and insular pattern which compatible with aGCTs. The mitotic activity was inconspicuous. (b) The recurrent tumor of the Pt #5 showed the similar histological findings as the primary tumor, though the mitotic activity was slightly higher compared to the primary tumor. (c) Primary and recurrent samples from patient #5 showed the moderate to strong p53 staining in the nucleus.</p

TaqMan based allelic discrimination assay.

<p>All samples gave fluorescent signal from both WT and mutant probes consistent with heterozygous c.402 C>G FOXL2 mutation. Each sample was duplicated and the black dot represents the negative control (H2O).</p

Patient characteristics.

<p>Patient characteristics.</p

Risk factors for recurrence (Univariate analysis).

<p>Risk factors for recurrence (Univariate analysis).</p

Risk factors for recurrence (multivariate analysis).

<p>Risk factors for recurrence (multivariate analysis).</p

Clinical findings of patients with recurrent disease.

<p>Clinical findings of patients with recurrent disease.</p

TIL and markers of immune function are associated with improved patient outcome.

<p>Kaplan-Meier analysis of disease-specific survival of high-grade serous ovarian carcinoma patients categorized by CD31, VEGF or the indicated immune markers: (A) CD31 and FoxP3, (B) CD31 and TIA-1, (C) CD31 and CD8, (D) VEGF and Granzyme B (GzmB), (E) VEGF and CD8. The statistical significance was determined using a log-rank test.</p

Hypoxia activates autophagy in CD8 T cells.

<p>(A) A representative Western blot indicating protein expression of HIF-1α, p62, LC3-II and β-actin is shown for OT-I CD8 T cells cultured under 1.5% oxygen. Cobalt chloride (CoCl₂) treatment was used as a positive control for HIF-1α protein expression. (B) Autophagy induction is shown by quantification of decreasing p62 levels without chloroquine (CQ) treatment and (C) LC3-II accumulation with CQ treatment under normoxia (norm) or 1.5% oxygen (hyp). The mean and standard error of the mean for the fold change of 3 independent experiments is reported. Statistical significance was determined using a one-sample t-test on log transformed values compared to a hypothetical mean of zero. *<i>p</i><0.05.</p

Markers of T Cell Infiltration and Function Associate with Favorable Outcome in Vascularized High-Grade Serous Ovarian Carcinoma

<div><p>Background</p><p>When T cells infiltrate the tumor environment they encounter a myriad of metabolic stressors including hypoxia. Overcoming the limitations imposed by an inadequate tumor vasculature that contributes to these stressors may be a crucial step to immune cells mounting an effective anti-tumor response. We sought to determine whether the functional capacity of tumor infiltrating lymphocytes (TIL) could be influenced by the tumor vasculature and correlated this with survival in patients with ovarian cancer.</p><p>Methodology and Principal Findings</p><p>In 196 high-grade serous ovarian tumors, we confirmed that the tumor vascularity as measured by the marker CD31 was associated with improved patient disease-specific survival. We also found that tumors positive for markers of TIL (CD8, CD4 and forkhead box P3 (FoxP3)) and T cell function (granzyme B and T-cell restricted intracellular antigen-1 (TIA-1)) correlated significantly with elevated vascularity. <i>In vitro</i>, hypoxic CD8 T cells showed reduced cytolytic activity, secreted less effector cytokines and upregulated autophagy. Survival analysis revealed that patients had a significant improvement in disease-specific survival when FoxP3 expressing cells were present in CD31-high tumors compared to patients with FoxP3 expressing cells in CD31-low tumors [HR: 2.314 (95% CI 1.049–5.106); <i>p</i> = 0.0377]. Patients with high vascular endothelial growth factor (VEGF) expressing tumors containing granzyme B positive cells had improved survival compared to patients with granzyme B positive cells in VEGF-low tumors [HR: 2.522 (95% CI 1.097–5.799); <i>p</i> = 0.0294].</p><p>Significance</p><p>Overall, this data provides a rationale for developing strategies aimed at improving the adaptability and function of TIL to hypoxic tumor conditions.</p></div