Data_Sheet_1.PDF

Background<p>Most pelvic high-grade serous (HGS) carcinomas have been proposed to arise from tubal primaries that progress rapidly to advanced disease. However, the temporal sequence of ovarian and peritoneal metastases is not well characterized.</p>Methods<p>To establish the sequence of metastases, phylogenetic relationships among the ovarian and peritoneal carcinomas were determined from single-nucleotide variations (SNVs) in nine tumor regions from each patient with pelvic HGS carcinomas. Somatic SNVs from each tumor sample were used to reconstruct phylogenies of samples from each patient. Variant allele frequencies were used to reconstruct subclone phylogenies in each tumor sample.</p>Results<p>We show that pelvic HGS carcinomas are highly heterogeneous, only sharing less than 4% of somatic SNVs among all nine carcinoma implants in one patient. TP53 mutations are found in all nine carcinoma implants in each patient. The phylogenetic analyses reveal that peritoneal metastases arose from early branching events that preceded branching events for ovarian carcinomas in some patients. Finally, subclone phylogenies indicate the presence of multiple subclones at each tumor implant and early tumor clones in peritoneal implants.</p>Conclusion<p>The genetic evidence that peritoneal implants arose before or concurrently with ovarian implants is consistent with the emerging concept of the extra-ovarian origin of pelvic HGS cancer. Our results challenge the concept of stepwise spatial progression from the fallopian primary to ovarian carcinomas to peritoneal dissemination and suggest an alternative progression model where peritoneal spreading of early clones occurs before or in parallel with ovarian metastases.</p

Tumor-infiltrating CD4⁺CD25⁺ cells up-regulate FoxP3 and TGF-β expression upon activation.

<p>(<b>A</b>) Shown are two dots plots of purified tumor-infiltrating CD4⁺ T cells stimulated with ConA or media alone. Cells are gated on CD4 and CD25. (<b>B</b>) Bar charts showing the mean (± SEM, <i>n</i>=3 unique patient samples) percentage of FoxP3⁺ and TGF-β T cells among the total CD4⁺CD25⁺ T cells stimulated with either ConA or media alone. P value obtained by a paired Student’s T test.</p

Patients with ovarian cancer have elevated levels of circulating FRα as compared to matched controls.

<p>Panel A shows the number of circulating folate receptor (FRs) extrapolated from the microfiltration assay assuming a 1∶1 molar ratio of FA∶FR in both the cases and matched controls Panel B shows the densitometric signal obtained using immunoblotting. P values in panels A and B are calculated using the Wilcoxon's matched pairs test and each bar represents the mean±s.e.m. Panel C: Lane 1, immunoprecipitation negative control; lane 2, negative control (MCF-7) lysate; lane 3, positive control (KB) lysate); lane 4, control serum sample; lane 5, matched ovarian cancer serum sample; lane 6, control serum sample; lane 7 matched ovarian cancer serum sample. Arrow = M_r 38000 protein; KD = kilodalton. Panel D shows the linear regression analysis correlating the microfiltration binding assay data and the immunoblotting data. Each data point represents a unique individual. Open symbols represent the healthy controls and closed symbols are patients.</p

Detection of circulating FRα using microfiltration and immunoblotting.

<p>Panel A: Ten µl aliquots of KB culture supernatant was diluted to 50 µL with binding buffer that contained increasing concentrations of ³H-FA pulsed, without (Total bound) or with 1000X cold FA. After incubation, unbound FA was removed by microfiltration. Shown are the CPM retained by the microfilter. Also shown is Background which is binding in the absence of KB supernatants and without excess cold FA. *p<0.05. Each data point is the mean (±SE) of 3 replicates. The inset dotted line is the linear regression line of the total bound presented with the corresponding p and r values. Panel B shows that the amount of specific binding is dependent on the input levels. In this case increasing amounts of serum containing a KB protein spike were examined. This experiment is representative of 2 different experiments. Panel C shows the FA concentration that was bound in either KB supernatants or a representative serum sample from a case and control, comparing the 30 KD and 10 cutoff microfilters. Experiment is representative of two experiments. Panel D shows a representative example of immunoblot analysis of FRα. Arrow = M_r 38000 protein; KD = kilodalton; IP = immunoprecipitation. Lane 1, IP w no serum; lane 2, IP of KB lysate; lane 3, IP of precleared serum with KB lysate spike; lane 4. IP of serum alone, lane 5, KB lysate no IP; lane 6, IP of KB lysate no serum.</p

Tumor and patient characteristics.

<p>Tumor and patient characteristics.</p

Inherited Variants in Regulatory T Cell Genes and Outcome of Ovarian Cancer

<div><p>Although ovarian cancer is the most lethal of gynecologic malignancies, wide variation in outcome following conventional therapy continues to exist. The presence of tumor-infiltrating regulatory T cells (Tregs) has a role in outcome of this disease, and a growing body of data supports the existence of inherited prognostic factors. However, the role of inherited variants in genes encoding Treg-related immune molecules has not been fully explored. We analyzed expression quantitative trait loci (eQTL) and sequence-based tagging single nucleotide polymorphisms (tagSNPs) for 54 genes associated with Tregs in 3,662 invasive ovarian cancer cases. With adjustment for known prognostic factors, suggestive results were observed among rarer histological subtypes; poorer survival was associated with minor alleles at SNPs in RGS1 (clear cell, rs10921202, p = 2.7×10⁻⁵), LRRC32 and TNFRSF18/TNFRSF4 (mucinous, rs3781699, p = 4.5×10⁻⁴, and rs3753348, p = 9.0×10⁻⁴, respectively), and CD80 (endometrioid, rs13071247, p = 8.0×10⁻⁴). Fo0r the latter, correlative data support a CD80 rs13071247 genotype association with CD80 tumor RNA expression (p = 0.006). An additional eQTL SNP in CD80 was associated with shorter survival (rs7804190, p = 8.1×10⁻⁴) among all cases combined. As the products of these genes are known to affect induction, trafficking, or immunosuppressive function of Tregs, these results suggest the need for follow-up phenotypic studies.</p> </div

Regulatory T Cell SNPs Associated with Overall Survival (p<0.001).

<p>Adjusted for study site (MAY+MAC, RPCI, POL, UKO+UKR, TBO, NCO, RMH, SEA), age at diagnosis (<50 years, 50–69 years, >70 years), tumor stage (I or II, III or IV, unknown), race (white, non-white, unknown), and tumor grade (low, high, unknown); linkage disequilibrium reduced to r²<0.95; MAF, minor allele frequency.</p

Distributions of Ovarian Cancer Clinical Characteristics by Study.

<p>Distributions of Ovarian Cancer Clinical Characteristics by Study.</p

<i>CD80</i> rs13071247 Genotype and Tumor Expression.

<p>Among N = 54 MAY+MAC invasive ovarian cancer cases, Agilent whole human genome 4×44 K expression array probe A_24_P155632; CD80 log₂ ratios of tumor versus reference RNA expression (y-axis) versus genotype (x-axis); dashes indicate median; each symbol represents a unique patient; data points are jittered so all data are visible.</p