Citations

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

AbstractPurpose/Objective(s)Bladder and prostate are unfavorable sites for rhabdomyosarcoma (B/P‐RMS), and represent a challenging location for radiotherapy.Materials/MethodsNineteen patients with B/P‐RMS were enrolled on a prospective registry protocol (2008‐2017) and treated with chemotherapy, proton beam therapy (PBT), and surgical resection (n = 8; 42%). Emphasis was given to treatment technique, disease‐related outcomes, and toxicity associated with PBT.ResultsThe majority of patients had bladder RMS (74%) of embryonal histology (95%), Group III (68%), and intermediate‐risk disease by Children's Oncology Group (COG) risk stratification (89%). Seven patients (37%) had primary tumors >5 cm in size. All patients were treated according to COG protocols. With a median follow‐up of 66.2 months, 5‐year overall survival (OS) and progression‐free survival (PFS) were 76%. Four patients (21%) experienced disease relapse, all presenting with local failure. The 5‐year local control (LC) rate was 76%. Tumor size predicted LC, with 5‐year LC for patients with >5 cm tumors being 43% versus 100% for those with ≤5 cm tumors (P = .006). Univariate analysis demonstrated an effect of tumor size on OS (tumor >5 cm, hazard ratio [HR] 17.7, P = .049) and PFS (HR 17.7, P = .049). Acute grade 2 toxicity was observed in two patients (11%, transient proctitis). Late grade 2+ toxicity was observed in three patients (16%; n = 1 grade 2 skeletal deformity; n = 3 transient grade 2 urinary incontinence; one patient experienced both).ConclusionsPBT for B/P‐RMS affords promising disease‐related outcomes with an acceptable toxicity profile. Higher local failure rates were observed for larger tumors, supporting dose‐escalation components of ongoing RMS clinical trials.

Abstract Identifying risk factors for women at high risk of symptom-detected breast cancers that were missed by screening would enable targeting of alternative prevention strategies. To identify breast cancer risk factors by mode of detection, we examined these associations in heavily-screened women from the Cancer Prevention Study (CPS)-II Nutrition Cohort. Among 76,406 women followed for a median of 13.8 years, 2,469 screen-detected and 1,189 symptom-detected breast cancer cases were diagnosed. Multivariable-adjusted associations were estimated using joint Cox proportional hazards regression models with person-time calculated contingent on screening. The mean (standard deviation) age at baseline was 62.0 (6.6) years for non-cases, 61.6 (6.1) for screen-detected cases, and 61.5 (6.2) for symptom-detected cases. Women with symptom-detected tumors were diagnosed at a slightly younger age (mean=69.4) and less likely to be diagnosed with a localized tumor (65.3%), than women with a screen-detected tumor (mean age at diagnosis=70.4 and localized tumors=83.3%). Factors associated with higher risks of symptom detected vs. screen detected breast cancer, included: current combined menopausal hormone use (HR=2.04, 95% CI 1.70 - 2.46 vs. HR=1.43, 95% CI 1.25 - 1.63), current estrogen only menopausal hormone use (HR=1.41, 95% CI 1.15 - 1.72 vs. HR=0.94, 95% CI 0.82 - 1.09), and history of benign breast disease (HR=1.89, 95% CI 1.67 - 2.13 vs. HR=1.49, 95% CI 1.36 - 1.62). The opposite pattern of higher risk of screen detected vs. symptom detected breast cancer was observed for greater adult weight gain (HR=2.32, 95% CI 1.91 - 2.82 vs. HR=1.42, 95% CI 1.10 - 1.84) and alcohol intake (HR=1.39, 95% CI 1.24 - 1.64 vs. HR=1.20, 95% CI 0.92 - 1.58). Our results suggest that understanding risk factors for symptom-detected cancers may help identify women who would benefit from more intensive screening to facilitate early detection. Citation Format: Mia M. Gaudet, Emily Deubler, W. Ryan Diver, Samantha Puvanesarajah, Alpa V. Patel, Ted Gansler, Dana Flanders, Mark Sherman, Susan Gapstur. Breast cancer risk factors by mode of detection among screened women in the Cancer Prevention Study-II [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4211.

Numerous social, economic and academic pressures can have a negative impact on representations of biomedical research. We review several of the forces playing an increasingly pernicious role in how health and science information is interpreted, shared and used, drawing discussions towards the role of narrative. In turn, we explore how aspects of narrative are used in different social contexts and communication environments, and present creative responses that may help counter the negative trends. As traditional methods of communication have in many ways failed the public, changes in approach are required, including the creative use of narratives.

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Background: Prospective, randomized trials are needed to determine optimal treatment approaches for palliative care problems such as malignant bowel obstruction (MBO). Randomization poses unique issues for such studies, especially with divergent treatment approaches and varying levels of equipoise. We report our experience accruing randomized patients to the Prospective Comparative Effectiveness Trial for Malignant Bowel Obstruction (SWOG S1316) study, comparing surgical and nonsurgical management of MBO. Methods: Patients with MBO who were surgical candidates and had treatment equipoise were accrued and offered randomization to surgical or nonsurgical management. Patients choosing nonrandomization were offered prospective observation. Trial details are listed on www.clinicaltrials.gov (NCT #02270450). An accrual algorithm was developed to enhance enrollment. Results: Accrual is ongoing with 176 patients enrolled. Most (89%) patients chose nonrandomization, opting for nonsurgical management. Of 25 sites that have accrued to this study, 6 enrolled patients on the randomization arm. Approximately 59% (20/34) of the randomization accrual goal has been achieved. Patient-related factors and clinician bias have been the most prevalent reasons for lack of randomization. An algorithm was developed from clinician experience to aid randomization. Using principles in this tool, repeated physician conversations discussing treatment options and goals of care, and a supportive team–approach has helped increase accrual. Conclusions: Experience gained from the S1316 study can aid future palliative care trials. Although difficult, it is possible to randomize patients to palliative studies by giving clinicians clear recommendations utilizing an algorithm of conversation, allotment of necessary time to discuss the trial, and encouragement to overcome internal bias.

Abstract Stromal tumor-infiltrating lymphocytes (sTILs) are often prominent in HER2-enriched and triple negative breast cancers and predict a favorable prognosis for these subtypes. Clinical and epidemiological determinants of sTILs have not been defined in large cohorts. Accordingly, we analyzed these associations for 702 eligible, postmenopausal invasive breast cancer cases from the Cancer Prevention Study (CPS)-II Nutrition cohort. CPS-II included 97,786 women who completed baseline and follow-up surveys since 1992. Women with self-reported breast cancer diagnoses were consented for medical record and tumor tissue retrieval. We analyzed pre-diagnostic data from the survey prior to diagnosis on personal history of benign breast disease (BBD), menopausal hormone use, alcohol intake, cigarette smoking status, waist circumference, body mass index, adult weight gain, nonsteroidal anti-inflammatory drugs, and physical activity. One pathologist (TG) evaluated sTILs using whole slide images of H&E stained sections according to recommendations by the International TILs Working Group 2014. sTIL levels were dichotomized: none/minimal (0-10%) and moderate/high (&gt;10%). We compared the sTIL levels by clinical and epidemiologic risk factors using chi-square statistics. Odds ratio (ORs) and 95% confidence intervals (CIs) for the associations of clinical and epidemiologic risk factors with sTILs were estimated with multivariable logistic regression. Mean age at diagnosis was 71.9 years; 44% of cancers were moderate grade, 74% were localized staged and 88% were luminal-like. Cancers with high/moderate sTIL levels (n=614), compared to none/minimal (n=88), were more likely to be high grade (54% vs. 20%; p-value &lt;0.001), node-positive (41% vs. 22%; p-value &lt;0.001), and non-luminal (36% vs. 8%; p-value &lt;0.001). In a model mutually-adjusted for age at diagnosis and clinical factors, all clinical factors were associated with moderate/high sTIL levels: node-positive (OR=2.15, 95% CI 1.28 – 3.58), high grade (OR=3.98, 95% CI 1.88 – 9.06), and non-luminal subtype (OR=3.65, 95% CI 2.00 – 6.59). Age at diagnosis was not associated with sTIL levels (per year: OR=1.01, 95% CI 0.97 – 1.05). Women with BBD were less likely to have moderate/high sTIL levels than minimal/none sTIL levels (38% vs. 54%; age-adjusted OR=0.50, 95% CI 0.31 – 0.79). Ever smokers also were less likely to have moderate/high sTIL levels (38% vs. 49%) with an OR=0.61 (95% 0.38 – 0.97). Other risk factors were not significantly associated with sTIL levels. In this large epidemiologic cohort, non-luminal cancers had more sTILs than luminal cancers. Women with a personal history of BBD and ever cigarette smokers had significant lower levels of sTILs. Our study is among the first to examine pre-diagnostic risk factors in relation to sTILs and provides the impetus for larger, population-based studies with phenotypic characterization of immune cells. Citation Format: Gaudet MM, Mellow MM, Puvanesarajah S, Gapstur SM, Sherman ME, Gansler T. Associations of epidemiologic and clinical features with intensity of immune infiltrates in postmenopausal breast cancer cases from the cancer prevention study-II cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-08-02.