Synchronous T-lymphoblastic lymphoma and neuroblastoma in a 3-yr-old with novel germline SMARCA4 and EZH2 variants.

T-lymphoblastic lymphoma (T-LLy) is the most common lymphoblastic lymphoma in children and often presents with a mediastinal mass. Lymphomatous suprarenal masses are possible but rare. Here, we discuss the case of a previously healthy 3-yr-old male who presented with mediastinal T-LLy with bilateral suprarenal masses. Following initial treatment, surgical biopsy of persisting adrenal masses revealed bilateral neuroblastoma (NBL). A clinical genetics panel for germline cancer predisposition did not identify any pathogenic variants. Combination large panel (864 genes) profiling analysis in the context of a precision oncology study revealed two novel likely pathogenic heterozygous variants: SMARCA4 c.1420-1G > T p.? and EZH2 c.1943G > C p.(Ile631Phefs*44). Somatic analysis revealed potential second hits/somatic variants in EZH2 (in the T-LLy) and a segmental loss in Chromosome 19p encompassing SMARCA4 (in the NBL). Synchronous cancers, especially at a young age, warrant genetic evaluation for cancer predisposition; enrollment in a precision oncology program assessing germline and tumor DNA can fulfill that purpose, particularly when standard first-line genetic testing is negative and in the setting of tumors that are not classic for common cancer predisposition syndromes.

Multiple Germline Events Contribute to Cancer Development in Patients with Li-Fraumeni Syndrome.

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer-predisposition disorder. Approximately 70% of individuals who fit the clinical definition of LFS harbor a pathogenic germline variant in the TP53 tumor suppressor gene. However, the remaining 30% of patients lack a TP53 variant and even among variant TP53 carriers, approximately 20% remain cancer-free. Understanding the variable cancer penetrance and phenotypic variability in LFS is critical to developing rational approaches to accurate, early tumor detection and risk-reduction strategies. We leveraged family-based whole-genome sequencing and DNA methylation to evaluate the germline genomes of a large, multi-institutional cohort of patients with LFS (n = 396) with variant (n = 374) or wildtype TP53 (n = 22). We identified alternative cancer-associated genetic aberrations in 8/14 wildtype TP53 carriers who developed cancer. Among variant TP53 carriers, 19/49 who developed cancer harbored a pathogenic variant in another cancer gene. Modifier variants in the WNT signaling pathway were associated with decreased cancer incidence. Furthermore, we leveraged the noncoding genome and methylome to identify inherited epimutations in genes including ASXL1, ETV6, and LEF1 that confer increased cancer risk. Using these epimutations, we built a machine learning model that can predict cancer risk in patients with LFS with an area under the receiver operator characteristic curve (AUROC) of 0.725 (0.633-0.810). Significance: Our study clarifies the genomic basis for the phenotypic variability in LFS and highlights the immense benefits of expanding genetic and epigenetic testing of patients with LFS beyond TP53. More broadly, it necessitates the dissociation of hereditary cancer syndromes as single gene disorders and emphasizes the importance of understanding these diseases in a holistic manner as opposed to through the lens of a single gene.

Vaginal Metastases of Wilms' Tumor in a Pediatric Patient: A Rare Case.

BACKGROUND: Wilms' tumor is the second most common pediatric abdominal cancer; however, it rarely involves the female reproductive tract. There are few cases reported in the literature describing uterine, ovarian, cervical, and vaginal involvement. CASE: We report the case of a 7-year-old girl presenting with a large renal mass with retroperitoneal nodal and lung metastases; she was diagnosed with stage 4 favorable histology Wilms' tumor. She was treated with surgery, chemotherapy, and radiation. She presented with vaginal bleeding 10 months after completing treatment; biopsy of a vaginal mass confirmed recurrence, and this was sent for molecular profiling, which did not identify an inherited cancer predisposition or targetable mutation. She was again treated with chemotherapy; examination redemonstrated a small vaginal mass, but re-biopsy of the lesion was negative for malignancy. Due to high risk of local relapse, ongoing chemotherapy and pelvic radiation ensued. End-of-treatment imaging and vaginoscopy showed no residual disease. SUMMARY AND CONCLUSION: Vaginal metastases of Wilms' tumor are very rare; this is the second reported case in the literature. Pediatric clinicians should have a strong suspicion for vaginal metastases in cancer patients presenting with vaginal bleeding, especially when their pubertal development does not suggest that bleeding would be secondary to menarche. Long-term gynecologic care for these patients is paramount to reduce morbidity from chemotherapy and pelvic radiation. Fertility preservation counselling should be made early, through referral to a specialist.

Rhabdomyosarcoma as the first presentation in Neurofibromatosis Type 1: case series and review of the literature.

Neurofibromatosis Type 1 (NF1) is a neurocutaneous syndrome characterized by multiple café-au-lait macules, neurofibromas, and predisposition to malignancies, including rhabdomyosarcomas (RMS). Somatic NF1 mutations occur in RMS and other cancers, and ∼1% of patients with RMS have NF1. We describe three patients who presented prior to one year of age with RMS and were subsequently diagnosed with NF1. Compared to sporadic RMS, patients with this cancer predisposition syndrome are diagnosed younger, genitourinary sites are more common, and tumors are almost exclusively the embryonal subtype. Genomic sequencing of the tumor was initiated in one patient, and we identified a second sequence variant in NF1. The identification of molecular drivers in tumors is changing the nature of pediatric oncology by informing therapeutics targeted to specific molecular pathways and selecting patients who are likely to harbor germline variants in cancer predisposition genes who would benefit from a Medical Genetics assessment.

The clinical utility of integrative genomics in childhood cancer extends beyond targetable mutations.

We conducted integrative somatic-germline analyses by deeply sequencing 864 cancer-associated genes, complete genomes and transcriptomes for 300 mostly previously treated children and adolescents/young adults with cancer of poor prognosis or with rare tumors enrolled in the SickKids Cancer Sequencing (KiCS) program. Clinically actionable variants were identified in 56% of patients. Improved diagnostic accuracy led to modified management in a subset. Therapeutically targetable variants (54% of patients) were of unanticipated timing and type, with over 20% derived from the germline. Corroborating mutational signatures (SBS3/BRCAness) in patients with germline homologous recombination defects demonstrates the potential utility of PARP inhibitors. Mutational burden was significantly elevated in 9% of patients. Sequential sampling identified changes in therapeutically targetable drivers in over one-third of patients, suggesting benefit from rebiopsy for genomic analysis at the time of relapse. Comprehensive cancer genomic profiling is useful at multiple points in the care trajectory for children and adolescents/young adults with cancer, supporting its integration into early clinical management.

Nature-inspired engineering of an artificial ligase enzyme by domain fusion.

The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature's concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature's successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.

A primary DICER1-sarcoma with KRAS and TP53 mutations in a child with suspected ECCL.

A child had been followed since infancy by our multi-disciplinary neuro-oncology clinic with annual magnetic resonance imaging (MRI) under the presumed diagnosis of encephalocraniocutaneous lipomatosis (ECCL), with clinical features including nevus psiloliparus, scalp lipoma, nodular skin tag on and coloboma of the eyelid, cortical atrophy and meningeal angiomatosis. At the age of 4, she was found to have a large temporoparietal lesion causing elevated intracranial pressure requiring surgical resection. Histopathological exam of the tumor was suggestive of an intracranial sarcoma. Sequencing analysis of the tumor revealed mutations in DICER1, KRAS and TP53. Subsequent germline testing confirmed DICER1 syndrome and revealed an insignificant FGFR1 variant at a low frequency. Methylation profile of the tumor showed the tumor clustered most closely with sarcoma (rhabdomyosarcoma-like), confirming this tumor to be a primary DICER1-sarcoma. Compared to the previously reported cases, our unique case of primary DICER1-sarcoma also demonstrated neurofilament and chromogranin positivity, and genomic instability with loss of chromosome 4p, 4q, 8p, 11p, and 19p, as well as gains in chromosome 7p, 9p, 9q, 13q, and 15q on copy variant analysis. The detailed sequencing and methylation information discovered in this unique case of DICER1-sarcoma will hopefully help further our understanding of this rare and emerging entity.

Heterogeneity of Circulating Tumor Cell-Associated Genomic Gains in Breast Cancer and Its Association with the Host Immune Response.

Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy-number gains. Through FISH, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21% to 69%, median 55.5%, n = 19). Furthermore, they are more prevalent in the metastases of patients who died from breast cancer after multiple rounds of treatment (70% to 100%, median 93%, samples n = 41). Diversity indices revealed that higher spatial heterogeneity for these regions within primary tumors is associated with increased dissemination and metastasis. An identified subclone with multiple regions gained (MRG clone) was enriched in a posttreatment primary breast carcinoma as well as multiple metastatic tumors and local breast recurrences obtained at autopsy, indicative of a distinct early subclone with the capability to resist multiple lines of treatment and eventually cause death. In addition, multiplex immunofluorescence revealed that tumor heterogeneity is significantly associated with the degree of infiltration of B lymphocytes in triple-negative breast cancer, a subtype with a large immune component. Collectively, these data reveal the functional potential of genetic subclones that comprise heterogeneous primary breast carcinomas and are selected for in CTCs and posttreatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment. SIGNIFICANCE: As breast cancers progress, they become more heterogeneous for multiple regions amplified in circulating tumor cells, and intratumoral spatial heterogeneity is associated with the immune landscape.

PacBio sequencing output increased through uniform and directional fivefold concatenation.

Advances in sequencing technology have allowed researchers to sequence DNA with greater ease and at decreasing costs. Main developments have focused on either sequencing many short sequences or fewer large sequences. Methods for sequencing mid-sized sequences of 600-5,000 bp are currently less efficient. For example, the PacBio Sequel I system yields ~ 100,000-300,000 reads with an accuracy per base pair of 90-99%. We sought to sequence several DNA populations of ~ 870 bp in length with a sequencing accuracy of 99% and to the greatest depth possible. We optimised a simple, robust method to concatenate genes of ~ 870 bp five times and then sequenced the resulting DNA of ~ 5,000 bp by PacBioSMRT long-read sequencing. Our method improved upon previously published concatenation attempts, leading to a greater sequencing depth, high-quality reads and limited sample preparation at little expense. We applied this efficient concatenation protocol to sequence nine DNA populations from a protein engineering study. The improved method is accompanied by a simple and user-friendly analysis pipeline, DeCatCounter, to sequence medium-length sequences efficiently at one-fifth of the cost.

Non-rhabdomyosarcoma soft tissue sarcomas diagnosed in patients at a young age. An overview of clinical, pathological, and molecular findings.

OBJECTIVE: Disease spectrum in pediatric sarcoma differs substantially from adults. We report a cohort of very young children with non-rhabdomyosarcoma soft tissue sarcoma (NRSTS) detailing their molecular features, treatment, and outcome. METHODS: We report features of consecutive children (age <2 years) with NRSTS (2000-2017). Archival pathological material was re-reviewed, with additional molecular techniques applied where indicated. RESULTS: Twenty-nine patients (16 females, 55%) were identified (median age 6 months; range 0-23). Most common diagnoses included infantile fibrosarcoma (IFS, n = 14, 48%), malignant rhabdoid tumor (MRT, n = 4, 14%), and undifferentiated sarcoma (n = 4, 14%). Twenty-seven of 29 (93%) had tumor molecular characterization to confirm diagnosis. Clinical presentation included a swelling/mass (n = 23, 79%). Disease extent was localized (n = 20, 69%), locoregional (n = 6, 21%), or metastatic (n = 3, 10%). Seventeen of 29 (59%) who underwent surgery achieved complete resection (R0). Other treatments included conventional chemotherapy (n = 26, 90%), molecularly targeted therapies (n = 3, 10%), and radiation (n = 5, 17%). At last follow-up (median 3 years; range 0.3-16.4), 23 (79%) were alive, disease-free and six (21%) had died of disease. All patients with IFS were alive and all those with MRT died. A cancer predisposition syndrome (CPS) was confirmed in three of 10 (30%) genetically tested patients. CONCLUSION: We recommend tumor molecular characterization in all young patients including evaluation for CPS to optimize treatment options and prognostication.

First report of t(5;11) KMT2A-MAML1 fusion in de novo infant acute lymphoblastic leukemia.

Infant acute lymphoblastic leukemia (ALL) comprises 2.5%-5% of pediatric ALL with inferior survival compared to older children. A majority of infants (80%) with ALL harbor KMT2A gene rearrangement, which portends a poor prognosis. Approximately 94 different partner genes have been identified to date. The common rearrangements include t(4;11)(q21;q23)KMT2A-AFF1,t(11;19) (q23;p13.3)KMT2A-MLLT1 and t(9;11)(p22;q23)KMT2A-MLLT3. We report a novel translocation t(5;11)(q35;q23)KMT2A-MAML1 in newly diagnosed infant precursor B-ALL. Long-term follow-up and a larger number of patients are needed to better understand its prognostic significance.

Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis.

BACKGROUND: Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling. METHODS: In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo. FINDINGS: CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ). INTERPRETATION: CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling. FUNDING: Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research.

An In Silico Investigation of Potential EGFR Inhibitors for the Clinical Treatment of Colorectal Cancer.

Colorectal cancer possesses the third highest diagnostic rate and is the second leading cause of cancer death in the USA as reported by NIH. Epidermal Growth Factor Receptor (EGFR), a transmembrane protein, participates in PLC gamma-1, RAS-RAF-MEK-MAPKs, phosphatidylinositol-3 kinase, Akt pathways and plays a key role in normal functioning of cell division, cell differentiation, apoptosis and migration. This protein is found to be overexpressed in more than 60% of the colorectal cancers. Overexpressed EGFR advances the tumorigenic properties through cell cycle dysregulation and activates signaling pathways linked to cancer such as WNT/ß-catenin, transforming growth factor ß (TGF-ß) and phosphoinositide-3-kinase (PI3K). Inhibiting the overexpressed EGFR protein has been proposed for the treatment and many inhibitors have been reported suppressing the activity of EGFR. However, patients in malignant state of cancer show resistance to those inhibitors, which open a wide space to research for the discovery of novel inhibitors. The present study employed Molecular Docking and Virtual Screening to find novel inhibitors with high affinity against EGFR. Molecular docking of existing inhibitors resulted in the compound titled as BGB-283 (PubChem CID-89670174) having the highest score, which was subjected to similarity search to retrieve the drugs with similar structure. The virtual screening concluded a compound SCHEMBL18435602 (PubChem CID-126517400) which revealed a better affinity with the target protein. A comparative study of both the compounds showed equivalent pharmacokinetic properties. These identified drugs have a high potential to act as EGFR inhibitors and can show promising results in the research of colorectal cancer.

The mutational landscape of accelerated- and blast-phase myeloproliferative neoplasms impacts patient outcomes.

There is a paucity of data regarding the impact of mutations on outcomes in accelerated-phase (AP) and blast-phase (BP) myeloproliferative neoplasms (MPNs). Moreover, it is unknown whether mutational status affects survival, as seen in chronic-phase MPNs. Therefore, we performed a retrospective analysis of all patients treated at our institution with AP/BP MPNs (N = 122; AP = 14; BP = 108) to comprehensively describe the mutational profile and correlate with clinical outcomes. Targeted sequencing with a 54-gene panel was performed. Forty-four patients were treated with intensive therapy, 27 with nonintensive therapy, and 51 with best supportive care (BSC). The most common mutation was JAK2V617F, occurring in 55% of subjects; CALR was found in 13% of patients and MPL in 6%. Thirty-two (26%) patients were triple negative. Other frequently mutated genes were ASXL1 (30%), TET2 (25%), SRSF2 (22%), RUNX1 (20%), and TP53 (17%). Mutations in 1, 2, 3, and ≥4 genes were seen in 15%, 13%, 25%, and 46% of patients, respectively. There was no difference in survival between patients treated with intensive vs nonintensive therapy, and the benefit of intensive therapy was limited to patients who were able to undergo transplantation. TP53 was the only individual mutation to correlate with shorter overall survival (hazard ratio, 1.89; P = .03). In the multivariate analysis, mutated TP53, ≥4 mutations, low albumin, increased peripheral blood blasts, ≥3 cytogenetic abnormalities, and BSC were associated with shorter survival. In conclusion, mutational data enhance the understanding of patients with AP/BP MPN who are likely to benefit from current therapeutic options.

Clinical Utility of Next-generation Sequencing in the Management of Myeloproliferative Neoplasms: A Single-Center Experience.

Although next-generation sequencing (NGS) has helped characterize the complex genomic landscape of myeloid malignancies, its clinical utility remains undefined. This has resulted in variable funding for NGS testing, limiting its accessibility. At our center, targeted sequencing (TAR-SEQ) using a 54-gene NGS myeloid panel is offered to all new patients referred for myeloid malignancies, as part of a prospective observational study. Here, we evaluated the diagnostic, prognostic, and potential therapeutic utility of clinical grade TAR-SEQ in the routine workflow of 179 patients with myeloproliferative neoplasms (MPN). Of 13 patients with triple negative (TN) MPN, who lacked driver mutations in JAK2, CALR, and MPL, TAR-SEQ confirmed clonal hematopoiesis in 8 patients. In patients with intermediate-risk myelofibrosis (MF), TAR-SEQ helped optimize clinical decisions in hematopoietic cell transplant (HCT)-eligible patients through identifying a high molecular risk (HMR) mutation profile. The presence of an HMR profile favored HCT in 9 patients with intermediate-1 risk MF. Absence of an HMR profile resulted in a delayed HCT strategy in 10 patients with intermediate-2 risk MF, 7 of which were stable at the last follow-up. Finally, TAR-SEQ identified patients with various targetable mutations in IDH1/2 (4%), spliceosome genes (28%), and EZH2 (7%). Some of these patients can be potential candidates for future targeted therapy trials. In conclusion, we have demonstrated that TAR-SEQ improves the characterization of TN MPN, can be integrated in clinical practice as an additional tool to refine decision making in HCT, and has the potential to identify candidates for future targeted therapy trials.

Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma.

Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development.

Negative Enrichment and Isolation of Circulating Tumor Cells for Whole Genome Amplification.

Circulating tumor cells (CTCs) are a rare population of cells found in the peripheral blood of patients with many types of cancer such as breast, prostate, colon, and lung cancers. Higher numbers of these cells in blood are associated with a poorer prognosis of patients. Genomic profiling of CTCs would help characterize markers specific for the identification of these cells in blood, and also define genomic alterations that give these cells a metastatic advantage over other cells in the primary tumor. Here, we describe an immunomagnetic method to enrich CTCs from the blood of patients with breast cancer, followed by single-cell laser capture microdissection to isolate single CTCs. Whole genome amplification of isolated CTCs allows for many downstream applications to be performed to aide in their characterization, such as whole genome or exome sequencing, Single Nucleotide Polymorphism (SNP) and copy number analysis, and targeted sequencing or quantitative Polymerase Chain Reaction (qPCR) for genomic analyses.

Molecular Profiling and Significance of Circulating Tumor Cell Based Genetic Signatures.

Cancer kills by metastasizing beyond the primary site. Early detection, surgical intervention and other treatments have improved the survival rates of patients with cancer, however, once metastasis occurs, responses to conventional therapies become significantly less effective, and this remains the leading cause of death. Circulating tumor cells (CTCs) are tumor cells that have preferentially disseminated from the primary tumor mass into the hematological system, and are en route to favorable distant sites where if they survive, can develop into metastases. They may be the earliest detectable cells with metastatic ability, and are gaining increasing attention because of their prognostic value in many types of cancers including breast, prostate, colon and lung. Recent technological advances have removed barriers that previously hindered the detection and isolation of these rare cells from blood, and have exponentially improved the genetic resolution at which we can characterize signatures that define CTCs. Some of the most significant observations from such examinations are described here. Firstly, aberrations that were thought to be unique to CTCs are detected at subclonal frequencies within primary tumors with measurable heterogeneity, indicating pre-existing genetic signatures for metastasis. Secondly, these subclonal events are enriched in CTCs and metastases, pointing towards the selection of a more 'fit' component of tumor cells with survival advantages. Lastly, this component of cancer cells may also be the chemoresistant portion that escapes systemic treatment, or acquires resistance during progression of the disease. The future of cancer management may include a standardized method of measuring intratumor heterogeneity of the primary as well as matched CTCs. This will help identify and target rare aberrations within primary tumors that make them more adept to disseminate, and also to monitor the development of treatment resistant subclones as cancer progresses.