Copy Number Analysis in Cancer Diagnostic Testing.

Accurate detection of copy number alterations (CNAs) has become increasingly important in clinical oncology for the purpose of diagnosis, prognostication, and disease management. Cytogenetic approaches for the detection of CNAs, including karyotype, fluorescence in situ hybridization (FISH), and chromosomal microarray, remain mainstays in clinical laboratories. Yet, with rapidly decreasing costs and improved accuracy of CNA detection using emerging technologies such as next-generation sequencing and optical genome mapping, we are approaching a new era of cytogenomics and molecular oncology. The aim of this review is to describe the benefits and limitations associated with the routine clinical application of available classic, emerging, and projected future technologies for the detection of CNAs in oncology.

Very Low Vitamin D in a Patient With a Novel Pathogenic Variant in the GC Gene That Encodes Vitamin D-Binding Protein.

Circulating plasma vitamin D metabolites are highly bound to vitamin D-binding protein (DBP), also known as group-specific component or Gc-globulin. DBP, encoded by the GC gene, is a member of the albumin family of globular serum transport proteins. We previously described a homozygous GC gene deletion in a patient with apparent severe vitamin D deficiency, fragility fractures, and ankylosing spondylitis. Here, we report an unrelated patient free of fractures or rheumatologic disease, but with very low 25-hydroxyvitamin D and 1,25-hydroxyvitamin D, as well as undetectable DBP measured by liquid chromatography-tandem mass spectrometry. A whole gene deletion was excluded by microarray, and Sanger sequencing of GC revealed a homozygous pathogenic variant affecting a canonical splice site (c0.702-1G > A). These findings indicate that loss of function variants in GC that eliminate DBP, and severely reduced total circulating vitamin D levels, do not necessarily result in significant metabolic bone disease. Together with our previous report, these cases support the free-hormone hypothesis, and suggest free vitamin D metabolites may serve as preferable indicators of bone and mineral metabolism, particularly when clinical suspicion of DBP deficiency is high.

A Pan-Canadian Validation Study for the Detection of EGFR T790M Mutation Using Circulating Tumor DNA From Peripheral Blood.

INTRODUCTION: Genotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. METHODS: In phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. RESULTS: All laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. CONCLUSIONS: Discrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible.

Clinical implementation of circulating tumour DNA testing for EGFR T790M for detection of treatment resistance in non-small cell lung cancer.

AIMS: Epidermal growth factor receptor (EGFR) T790M mutations can be detected in the circulating tumour DNA from plasma of patients with non-small cell lung cancer (NSCLC) to triage patients for osimertinib eligibility and monitor patients longitudinally for development of T790M-mediated resistance. METHODS: Using droplet digital PCR (ddPCR), we examined the EGFR T790M status of 343 sequential patients with NSCLC and correlated mutational status with demographic and clinical features. Where available, serial T790M blood test results were assessed to identify clinical triggers and timing of repeat testing. RESULTS: Of the 343 patients with liquid biopsy test results, 24% were T790M positive. No clear clinical correlation with a T790M positive test result was identified in this study, although the number of metastatic sites did correlate significantly with the presence of EGFR sensitising mutations (L858R or exon 19 deletion) in patient plasma, as a measure of tumour DNA shedding. Of the 59 serial blood tests from patients that initially tested negative, 14% were positive on sequential testing, at a time interval up to 6 months after an initially negative blood test. CONCLUSIONS: The ddPCR test for EGFR T790M mutations effectively triaged 24% of patients for treatment with osimertinib, avoiding the need for invasive tissue biopsy in these patients. Our findings suggest that initial and repeat ctDNA testing can be used to monitor for acquired EGFR T790M resistance for NSCLC.

Heterogenous loss of mismatch repair (MMR) protein expression: a challenge for immunohistochemical interpretation and microsatellite instability (MSI) evaluation.

Immunohistochemistry (IHC) for mismatch repair (MMR) proteins is used to identify MMR status: being diffusely positive (intact/retained nuclear staining) or showing loss of nuclear tumour staining (MMR protein deficient). Four colonic adenocarcinomas and a gastric adenocarcinoma with associated dysplasia that displayed heterogenous IHC staining patterns in at least one of the four MMR proteins were characterised by next-generation sequencing (NGS). In order to examine a potential molecular mechanism for these staining patterns, the respective areas were macrodissected, analysed for microsatellite instability (MSI) and investigated by NGS and multiplex ligation-dependent probe amplification (MLPA) analysis of MLH1, MSH2, MSH6 and PMS2 genes, including MLH1 methylation analysis. One colonic adenocarcinoma showed heterogenous MSH6 IHC staining and molecular analysis demonstrated increasing allelic burden of two MSH6 frameshift variants (c.3261delC and c.3261dupC) in areas with MSH6 protein loss compared to areas where MSH6 was retained. Two colonic adenocarcinomas with heterogenous MLH1 staining showed no differences in sequence variants. In one of these cases, however, MLH1 was hypermethylated in the area of MLH1 loss. Another colon carcinoma with heterogenous PMS2 staining (but with retained MSH6) showed both MSH6 c.3261dupC and 3260_3261dupCC where PMS2 protein was lost and only c.3261dupC where PMS2 was retained. The gastric carcinoma showed complete loss of MSH6 in dysplastic foci, while the underlying invasive carcinoma showed retention of MSH6. Both these areas, however, were MSI-high and showed the same MSH6 variant: c.3261delC. The gastric dysplasia additionally showed MSH6 c.3261dupC. In four of the five cases where MMR protein was lost, these areas were MSI-high. Heterogenous MMR IHC (focal and/or zonal within the same tumour or between invasive and dysplastic preinvasive areas) is not always due to artefact and is invariably related to MSI-high status in the areas of loss. An interesting aspect to this study is the presence of MSH6 somatic mutations irrespective of whether MSH6 IHC staining was intact or lost.

Hyperthermia-mediated drug delivery induces biological effects at the tumor and molecular levels that improve cisplatin efficacy in triple negative breast cancer.

Triple negative breast cancer is an aggressive disease that accounts for at least 15% of breast cancer diagnoses, and a disproportionately high percentage of breast cancer related morbidity. Intensive research efforts are focused on the development of more efficacious treatments for this disease, for which therapeutic options remain limited. The high incidence of mutations in key DNA repair pathways in triple negative breast cancer results in increased sensitivity to DNA damaging agents, such as platinum-based chemotherapies. Hyperthermia has been successfully used in breast cancer treatment to sensitize tumors to radiation therapy and chemotherapy. It has also been used as a mechanism to trigger drug release from thermosensitive liposomes. In this study, mild hyperthermia is used to trigger release of cisplatin from thermosensitive liposomes in the vasculature of human triple negative breast cancer tumors implanted orthotopically in mice. This heat-triggered liposomal formulation of cisplatin resulted in significantly delayed tumor growth and improved overall survival compared to treatment with either non-thermosensitive liposomes containing cisplatin or free cisplatin, as was observed in two independent tumor models (i.e. MDA-MB-231 and MDA-MB-436). The in vitro sensitivity of the cell lines to cisplatin and hyperthermia alone and in combination was characterized extensively using enzymatic assays, clonogenic assays, and spheroid growth assays. Evaluation of correlations between the in vitro and in vivo results served to identify the in vitro approach that is most predictive of the effects of hyperthermia in vivo. Relative expression of several heat shock proteins and the DNA damage repair protein BRCA1 were assayed at baseline and in response to hyperthermia both in vitro and in vivo. Interestingly, delivery of cisplatin in thermosensitive liposomes in combination with hyperthermia resulted in the most significant tumor growth delay, relative to free cisplatin, in the less cisplatin-sensitive cell line (i.e. MDA-MB-231). This work demonstrates that thermosensitive cisplatin liposomes used in combination with hyperthermia offer a novel method for effective treatment of triple negative breast cancer.

MicroRNAs in extracellular vesicles: potential cancer biomarkers.

Extracellular vesicles (EV) are small membrane-bound structures that are secreted by various cell types, including tumor cells. Recent studies have shown that EVs are important for cell-to-cell communication, locally and distantly; horizontally transferring DNA, mRNA, microRNA (miRNA), proteins and lipids. In the context of cancer biology, tumor-derived EVs are capable of modifying the microenvironment, promoting tumor progression, immune evasion, angiogenesis and metastasis. miRNAs contained within EVs are functionally associated with cancer progression, metastasis and aggressive tumor phenotypes. These factors, along with their stability in bodily fluids, have led to extensive investigations on the potential role of circulating EV-derived miRNAs as tumor biomarkers. In this review, we summarize the current understanding of circulating EV miRNAs in human cancer, and discuss their clinical utility and challenges in functioning as biomarkers.

Optimizing computed tomography simulation wait times in a busy radiation medicine program.

PURPOSE: An audit was conducted of patient schedules for computed tomography simulation (CT-Sim) scans within the Radiation Medicine Program at the Princess Margaret Cancer Centre to investigate opportunities for improved efficiencies, enhancing process, reducing rescanning rates, and decreasing wait times. METHODS AND MATERIALS: A 3-phased approach was undertaken to evaluate the current practice in the CT-Sim facility with a view toward implementing improvements. The first phase involved a review and assessment of the validity of current guidelines and protocols associated with 16 different disease sites. The second phase incorporated the use of a patient record and verification program MOSAIQ to capture the duration of each appointment. The last phase allocated additional time for patient-centered care and staff engagement. RESULTS: The audit revealed that efficiency could be achieved through staff training, updating protocols, and improving process coordination. With the exception of sarcoma, pediatric, and palliative patients who require unique management approaches, the duration for each CT-Sim appointment was successfully shortened for all disease sites by 22% to 33%, corresponding to a reduction of 10 to 15 minutes per appointment. Rescanning rates for patients requiring self-administered preparations before CT-Sim procedures were also significantly reduced by enhancing processes to increase patient compliance. Implementation of procedural changes resulted in an overall net gain of 3060 minutes, equivalent to 102 additional 30-minute CT-Sim appointment slots available for each month. CONCLUSIONS: This retrospective evaluation, review, and optimization of CT-Sim guidelines and practices identified opportunities to shorten appointment timeslots, and reduce rescanning rates for CT-Sim procedures, thereby significantly shortening wait times and improving access to service for our patients.

HPV Associated Head and Neck Cancer.

Head and neck cancers (HNCs) are a highly heterogeneous group of tumours that are associated with diverse clinical outcomes. Recent evidence has demonstrated that human papillomavirus (HPV) is involved in up to 25% of HNCs; particularly in the oropharyngeal carcinoma (OPC) subtype where it can account for up to 60% of such cases. HPVs are double-stranded DNA viruses that infect epithelial cells; numerous HPV subtypes, including 16, 18, 31, 33, and 35, drive epithelial cell transformation and tumourigenesis. HPV positive (HPV+) HNC represents a distinct molecular and clinical entity from HPV negative (HPV-) disease; the biological basis for which remains to be fully elucidated. HPV positivity is strongly correlated with a significantly superior outcome; indicating that such tumours should have a distinct management approach. This review focuses on the recent scientific and clinical investigation of HPV+ HNC. In particular, we discuss the importance of molecular and clinical evidence for defining the role of HPV in HNC, and the clinical impact of HPV status as a biomarker for HNC.

MicroRNAs in nasopharyngeal carcinoma.

It is becoming increasingly evident that aberrantly expressed microRNAs (miRNAs) are responsible for a number of disease processes, including cancer initiation and progression. miRNAs have been implicated as key players in numerous neoplasms, including nasopharyngeal carcinoma (NPC). Functionally, deregulation of miRNAs that act either as tumour suppressors or oncogenes results in numerous cancer-associated phenomena, including changes in proliferation, migration, and cell survival. Furthermore, miRNA expression has been associated with chemoresistant or radioresistant phenotypes; highlighting the importance of miRNAs in mediating oncogenic processes. Prognostic and predictive miRNA signatures have been defined for a variety of cancer types, including NPC, whereby these signatures offer a potentially important clinical tool for assessing the disease state, as well as predicting treatment response and clinical outcome. Therefore, further examination and validation of miRNAs that are deregulated in NPC will provide insight into the fundamental drivers of this disease, which will aid in the identification of novel targeted treatments. This review summarizes recent advances in the study of miRNAs in NPC, with specific discussion on the role of miRNAs in NPC pathogenesis and the potential utility of miRNAs as prognostic biomarkers. Our increasing understanding of the role of miRNAs in NPC tumorigenesis and their application as novel biomarkers will undoubtedly prove useful in the stratification of future patients into clinically relevant treatment classifications, thereby improving and personalizing disease management.

Preclinical imaging and translational animal models of cancer for accelerated clinical implementation of nanotechnologies and macromolecular agents.

The majority of animal models of cancer have performed poorly in terms of predicting clinical performance of new therapeutics, which are most often first evaluated in patients with advanced, metastatic disease. The development and use of metastatic models of cancer may enhance clinical translatability of preclinical studies focused on the development of nanotechnology-based drug delivery systems and macromolecular therapeutics, potentially accelerating their clinical implementation. It is recognized that the development and use of such models are not without challenge. Preclinical imaging tools offer a solution by allowing temporal and spatial characterization of metastatic lesions. This paper provides a review of imaging methods applicable for evaluation of novel therapeutics in clinically relevant models of advanced cancer. An overview of currently utilized models of oncology in small animals is followed by image-based development and characterization of visceral metastatic cancer models. Examples of imaging tools employed for metastatic lesion detection, evaluation of anti-tumor and anti-metastatic potential and biodistribution of novel therapies, as well as the co-development and/or use of imageable surrogates of response, are also discussed. While the focus is on development of macromolecular and nanotechnology-based therapeutics, examples with small molecules are included in some cases to illustrate concepts and approaches that can be applied in the assessment of nanotechnologies or macromolecules.

Integration of imaging into clinical practice to assess the delivery and performance of macromolecular and nanotechnology-based oncology therapies.

Functional and molecular imaging has become increasingly used to evaluate interpatient and intrapatient tumor heterogeneity. Imaging allows for assessment of microenvironment parameters including tumor hypoxia, perfusion and proliferation, as well as tumor metabolism and the intratumoral distribution of specific molecular markers. Imaging information may be used to stratify patients for targeted therapies, and to define patient populations that may benefit from alternative therapeutic approaches. It also provides a method for non-invasive monitoring of treatment response at earlier time-points than traditional cues, such as tumor shrinkage. Further, companion diagnostic imaging techniques are becoming progressively more important for development and clinical implementation of targeted therapies. Imaging-based companion diagnostics are likely to be essential for the validation and FDA approval of targeted nanotherapies and macromolecular medicines. This review describes recent clinical advances in the use of functional and molecular imaging to evaluate the tumor microenvironment. Additionally, this article focuses on image-based assessment of distribution and anti-tumor effect of nano- and macromolecular systems.

CNS-PNETs with C19MC amplification and/or LIN28 expression comprise a distinct histogenetic diagnostic and therapeutic entity.

Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.

Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR.

Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.

A novel C19MC amplified cell line links Lin28/let-7 to mTOR signaling in embryonal tumor with multilayered rosettes.

BACKGROUND: Embryonal tumor with multilayered rosettes (ETMR) is an aggressive central nervous system primitive neuroectodermal tumor (CNS-PNET) variant. ETMRs have distinctive histology, amplification of the chromosome 19 microRNA cluster (C19MC) at chr19q13.41-42, expression of the RNA binding protein Lin28, and dismal prognosis. Functional and therapeutic studies of ETMR have been limited by a lack of model systems. METHODS: We have established a first cell line, BT183, from a case of ETMR and characterized its molecular and cellular features. LIN28 knockdown was performed in BT183 to examine the potential role of Lin28 in regulating signaling pathway gene expression in ETMR. Cell line findings were corroborated with immunohistochemical studies in ETMR tissues. A drug screen of 73 compounds was performed to identify potential therapeutic targets. RESULTS: The BT183 line maintains C19MC amplification, expresses C19MC-encoded microRNAs, and is tumor initiating. ETMRs, including BT183, have high LIN28 expression and low let-7 miRNA expression, and show evidence of mTOR pathway activation. LIN28 knockdown increases let-7 expression and decreases expression of IGF/PI3K/mTOR pathway components. Pharmacologic inhibition of the mTOR pathway reduces BT183 cell viability. CONCLUSIONS: BT183 retains key genetic and histologic features of ETMR. In ETMR, Lin28 is not only a diagnostic marker but also a regulator of genes involved in growth and metabolism. Our findings indicate that inhibitors of the IGF/PI3K/mTOR pathway may be promising novel therapies for these fatal embryonal tumors. As the first patient-derived cell line of these rare tumors, BT183 is an important, unique reagent for investigating ETMR biology and therapeutics.

Mutational analysis of the Sinorhizobium meliloti short-chain dehydrogenase/reductase family reveals substantial contribution to symbiosis and catabolic diversity.

The short-chain dehydrogenase/reductase (SDR) family is one of the largest and most ubiquitous protein families in bacterial genomes. Despite there being a few well-characterized examples, the substrate specificities or functions of most members of the family are unknown. In this study, we carried out a large-scale mutagenesis of the SDR gene family in the alfalfa root nodule symbiont Sinorhizobium meliloti. Subsequent phenotypic analysis revealed phenotypes for mutants of 21 of the SDR-encoding genes. This brings the total number of S. meliloti SDR-encoding genes with known function or associated phenotype to 25. Several of the mutants were deficient in the utilization of specific carbon sources, while others exhibited symbiotic deficiencies on alfalfa (Medicago sativa), ranging from partial ineffectiveness to complete inability to form root nodules. Five of the mutants had both symbiotic and carbon utilization phenotypes. These results clearly demonstrate the importance of the SDR family in both symbiosis and saprotrophy, and reinforce the complex nature of the interaction of S. meliloti with its plant hosts. Further analysis of the genes identified in this study will contribute to the overall understanding of the biology and metabolism of S. meliloti in relation to its interaction with alfalfa.