Autoantibodies, antigen-autoantibody complexes and antigens complement CA125 for early detection of ovarian cancer.

BACKGROUND: Multiple antigens, autoantibodies (AAb), and antigen-autoantibody (Ag-AAb) complexes were compared for their ability to complement CA125 for early detection of ovarian cancer. METHODS: Twenty six biomarkers were measured in a single panel of sera from women with early stage (I-II) ovarian cancers (n = 64), late stage (III-IV) ovarian cancers (186), benign pelvic masses (200) and from healthy controls (502), and then split randomly (50:50) into a training set to identify the most promising classifier and a validation set to compare its performance to CA125 alone. RESULTS: Eight biomarkers detected ≥ 8% of early stage cases at 98% specificity. A four-biomarker panel including CA125, HE4, HE4 Ag-AAb and osteopontin detected 75% of early stage cancers in the validation set from among healthy controls compared to 62% with CA125 alone (p = 0.003) at 98% specificity. The same panel increased sensitivity for distinguishing early-stage ovarian cancers from benign pelvic masses by 25% (p = 0.0004) at 95% specificity. From 21 autoantibody candidates, 3 AAb (anti-p53, anti-CTAG1 and annt-Il-8) detected 22% of early stage ovarian cancers, potentially lengthening lead time prior to diagnosis. CONCLUSION: A four biomarker panel achieved greater sensitivity at the same specificity for early detection of ovarian cancer than CA125 alone.

Pertuzumab Plus Trastuzumab in Patients With Endometrial Cancer With ERBB2/3 Amplification, Overexpression, or Mutation: Results From the TAPUR Study.

PURPOSE: The TAPUR Study is a pragmatic basket trial evaluating antitumor activity of commercially available targeted agents in patients with advanced cancers harboring potentially actionable genomic alterations. Data from a cohort of patients with endometrial cancer (EC) with ERBB2 or ERBB3 (ERBB2/3) amplification, overexpression, or mutation treated with pertuzumab plus trastuzumab (P + T) are reported. METHODS: Eligible patients had advanced EC, no standard treatment options, measurable disease (RECIST v1.1), Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, and tumors with ERBB2/3 amplification, overexpression, or mutation. Simon's two-stage design was used with a primary end point of disease control (DC), defined as objective response (OR) or stable disease (SD) of at least 16 weeks (SD16+) duration. Secondary end points include safety, duration of response, duration of SD, progression-free survival (PFS), and overall survival (OS). RESULTS: Twenty-eight patients were enrolled from March 2017 to November 2019; all patients were evaluable for efficacy and toxicity. Seventeen patients had tumors with ERBB2/3 amplification and/or overexpression, eight with both ERBB2 amplification and ERBB2/3 mutations, and three with only ERBB2 mutations. Ten patients had DC (two partial response and eight SD16+); all 10 had ERBB2 amplification, and 6 of the 10 patients with DC had >1 ERBB2/3 alteration. DC and OR rates were 37% (95% CI, 21 to 50) and 7% (95% CI, 1 to 24), respectively; the median PFS and median OS were 16 weeks (95% CI, 10-28) and 61 weeks (95% CI, 24-105), respectively. One patient experienced a grade 3 serious adverse event (muscle weakness) at least possibly related to P + T. CONCLUSION: P + T has antitumor activity in heavily pretreated patients with EC with ERBB2 amplification and warrants additional study.

Cancer case trends following the onset of the COVID-19 pandemic: A community-based observational study with extended follow-up.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has impacted health care delivery worldwide. Cancer is a leading cause of death, and the impact of the pandemic on cancer diagnoses is an important public health concern. METHODS: This cross-sectional study retrospectively analyzed the electronic medical records of 80,138 cancer patients diagnosed between January 1, 2019, and May 31, 2021. Outcome measures included weekly number of new cancer cases and trends in weekly cancer cases, before and after the pandemic; patient demographics; and positive COVID-19 test rates. RESULTS: Beginning March 4, 2020, defined as the onset of the pandemic, weekly cancer cases declined precipitously (-110.0 cases per week [95% confidence interval, -190.2 to -29.8]) for 4 weeks, followed by a moderate recovery (+23.7 cases per week [9.1 to 38.4]) of 10 weeks duration. Thereafter, weekly cancer cases trended slowly back toward pre-COVID-19 baseline levels. Following the pandemic onset, there was a cumulative year-over-year decline in cancer cases overall of 7.3%, including a 20.2%, 14.3%, and 12.8% decline in nonmelanoma skin cancer, breast cancer, and prostate cancer, respectively. Changes in case volumes were accompanied by variations in patient characteristics, including region, age, gender, race, insurance coverage, and COVID-19 positive test rates (P < .01 for all). Among patients tested for COVID-19, 5.3% had a positive result. CONCLUSIONS: The data in this study demonstrate a substantial reduction in cancer diagnoses following the onset of COVID-19, which appear to reach expected pre-COVID norms 12 months later. The largest reduction was noted among cancers that are typically screen-detected or identified as part of a routine wellness examination.

Quantitative global lipidomics analysis of patients with ovarian cancer versus benign adnexal mass.

Altered lipid metabolism has emerged as an important feature of ovarian cancer (OC), yet the translational potential of lipid metabolites to aid in diagnosis and triage remains unproven. We conducted a multi-level interrogation of lipid metabolic phenotypes in patients with adnexal masses, integrating quantitative lipidomics profiling of plasma and ascites with publicly-available tumor transcriptome data. Using Sciex Lipidyzer, we assessed concentrations of > 500 plasma lipids in two patient cohorts-(i) a pilot set of 100 women with OC (50) or benign tumor (50), and (ii) an independent set of 118 women with malignant (60) or benign (58) adnexal mass. 249 lipid species and several lipid classes were significantly reduced in cases versus controls in both cohorts (FDR < 0.05). 23 metabolites-triacylglycerols, phosphatidylcholines, cholesterol esters-were validated at Bonferroni significance (P < 9.16 × 10-5). Certain lipids exhibited greater alterations in early- (diacylglycerols) or late-stage (lysophospholipids) cases, and multiple lipids in plasma and ascites were positively correlated. Lipoprotein receptor gene expression differed markedly in OC versus benign tumors. Importantly, several plasma lipid species, such as DAG(16:1/18:1), improved the accuracy of CA125 in differentiating early-stage OC cases from benign controls, and conferred a 15-20% increase in specificity at 90% sensitivity in multivariate models adjusted for age and BMI. This study provides novel insight into systemic and local lipid metabolic differences between OC and benign disease, further implicating altered lipid uptake in OC biology, and advancing plasma lipid metabolites as a complementary class of circulating biomarkers for OC diagnosis and triage.

Biomarkers and Strategies for Early Detection of Ovarian Cancer.

Early detection of ovarian cancer remains an important unmet medical need. Effective screening could reduce mortality by 10%-30%. Used individually, neither serum CA125 nor transvaginal sonography (TVS) is sufficiently sensitive or specific. Two-stage strategies have proven more effective, where a significant rise above a woman's baseline CA125 prompts TVS and an abnormal sonogram prompts surgery. Two major screening trials have documented that this strategy has adequate specificity, but sensitivity for early-stage (I-II) disease must improve to have a greater impact on mortality. To improve the first stage, different panels of protein biomarkers have detected cases missed by CA125. Autoantibodies against TP53 have detected 20% of early-stage ovarian cancers 8 months before elevation of CA125 and 22 months before clinical diagnosis. Panels of autoantibodies and antigen-autoantibody complexes are being evaluated with the goal of detecting >90% of early-stage ovarian cancers, alone or in combination with CA125, while maintaining 98% specificity in control subjects. Other biomarkers, including micro-RNAs, ctDNA, methylated DNA, and combinations of ctDNA alterations, are being tested to provide an optimal first-stage test. New technologies are also being developed with greater sensitivity than TVS to image small volumes of tumor.See all articles in this CEBP Focus section, "NCI Early Detection Research Network: Making Cancer Detection Possible."

Prophylactic In Vivo Hematopoietic Stem Cell Gene Therapy with an Immune Checkpoint Inhibitor Reverses Tumor Growth in Syngeneic Mouse Tumor Models.

Population-wide testing for cancer-associated mutations has established that more than one-fifth of ovarian and breast carcinomas are associated with inherited risk. Salpingo-oophorectomy and/or mastectomy are currently the only effective options offered to women with high-risk germline mutations. Our goal here is to develop a long-lasting approach that provides immunoprophylaxis for mutation carriers. Our approach leverages the fact that at early stages, tumors recruit hematopoietic stem/progenitor cells (HSPC) from the bone marrow and differentiate them into tumor-supporting cells. We developed a technically simple technology to genetically modify HSPCs in vivo. The technology involves HSPC mobilization and intravenous injection of an integrating HDAd5/35++ vector. In vivo HSPC transduction with a GFP-expressing vector and subsequent implantation of syngeneic tumor cells showed >80% GFP marking in tumor-infiltrating leukocytes. To control expression of transgenes, we developed a miRNA regulation system that is activated only when HSPCs are recruited to and differentiated by the tumor. We tested our approach using the immune checkpoint inhibitor anti-PD-L1-γ1 as an effector gene. In in vivo HSPC-transduced mice with implanted mouse mammary carcinoma (MMC) tumors, after initial tumor growth, tumors regressed and did not recur. Conventional treatment with an anti-PD-L1 mAb had no significant antitumor effect, indicating that early, self-activating expression of anti-PD-L1-γ1 can overcome the immunosuppressive environment in MMC tumors. The efficacy and safety of this approach was further validated in an ovarian cancer model with typical germline mutations (ID8 p53-/- brca2-/-), both in a prophylactic and therapeutic setting. This HSPC gene therapy approach has potential for clinical translation. SIGNIFICANCE: Considering the limited prophylactic options that are currently offered to women with high-risk germ-line mutations, the in vivo HSPC gene therapy approach is a promising strategy that addresses a major medical problem.

Engineered Adoptive T-cell Therapy Prolongs Survival in a Preclinical Model of Advanced-Stage Ovarian Cancer.

Adoptive T-cell therapy using high-affinity T-cell receptors (TCR) to target tumor antigens has potential for improving outcomes in high-grade serous ovarian cancer (HGSOC) patients. Ovarian tumors develop a hostile, multicomponent tumor microenvironment containing suppressive cells, inhibitory ligands, and soluble factors that facilitate evasion of antitumor immune responses. Developing and validating an immunocompetent mouse model of metastatic ovarian cancer that shares antigenic and immunosuppressive qualities of human disease would facilitate establishing effective T-cell therapies. We used deep transcriptome profiling and IHC analysis of human HGSOC tumors and disseminated mouse ID8VEGF tumors to compare immunologic features. We then evaluated the ability of CD8 T cells engineered to express a high-affinity TCR specific for mesothelin, an ovarian cancer antigen, to infiltrate advanced ID8VEGF murine ovarian tumors and control tumor growth. Human CD8 T cells engineered to target mesothelin were also evaluated for ability to kill HLA-A2+ HGSOC lines. IHC and gene-expression profiling revealed striking similarities between tumors of both species, including processing/presentation of a leading candidate target antigen, suppressive immune cell infiltration, and expression of molecules that inhibit T-cell function. Engineered T cells targeting mesothelin infiltrated mouse tumors but became progressively dysfunctional and failed to persist. Treatment with repeated doses of T cells maintained functional activity, significantly prolonging survival of mice harboring late-stage disease at treatment onset. Human CD8 T cells engineered to target mesothelin were tumoricidal for three HGSOC lines. Treatment with engineered T cells may have clinical applicability in patients with advanced-stage HGSOC.

False-positive screening events and worry influence decisions about surgery among high-risk women.

OBJECTIVE: Studies of cancer screening have found that false positive screening events (FPSE) can affect worry about cancer risk and screening program use, we sought to further explore this. METHOD: In a study of 1,100 women at high risk for ovarian cancer who participated in a previously published randomized controlled trial (RCT), we sought to explore whether worry might also influence the use of risk-reducing surgical procedures by women. Participants included 234 women with BRCA1/2 mutations and 866 women with high-risk pedigrees. We followed the women for up to 6 years. RESULTS: Worry predicted risk reducing prophylactic bilateral salpingo-oophorectomy (pBSO) for both mutation carriers (HR = 1.74; p = .02), and women with high-risk pedigree (HR = 3.41; p < .001). FPSE also predicted subsequent pBSO among women with a high-risk pedigree (HR 2.31; p < .01). While screening may reduce worry among those who never receive a positive result, FPSE increase worry at least temporarily. Worry about ovarian cancer risk predicted use of preventative pBSO among high-risk women including those with BRCA1/2 mutations enrolled in an ovarian cancer-screening program. FPSE also predicted risk-reducing ovarian surgery among high-risk women without a known mutation at the time of screening program enrollment. CONCLUSIONS: Physicians who offer screening should know that false positive results may increase use of pBSO, how this should effect clinical practice is unclear. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Control of Tumor Initiation by NKG2D Naturally Expressed on Ovarian Cancer Cells.

Cancer cells may co-opt the NKG2D lymphocyte receptor to complement the presence of its ligands for autonomous stimulation of oncogenic signaling. Previous studies raise the possibility that cancer cell NKG2D may induce high malignancy traits, but its full oncogenic impact is unknown. Using epithelial ovarian cancer as model setting, we show here that ex vivo NKG2D+ cancer cells have stem-like capacities, and provide formal in vivo evidence linking NKG2D stimulation with the development and maintenance of these functional states. NKG2D+ ovarian cancer cell populations harbor substantially greater capacities for self-renewing in vitro sphere formation and in vivo tumor initiation in immunodeficient (NOD scid gamma) mice than NKG2D- controls. Sphere formation and tumor initiation are impaired by NKG2D silencing or ligand blockade using antibodies or a newly designed pan ligand-masking NKG2D multimer. In further support of pathophysiological significance, a prospective study of 47 high-grade serous ovarian cancer cases revealed that the odds of disease recurrence were significantly greater and median progression-free survival rates higher among patients with above and below median NKG2D+ cancer cell frequencies, respectively. Collectively, our results define cancer cell NKG2D as an important regulator of tumor initiation in ovarian cancer and presumably other malignancies and thus challenge current efforts in immunotherapy aimed at enhancing NKG2D function.

Early Detection of Ovarian Cancer using the Risk of Ovarian Cancer Algorithm with Frequent CA125 Testing in Women at Increased Familial Risk - Combined Results from Two Screening Trials.

Purpose: Women at familial/genetic ovarian cancer risk often undergo screening despite unproven efficacy. Research suggests each woman has her own CA125 baseline; significant increases above this level may identify cancers earlier than standard 6- to 12-monthly CA125 > 35 U/mL.Experimental Design: Data from prospective Cancer Genetics Network and Gynecologic Oncology Group trials, which screened 3,692 women (13,080 woman-screening years) with a strong breast/ovarian cancer family history or BRCA1/2 mutations, were combined to assess a novel screening strategy. Specifically, serum CA125 q3 months, evaluated using a risk of ovarian cancer algorithm (ROCA), detected significant increases above each subject's baseline, which triggered transvaginal ultrasound. Specificity and positive predictive value (PPV) were compared with levels derived from general population screening (specificity 90%, PPV 10%), and stage-at-detection was compared with historical high-risk controls.Results: Specificity for ultrasound referral was 92% versus 90% (P = 0.0001), and PPV was 4.6% versus 10% (P > 0.10). Eighteen of 19 malignant ovarian neoplasms [prevalent = 4, incident = 6, risk-reducing salpingo-oophorectomy (RRSO) = 9] were detected via screening or RRSO. Among incident cases (which best reflect long-term screening performance), three of six invasive cancers were early-stage (I/II; 50% vs. 10% historical BRCA1 controls; P = 0.016). Six of nine RRSO-related cases were stage I. ROCA flagged three of six (50%) incident cases before CA125 exceeded 35 U/mL. Eight of nine patients with stages 0/I/II ovarian cancer were alive at last follow-up (median 6 years).Conclusions: For screened women at familial/genetic ovarian cancer risk, ROCA q3 months had better early-stage sensitivity at high specificity, and low yet possibly acceptable PPV compared with CA125 > 35 U/mL q6/q12 months, warranting further larger cohort evaluation. Clin Cancer Res; 23(14); 3628-37. ©2017 AACR.

The effect of referral for genetic counseling on genetic testing and surgical prevention in women at high risk for ovarian cancer: Results from a randomized controlled trial.

BACKGROUND: Guidelines recommend genetic counseling and testing for women who have a pedigree suggestive of an inherited susceptibility for ovarian cancer. The authors evaluated the effect of referral to genetic counseling on genetic testing and prophylactic oophorectomy in a randomized controlled trial. METHODS: Data from an electronic mammography reporting system identified 12,919 women with a pedigree that included breast cancer, of whom 625 were identified who had a high risk for inherited susceptibility to ovarian cancer using a risk-assessment questionnaire. Of these, 458 women provided informed consent and were randomized 1:1 to intervention consisting of a genetic counseling referral (n = 228) or standard clinical care (n = 230). RESULTS: Participants were predominantly aged 45 to 65 years, and 30% and 20% reported a personal history of breast cancer or a family history of ovarian cancer, respectively. Eighty-five percent of women in the intervention group participated in a genetic counseling session. Genetic testing was reported by 74 (33%) and 20 (9%) women in the intervention and control arms (P < .005), respectively. Five women in the intervention arm and 2 in the control arm were identified as germline mutation carriers. Ten women in the intervention arm and 3 in the control arm underwent prophylactic bilateral salpingo-oophorectomy (P < .05). CONCLUSIONS: Routine referral of women at high risk for ovarian cancer to genetic counseling promotes genetic testing and prophylactic surgery. The findings from the current randomized controlled trial demonstrate the value of implementing strategies that target women at high risk for ovarian cancer to ensure they are offered access to recommended care. CA Cancer J Clin 2016. © 2016 American Cancer Society, Inc. Cancer 2016;122:3509-3518. © 2016 American Cancer Society.

Extracting a low-dimensional description of multiple gene expression datasets reveals a potential driver for tumor-associated stroma in ovarian cancer.

Patterns in expression data conserved across multiple independent disease studies are likely to represent important molecular events underlying the disease. We present the INSPIRE method to infer modules of co-expressed genes and the dependencies among the modules from multiple expression datasets that may contain different sets of genes. We show that INSPIRE infers more accurate models than existing methods to extract low-dimensional representation of expression data. We demonstrate that applying INSPIRE to nine ovarian cancer datasets leads to a new marker and potential driver of tumor-associated stroma, HOPX, followed by experimental validation. The implementation of INSPIRE is available at http://inspire.cs.washington.edu .

Targeted single molecule mutation detection with massively parallel sequencing.

Next-generation sequencing (NGS) technologies have transformed genomic research and have the potential to revolutionize clinical medicine. However, the background error rates of sequencing instruments and limitations in targeted read coverage have precluded the detection of rare DNA sequence variants by NGS. Here we describe a method, termed CypherSeq, which combines double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment to vastly improve NGS-based rare variant detection. The CypherSeq methodology involves the ligation of sample DNA into circular vectors, which contain double-stranded barcodes for computational error correction and adapters for library preparation and sequencing. CypherSeq is capable of detecting rare mutations genome-wide as well as those within specific target genes via RCA-based enrichment. We demonstrate that CypherSeq is capable of correcting errors incurred during library preparation and sequencing to reproducibly detect mutations down to a frequency of 2.4 × 10(-7) per base pair, and report the frequency and spectra of spontaneous and ethyl methanesulfonate-induced mutations across the Saccharomyces cerevisiae genome.

Identification of novel candidate plasma metabolite biomarkers for distinguishing serous ovarian carcinoma and benign serous ovarian tumors.

OBJECTIVE: Serous ovarian carcinoma (OC) represents a leading cause of cancer-related death among U.S. women. Non-invasive tools have recently emerged for discriminating benign from malignant ovarian masses, but evaluation remains ongoing, without widespread implementation. In the last decade, metabolomics has matured into a new avenue for cancer biomarker development. Here, we sought to identify novel plasma metabolite biomarkers to distinguish serous ovarian carcinoma and benign serous ovarian tumor. METHODS: Using liquid chromatography-mass spectrometry, we conducted global and targeted metabolite profiling of plasma isolated at the time of surgery from 50 serous OC cases and 50 serous benign controls. RESULTS: Global lipidomics analysis identified 34 metabolites (of 372 assessed) differing significantly (P<0.05) between cases and controls in both training and testing sets, with 17 candidates satisfying FDR q<0.05, and two reaching Bonferroni significance. Targeted profiling of ~150 aqueous metabolites identified a single amino acid, alanine, as differentially abundant (P<0.05). A multivariate classification model built using the top four lipid metabolites achieved an estimated AUC of 0.85 (SD=0.07) based on Monte Carlo cross validation. Evaluation of a hybrid model incorporating both CA125 and lipid metabolites was suggestive of increased classification accuracy (AUC=0.91, SD=0.05) relative to CA125 alone (AUC=0.87, SD=0.07), particularly at high fixed levels of sensitivity, without reaching significance. CONCLUSIONS: Our results provide insight into metabolic changes potentially correlated with the presence of serous OC versus benign ovarian tumor and suggest that plasma metabolites may help differentiate these two conditions.

Identifying post-menopausal women at elevated risk for epithelial ovarian cancer.

OBJECTIVE: We developed and validated a hybrid risk classifier combining serum markers and epidemiologic risk factors to identify post-menopausal women at elevated risk for invasive fallopian tube, primary peritoneal, and ovarian epithelial carcinoma. METHODS: To select epidemiologic risk factors for use in the classifier, Cox proportional hazards analyses were conducted using 74,786 Women's Health Initiative (WHI) Observational Study (OS) participants. To construct a combination classifier, 210 WHI OS cases and 536 matched controls with serum marker measurements were analyzed; validation employed 143 cases and 725 matched controls from the WHI Clinical Trial (CT) with similar data. RESULTS: Analyses identified a combination risk classifier composed of two elevated-risk groups: 1) women with CA125 or HE4 exceeding a 98% specificity threshold; and 2) women with intact fallopian tubes, prior use of menopausal hormone therapy for at least two years, and either a first degree relative with breast or ovarian cancer or a personal history of breast cancer. In the WHI OS population, it classified 13% of women as elevated risk, identifying 30% of ovarian cancers diagnosed up to 7.8years post-enrollment (Hazard Ratio [HR]=2.6, p<0.001). In the WHI CT validation population, it classified 8% of women as elevated risk, identifying 31% of cancers diagnosed within 7years of enrollment (HR=4.6, p<0.001). CONCLUSION: CA125 and HE4 contributed significantly to a risk prediction classifier combining serum markers with epidemiologic risk factors. The hybrid risk classifier may be useful to identify post-menopausal women who would benefit from timely surgical intervention to prevent epithelial ovarian cancer.

Use of CA125 and HE4 serum markers to predict ovarian cancer in elevated-risk women.

BACKGROUND: Serum markers are used before pelvic imaging to improve specificity and positive predictive value (PPV) of ovarian cancer multimodal screening strategies. METHODS: We conducted a randomized controlled pilot trial to estimate surgical PPV of a "2 of 3 tests positive" screening rule, and to compare use of HE4 as a first-line (Arm 1) versus a second-line (Arm 2) screen, in women at high and elevated risk for epithelial ovarian cancer (EOC) at five study sites. Semiannual screening was offered to 208 women ages 25 to 80 years with deleterious BRCA germline mutations and to 834 women ages 35 to 80 years with pedigrees suggesting inherited susceptibility. Annual screening was offered to 130 women ages 45 to 80 years (Risk Group 3) with epidemiologic and serum marker risk factors. Rising marker levels were identified using the parametric empirical Bayes algorithm. RESULTS: Both strategies yielded surgical PPV above 25%. Protocol-indicated surgery was performed in 6 women, identifying two ovarian malignancies and yielding a surgical PPV in both arms combined of 33% (95% confidence interval: 4%-78%), 25% in Arm 1 and 50% in Arm 2. Surgical consultation was recommended for 37 women (26 in Arm 1 and 11 in Arm 2). On the basis of 12 women with at least 2 of 3 tests positive (CA125, HE4, or imaging), an intent-to-treat analysis yielded PPV of 14% in Arm 1 and 20% in Arm 2. CONCLUSIONS: Positive screens were more frequent when HE4 was included in the primary screen. IMPACT: HE4 may be useful as a confirmatory screen when rising CA125 is used alone as a primary screen.

Ultrasound molecular imaging in a human CD276 expression-modulated murine ovarian cancer model.

PURPOSE: To develop a mouse ovarian cancer model that allows modulating the expression levels of human vascular targets in mouse xenograft tumors and to test whether expression of CD276 during tumor angiogenesis can be visualized by molecularly targeted ultrasound in vivo. EXPERIMENTAL DESIGN: CD276-expressing MILE SVEN 1 (MS1) mouse endothelial cells were engineered and used for coinjection with 2008 human ovarian cancer cells for subcutaneous xenograft tumor induction in 15 nude mice. Fourteen control mice were injected with 2008 cells only. After confirming their binding specificity in flow chamber cell attachment studies, anti-CD276 antibody-functionalized contrast microbubbles were used for in vivo CD276-targeted contrast-enhanced ultrasound imaging. RESULTS: CD276-targeted ultrasound imaging signal was significantly higher (P = 0.006) in mixed MS1/2008 tumors than in control tumors. Compared with control microbubbles, the ultrasound signal using CD276-targeted microbubbles was significantly higher (P = 0.002), and blocking with purified anti-CD276 antibody significantly decreased (P = 0.0096) the signal in mixed MS1/2008 tumors. Immunofluorescence analysis of the tumor tissue confirmed higher quantitative immunofluorescence signal in mixed MS1/2008 tumors than in control 2008 only tumors, but showed not significantly different (P = 0.54) microvessel density. CONCLUSIONS: Our novel small animal model allows for modulating the expression of human tumor-associated vascular endothelial imaging targets in a mouse host and these expression differences can be visualized noninvasively by ultrasound molecular imaging. The animal model can be applied to other human vascular targets and may facilitate the preclinical development of new imaging probes such as microbubbles targeted at human vascular markers not expressed in mice.

Digital genomic quantification of tumor-infiltrating lymphocytes.

Infiltrating T lymphocytes are frequently found in malignant tumors and are suggestive of a host cancer immune response. Multiple independent studies have documented that the presence and quantity of tumor-infiltrating lymphocytes (TILs) are strongly correlated with increased survival. However, because of methodological factors, the exact effect of TILs on prognosis has remained enigmatic, and inclusion of TILs in standard prognostic panels has been limited. For example, some reports enumerate all CD3(+) cells, some count only cytotoxic CD8(+) T cells, and the criteria used to score tumors as TIL-positive or TIL-negative are inconsistent among studies. To address this limitation, we introduce a robust digital DNA-based assay, termed QuanTILfy, to count TILs and assess T cell clonality in tissue samples, including tumors. We demonstrate the clonal specificity of this approach by the diagnosis of T cell acute lymphoblastic leukemia and the accurate, sensitive, and highly reproducible measurement of TILs in primary and metastatic ovarian cancer. Our experiments demonstrate an association between higher TIL counts and improved survival among women with ovarian cancer, and are consistent with previous observations that the immune response against ovarian cancer is a meaningful and independent prognostic factor. Surprisingly, the TIL repertoire is diverse for all tumors in the study with no notable oligoclonal expansions. Furthermore, because variability in the measurement and characterization of TILs has limited their clinical utility as biomarkers, these results highlight the significant translational potential of a robust, standardizable DNA-based assay to assess TILs in a variety of cancer types.

High-throughput sequencing of T-cell receptors reveals a homogeneous repertoire of tumour-infiltrating lymphocytes in ovarian cancer.

The cellular adaptive immune system mounts a response to many solid tumours mediated by tumour-infiltrating T lymphocytes (TILs). Basic measurements of these TILs, including total count, show promise as prognostic markers for a variety of cancers, including ovarian and colorectal. In addition, recent therapeutic advances are thought to exploit this immune response to effectively fight melanoma, with promising studies showing efficacy in additional cancers. However, many of the basic properties of TILs are poorly understood, including specificity, clonality, and spatial heterogeneity of the T-cell response. We utilize deep sequencing of rearranged T-cell receptor beta (TCRB) genes to characterize the basic properties of TILs in ovarian carcinoma. Due to somatic rearrangement during T-cell development, the TCR beta chain sequence serves as a molecular tag for each T-cell clone. Using these sequence tags, we assess similarities and differences between infiltrating T cells in discretely sampled sections of large tumours and compare to T cells from peripheral blood. Within the limits of sensitivity of our assay, the TIL repertoires show strong similarity throughout each tumour and are distinct from the circulating T-cell repertoire. We conclude that the cellular adaptive immune response within ovarian carcinomas is spatially homogeneous and distinct from the T-cell compartment of peripheral blood.