Clinical management of TP53 mosaic variants found on germline genetic testing.

BACKGROUND: Germline heterozygous TP53 pathogenic variants (PVs) cause Li Fraumeni Syndrome (LFS, OMIM#151623). TP53 PVs at lower-than-expected variant allele frequencies (VAF) may reflect postzygotic mosaicism (PZM) or clonal hematopoiesis (CH); however, no guidelines exist for workup and clinical management. PATIENTS AND METHODS: Retrospective analysis of probands who presented to an academic cancer genetics program with a TP53 PV result on germline genetic testing. RESULTS: Twenty-one of 125 unrelated probands (17 %) were found to harbor a TP53 PV with VAF<30 % or a designation of "mosaic". A diagnosis of PZM was made in nine (43 %) due to a clinical phenotype consistent with LFS with (n = 8) or without (n = 1) positive ancillary tissue testing. Twelve patients (57 %) were diagnosed with presumed CH (pCH) due to a diagnosis of a myeloproliferative neoplasm, negative ancillary tissue testing, clinical phenotype not meeting LFS criteria, no cancer, and/or no first cancer age<50. Of the 19 patients with biological offspring, nine had either partial or complete offspring testing, all negative. CONCLUSIONS: Determining the etiology of low VAF TP53 PVs requires ancillary tissue testing and incorporation of clinical phenotype. Discerning PZM versus CH is important to provide optimal care and follow-up.

Loss of heterozygosity does not occur in BRCA1/2 mutant pediatric solid and central nervous system tumors.

Utilization of tumor-only sequencing has expanded in pediatric cancer patients, which can lead to identification of pathogenic variants in genes that may be germline and/or have uncertain relevance to the tumor in question, such as the homologous recombination (HR) pathway genes BRCA1/2. We identified patients with pathogenic BRCA1/2 mutations from somatic tumor sequencing, and performed additional germline sequencing to assess for the presence of loss of heterozygosity (LOH). Of seven patients identified, four (57.1%) mutations were found in the germline and none had associated LOH. Our data suggest that BRCA1/2 mutations identified in this context are likely incidental findings.

Clinical Targeted Next-Generation Panel Sequencing Reveals MYC Amplification Is a Poor Prognostic Factor in Osteosarcoma.

PURPOSE: Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS: In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS: In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS (P = .04). The most frequently altered genes in the primary cohort were TP53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort (P = .015) and the validation cohort (P = .012). CONCLUSION: The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Rare FGFR Oncogenic Alterations in Sequenced Pediatric Solid and Brain Tumors Suggest FGFR Is a Relevant Molecular Target in Childhood Cancer.

PURPOSE: Multiple FGFR inhibitors are currently in clinical trials enrolling adults with different solid tumors, while very few enroll pediatric patients. We determined the types and frequency of FGFR alterations (FGFR1-4) in pediatric cancers to inform future clinical trial design. METHODS: Tumors with FGFR alterations were identified from two large cohorts of pediatric solid tumors subjected to targeted DNA sequencing: The Dana-Farber/Boston Children's Profile Study (n = 888) and the multi-institution GAIN/iCAT2 (Genomic Assessment Improves Novel Therapy) Study (n = 571). Data from the combined patient population of 1,395 cases (64 patients were enrolled in both studies) were reviewed and cases in which an FGFR alteration was identified by OncoPanel sequencing were further assessed. RESULTS: We identified 41 patients with tumors harboring an oncogenic FGFR alteration. Median age at diagnosis was 8 years (range, 6 months-26 years). Diagnoses included 11 rhabdomyosarcomas, nine low-grade gliomas, and 17 other tumor types. Alterations included gain-of-function sequence variants (n = 19), amplifications (n = 10), oncogenic fusions (FGFR3::TACC3 [n = 3], FGFR1::TACC1 [n = 1], FGFR1::EBF2 [n = 1], FGFR1::CLIP2 [n = 1], and FGFR2::CTNNA3 [n = 1]), pathogenic-leaning variants of uncertain significance (n = 4), and amplification in combination with a pathogenic-leaning variant of uncertain significance (n = 1). Two novel FGFR1 fusions in two different patients were identified in this cohort, one of whom showed a response to an FGFR inhibitor. CONCLUSION: In summary, activating FGFR alterations were found in approximately 3% (41/1,395) of pediatric solid tumors, identifying a population of children with cancer who may be eligible and good candidates for trials evaluating FGFR-targeted therapy. Importantly, the genomic and clinical data from this study can help inform drug development in accordance with the Research to Accelerate Cures and Equity for Children Act.

Rapid and highly sensitive approach for multiplexed somatic fusion detection.

Somatic gene translocations are key to making an accurate diagnosis in many cancers including many pediatric sarcomas. Currently available molecular diagnostic approaches to identifying somatic pathognomonic translocations have limitations such as minimal multiplexing, high cost, complex computational requirements, or slow turnaround times. We sought to develop a new fusion-detection assay optimized to mitigate these challenges. To accomplish this goal, we developed a highly sensitive multiplexed digital PCR-based approach that can identify the gene partners of multiple somatic fusion transcripts. This assay was validated for specificity with cell lines and synthetized DNA fragments. Assay sensitivity was optimized using a tiered amplification approach for fusion detection from low input and/or degraded RNA. The assay was then tested for the potential application of fusion detection from FFPE tissue and liquid biopsy samples. We found that this multiplexed PCR approach was able to accurately identify the presence of seven different targeted fusion transcripts with a turnaround time of 1 to 2 days. The addition of a tiered amplification step allowed the detection of targeted fusions from as little as 1 pg of RNA input. We also identified fusions from as little as two unstained slides of FFPE tumor biopsy tissue, from circulating tumor cells collected from tumor-bearing mice, and from liquid biopsy samples from patients with known fusion-positive cancers. We also demonstrated that the assay could be easily adapted for additional fusion targets. In summary, this novel assay detects multiple somatic fusion partners in biologic samples with low tumor content and low-quality RNA in less than two days. The assay is inexpensive and could be applied to surgical and liquid biopsies, particularly in places with inadequate resources for more expensive and expertise-dependent assays such as next-generation sequencing.

Text-only and picture conversation aids both supported shared decision making for breast cancer surgery: Analysis from a cluster randomized trial.

OBJECTIVES: To determine if two encounter conversation aids for early-stage breast cancer surgery increased observed and patient-reported shared decision making (SDM) compared with usual care and if observed and patient-reported SDM were associated. METHODS: Surgeons in a cluster randomized trial at four cancer centers were randomized to use an Option Grid, Picture Option Grid, or usual care. We used bivariate statistics, linear regression, and multilevel models to evaluate the influence of trial arm, patient socioeconomic status and health literacy on observed SDM (via OPTION-5) and patient-reported SDM (via collaboRATE). RESULTS: From 311 recordings, OPTION-5 scores were 73/100 for Option Grid (n = 40), 56.3/100 for Picture Option Grid (n = 144), and 41.0/100 for usual care (n = 127; p < 0.0001). Top collaboRATE scores were 81.6 % for Option Grid, 80.0 % for Picture Option Grid, and 56.4 % for usual care (p < 0.001). Top collaboRATE scores correlated with an 8.60 point (95 %CI 0.66, 13.7) higher OPTION-5 score (p = 0.008) with no correlation in the multilevel analysis. Patients of lower socioeconomic status had lower OPTION-5 scores before accounting for clustering. CONCLUSIONS: Both conversation aids led to meaningfully higher observed and patient-reported SDM. Observed and patient-reported SDM were not strongly correlated. PRACTICE IMPLICATIONS: Healthcare providers could implement these conversation aids in real-world settings.

Making the most of small samples: Optimization of tissue allocation of pediatric solid tumors for clinical and research use.

INTRODUCTION: Tissue from pediatric solid tumors is in high demand for use in high-impact research studies, making the allocation of tissue from an anatomic pathology laboratory challenging. We designed, implemented, and assessed an interdepartmental process to optimize tissue allocation of pediatric solid tumors for both clinical care and research. METHODS: Oncologists, pathologists, surgeons, interventional radiologists, pathology technical staff, and clinical research coordinators participated in the workflow design. Procedures were created to address patient identification and consent, prioritization of protocols, electronic communication of requests, tissue preparation, and distribution. Pathologists were surveyed about the value of the new workflow. RESULTS: Over a 5-year period, 644 pediatric solid tumor patients consented to one or more studies requesting archival or fresh tissue. Patients had a variety of tumor types, with many rare and singular diagnoses. Sixty-seven percent of 1768 research requests were fulfilled. Requests for archival tissue were fulfilled at a significantly higher rate than those for fresh tissue (P > .001), and requests from resection specimens were fulfilled at a significantly higher rate than those from biopsies (P > .0001). In an anonymous survey, seven of seven pathologists reported that the process had improved since the introduction of the electronic communication model. CONCLUSIONS: A collaborative and informed model for tissue allocation is successful in distributing archival and fresh tissue for clinical research studies. Our workflows and policies have gained pathologists' approval and streamlined our processes. As clinical and research programs evolve, a thoughtful tissue allocation process will facilitate ongoing research.

DICER1-associated central nervous system sarcoma in children: comprehensive clinicopathologic and genetic analysis of a newly described rare tumor.

The spectrum of neoplasms associated with DICER1 variants continues to expand, with the recent addition of primary "DICER1-associated central nervous system sarcoma" (DCS). DCS is a high-grade malignancy predominantly affecting pediatric patients. Six pediatric DCS were identified through a combination of clinical diagnostic studies, archival inquiry, and interinstitutional collaboration. Clinical, histologic, immunohistologic, and molecular features were examined. Genomic findings in the 6 DCS were compared with those in 14 additional DICER1-associated tumors sequenced with the same assay. The six patients presented at ages 3-15 years with CNS tumors located in the temporal (n = 2), parietal (n = 1), fronto-parietal (n = 1), and frontal (n = 2) lobes. All underwent surgical resection. Histologic examination demonstrated high-grade malignant spindle cell tumors with pleuropulmonary blastoma-like embryonic "organoid" features and focal rhabdomyoblastic differentiation; immature cartilage was seen in one case. Immunohistochemically, there was patchy desmin and myogenin staining, and patchy loss of H3K27me3, and within eosinophilic cytoplasmic globules, alfa-fetoprotein staining. Biallelic DICER1 variants were identified in all cases, with germline variants in two of five patients tested. DCS demonstrated genomic alterations enriched for Ras pathway activation and TP53 inactivation. Tumor mutational burden was significantly higher in the 6 DCS tumors than in 14 other DICER1-associated tumors examined (mean 12.9 vs. 6.8 mutations/Mb, p = 0.035). Postoperative care included radiation (n = 5) and chemotherapy (n = 3); at the last follow-up, three patients were alive without DCS, and three had died of disease. Our analysis expands the clinical, histologic, immunohistological, and molecular spectrum of DCS, identifying distinctive features that can aid in the diagnosis, multidisciplinary evaluation, and treatment of DCS.

Genomic and Immunologic Characterization of INI1-Deficient Pediatric Cancers.

PURPOSE: Several aggressive pediatric cancers harbor alterations in SMARCB1, including rhabdoid tumors, epithelioid sarcoma, and chordoma. As tumor profiling has become more routine in clinical care, we investigated the relationship between SMARCB1 genetic variants identified by next-generation sequencing (NGS) and INI1 protein expression. Therapeutic approaches for INI1-deficient tumors are limited. Early reports suggest a potential role for immune checkpoint inhibition in these patients. Thus, we also investigated PD-L1 and CD8 expression in INI1-negative pediatric brain and solid tumors. EXPERIMENTAL DESIGN: We performed immunohistochemistry (IHC) for INI1 and immune markers (PD-L1, CD8, and CD163) and NGS on tumor samples from 43 pediatric patients who had tumors with INI1 loss on previous IHC or SMARCB1 genomic alterations on prior somatic sequencing. RESULTS: SMARCB1 two-copy deletions and inactivating mutations on NGS were associated with loss of INI1 protein expression. Single-copy deletion of SMARCB1 was not predictive of INI1 loss in tumor histologies not known to be INI1-deficient. In the 27 cases with INI1 loss and successful tumor sequencing, 24 (89%) had a SMARCB1 alteration detected. In addition, 47% (14/30) of the patients with INI1-negative tumors had a tumor specimen that was PD-L1 positive and 60% (18/30) had positive or rare CD8 staining. We report on 3 patients with INI1-negative tumors with evidence of disease control on immune checkpoint inhibitors. CONCLUSIONS: A significant proportion of the INI1-negative tumors express PD-L1, and PD-L1 positivity was associated with extracranial tumor site. These results suggest that clinical trials of immune checkpoint inhibitors are warranted in INI1-negative pediatric cancers.

Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition.

Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of SMARCB1 for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor SMARCB1 loss, which also require expression of the E2 ubiquitin-conjugating enzyme, UBE2C. Our studies identify a synthetic lethal relationship between SMARCB1-deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors.

Seasonal malaria chemoprevention packaged with malnutrition prevention in northern Nigeria: A pragmatic trial (SMAMP study) with nested case-control.

Integrating seasonal malaria chemoprevention (SMC), recommended by the WHO since 2012 to prevent malaria infection, with nutrition interventions may improve health outcomes and operational efficiencies. This study assessed the effects of co-packaging interventions on distribution coverage, nutrition, and clinical malaria outcomes in northern Nigeria. From August to November 2014, community volunteers delivered sulfadoxine-pyrimethamine and amodiaquine (SP-AQ) door-to-door each month to approximately 7,000 children aged 6-24 months in seven wards of Madobi, Kano State, Nigeria. In three of the wards children additionally received a lipid-based nutrient supplement (LNS-medium quantity), Plumpy Doz. Coverage, adherence, and anthropometric outcomes were assessed through baseline, midline, and endline household surveys. A facility-based case-control study was also conducted to estimate impact on clinical malaria outcomes. Coverage of SP-AQ was similar between arms at 89% (n = 2,409 child-months [88-90%]) in the SP-AQ only arm and 90% (n = 1,947 child-months [88-92%]) in the SP-AQ plus LNS arm (p = 0.52). Coverage of LNS was 83% (n = 2,409 child-months [81-84%]). Whilst there were marked changes in anthropometric status between baseline, midline and endline, these were largely accounted for by socioeconomic status and must be interpreted with care due to possible measurement issues, especially length-based indices. Overall nutritional status of our most robust measure, weight-for-age, does appear to have improved by endline, but was similar in the two study arms, suggesting no additional benefit of the LNS. While the odds of clinical malaria among those who received the intended intervention were lower in each study arm compared to children who did not receive interventions (SP-AQ only OR = 0.23 [0.09-0.6]; SP-AQ plus LNS OR = 0.22 [0.09-0.55]), LNS was not shown to have an additional impact. Coverage of SMC was high regardless of integrating LNS delivery into the SMC campaign. Supplementation with LNS did not appear to impact nutritional outcomes, but appeared to enhance the impact of SP-AQ on clinical odds of malaria. These results indicate that combining nutritional interventions with seasonal malaria chemoprevention in high-risk areas can be done successfully, warranting further exploration with other products or dosing. Trial Registration: ISRCTN 11413895.

Adapting the Breast Cancer Surgery Decision Quality Instrument for Lower Socioeconomic Status: Improving Readability, Acceptability, and Relevance.

Introduction. Breast cancer is the second most common malignancy in women. The Decision Quality Instrument (DQI) measures the extent to which patients are informed and involved in breast surgery decisions and receive treatment that aligns with their preferences. There are limited data on the performance of the DQI in women of lower socioeconomic status (SES). Our aims were to 1) examine (and if necessary adapt) the readability, usability, and acceptability of the DQI and 2) explore whether it captures factors important to breast cancer surgery decisions among women of lower SES (relevance). Methods. We conducted semistructured cognitive interviews with women of lower SES (based on insurance status, income, and education) who had completed early-stage breast cancer treatments at three cancer centers. We used a two-step thematic analysis with dual independent coding. The study team (including Patient Partners and a Community Advisory Board) reviewed and refined suggested changes. The revised DQI was presented in two focus groups of breast cancer survivors. Results. We conducted 39 interviews. Participants found most parts of the DQI to be helpful and easy to understand. We made the following suggested changes: 1) added a glossary of key terms, 2) added two answer choices and an open text question in the goals and concerns subscale, 3) reworded the treatment intention question, and 4) revised the knowledge subscale instructions since several women disliked the wording and were unsure of what was expected. Discussion. The readability, usability, acceptability, and relevance of a measure that was primarily developed and validated in women of higher SES required adaptation for optimal use by women of lower SES. Further research will test these adaptations in lower SES populations.

Detection of Somatic Structural Variants Enables Quantification and Characterization of Circulating Tumor DNA in Children With Solid Tumors.

Objective: Liquid biopsies are being rapidly used in adult cancers as new biomarkers of disease. Circulating tumor DNA (ctDNA) levels have been reported to be proportional to disease burden, correlate with treatment response, and predict relapse. However, little is known about how frequently ctDNA is detectable in pediatric patients with solid tumors. Therefore, we developed a next-generation sequencing approach to detect and quantify ctDNA in the blood of patients with the most common pediatric solid tumors. Methods: Detection of ctDNA requires assays sensitive to somatic events typically observed in the cancer type being studied. In pediatric solid tumors, structural variants are more common than recurrent point mutations. We adapted an ultralow passage whole-genome sequencing approach to capture copy number variants and a hybrid capture sequencing assay to detect translocations in liquid biopsy samples from pediatric patients. Results: Copy number changes seen by ultralow passage whole-genome sequencing enabled detection of ctDNA in patients with osteosarcoma, neuroblastoma, alveolar rhabdomyosarcoma, and Wilms tumor. In Ewing sarcoma, detection of the EWSR1 translocation was a more sensitive approach. For patients with samples collected at multiple time points, changes in ctDNA levels corresponded to treatment response. We also found that disease-specific genomic biomarkers of prognosis were detectable in ctDNA. Conclusion: This study demonstrates that liquid biopsy approaches that detect somatic structural variants are well suited to pediatric solid tumors. We show that children with the most common solid tumor malignancies have detectable levels of ctDNA, which may be used to track disease response and identify genomic subclassifiers of disease. Efforts to profile larger collections of clinically annotated specimens are under way to validate the clinical use of these assays.

Blood collection in cell-stabilizing tubes does not impact germline DNA quality for pediatric patients.

OBJECTIVES: Liquid biopsy technologies allow non-invasive tumor profiling for patients with solid tumor malignancies by sequencing circulating tumor DNA. These studies may be useful in risk-stratification, monitoring for relapse, and understanding tumor evolution. The quality of DNA obtained for these studies is improved when blood samples are collected in tubes that stabilizing white blood cells (WBC). However, ongoing germline research in pediatric oncology generally requires obtaining blood samples in EDTA tubes, which do not contain a WBC-stabilizing preservative. In this study, we explored whether blood samples collected in WBC-stabilizing tubes could be used for both liquid biopsy and germline studies simultaneously, minimizing blood collection volumes for pediatric patients. METHODS: Blood was simultaneously collected from three patients in both EDTA and Streck Cell-Free DNA BCT® tubes. Germline DNA was extracted from all blood samples and subjected to whole-exome sequencing and microarray profiling. RESULTS: Quality control metrics of DNA quality, sequencing library preperation and whole-exome sequencing alignment were virtually identical regardless of the sample collection method. There was no discernable difference in patterns of variant calling for paired samples by either whole-exome sequencing or microarray analysis. CONCLUSION: Our study demonstrates that high-quality genomic studies may be performed from germline DNA obtained in Streck tubes. Therefore, these tubes may be used to simultaneously obtain samples for both liquid biopsy and germline studies in pediatric patients when the volume of blood available for research studies may be limited.

The association of area-level social class and tobacco use with adverse breast cancer characteristics among white and black women: evidence from Maryland, 1992-2003.

BACKGROUND: In breast cancer, worse disease characteristics are associated with fewer social resources and black race. However, it is unknown whether social gradients have similar impact across race, and whether behaviors, including tobacco use, may explain a portion of the social gradient. METHODS: We modeled relationships between area-level social class, tobacco spending and tumor characteristics, using 50,062 white and black cases diagnosed from 1992-2003 in Maryland, a racially and economically diverse state on the east coast of the United States. Multi-level models estimated the effect of area-level social class and tobacco consumption on tumor grade, size, and stage at diagnosis. RESULTS: Adjusting for race, age and year of diagnosis, higher social class was associated with lower risk for tumors with histological grade 3 or 4 (O.R. 0.96, 95% C.I. 0.94,0.99), those diagnosed at SEER stage 2 or later (O.R. 0.89, 95% C.I. 0.86, 0.91), and tumor size >2 cm (O.R. 0.87, 95% C.I. 0.84, 0.90). Higher tobacco spending was associated with higher risk for higher grade (O.R. 1.01, 1.00, 1.03) and larger tumors (O.R. 1.03, 95% C.I. 1.01, 1.06), but was not statistically significantly related to later stage (O.R. 1.00, 95% C.I. 0.98, 1.02). Social class was less protective for black women, but tobacco effects were not race-specific. CONCLUSIONS: Results suggest that in one U.S. geographic area, there is a differential protection from social class for black and white women, supporting use of intersectionality theory in breast cancer disparities investigations. Area-level tobacco consumption may capture cases' direct use and second hand smoke exposure, but also may identify neighborhoods with excess cancer-related behavioral or environmental exposures, beyond those measured by social class. Given the growing global burden of both tobacco addiction and aggressive breast cancer, similar investigations across diverse geographic areas are warranted.