Incidence of NUT carcinoma in Western Australia from 1989 to 2014: a review of pediatric and adolescent cases from Perth Children's Hospital.

BACKGROUND: NUT carcinoma (NC), previously known as NUT midline carcinoma, is a rare and very aggressive cancer that occurs in both children and adults. NC is largely chemoresistant, with an overall survival of less than 7 months. Because the carcinoma is not restricted to a particular organ, diagnosis is often a challenge. In the absence of a clearly determined incidence for NC, we sought to study the diagnosis of patients in a well-defined population. METHODS: We systematically reviewed records of all patients that presented to the Oncology Department of the Princess Margaret Hospital for Children from 1989 to 2014. This institution in the geographically isolated state of Western Australia has a catchment population of around 2 million. We then identified all high grade undifferentiated sarcomas or carcinomas in the 0-16 year age group. RESULTS: Over 26 years, we found 14 patients of 16 years or younger with undifferentiated malignant tumors. Of these, five tumors were positive by immunohistochemistry for the NUT/NUTM1 (Nuclear Protein in Testis) protein and/or the translocation t(15;19). Three patients presented with thoracic tumors, one with a para-spinal tumor, and one had an upper airway nasopharyngeal carcinoma. In all five cases, there was an initial response to therapy and then progression. This 26-year survey was conducted in a geographically isolated state with a well-defined population, and we determined an estimated incidence of NC of around 0.41 per million child years (0-16 yrs. of age) at risk. From three patients it was feasible to derive cell lines for further genetic analyses and drug screening. CONCLUSIONS: For the first time, the incidence of NC could be determined in a well-defined geographic area. The calculated rate of NC incidence is consistent with a history of under-recognition for this malignancy. These findings indicate that improved diagnostic detection of NC would enable better management and counselling of patients. Our findings emphasize the heterogeneity of NC, and they highlight the need to develop personalised therapy options, and to consider a diagnosis of NC in undifferentiated malignant tumors.

Case Report: Long-Term Survival of a Pediatric Patient With an Intra-Abdominal Undifferentiated Carcinoma of Unknown Primary.

An 8-year and 10-month-old boy presented following 2 weeks of abdominal pain, vomiting, constipation, and rectal pain. A diffuse lower-abdominal mass was felt upon palpation, with radiological findings confirming the presence of a large, multilobulated intraperitoneal mass with mesenteric lymphadenopathy and hepatic metastatic disease. A biopsy of the mass revealed anatomical pathological findings consistent with a diagnosis of intra-abdominal undifferentiated carcinoma of unknown primary (CUP). The patient was treated with six cycles of carboplatin and gemcitabine prior to surgery. Following incomplete resection of the tumor, four further cycles were administered resulting in resolution of the pelvic mass, but progression in the right and left lobes of the liver. Therapy was accordingly adjusted, with administration of six cycles of ifosfamide and doxorubicin followed by 1 year of metronomic vinorelbine and cyclophosphamide maintenance therapy. The patient remains in remission 7 years from completion of therapy. Whole exome sequencing revealed missense mutations in the DNA-repair and chromatin-remodeling genes FANCM and SMARCD2, and a tumor-derived cell line revealed a complex karyotype suggesting chromosomal instability. CUP is an extremely rare diagnosis in the pediatric population, previously reported during adolescence. This report provides detailed characterization of CUP in a young child and in the absence of defined therapeutic guidelines for pediatric CUP, the successful treatment strategy described should be considered for similar cases.

Molecular-genetic profiling and high-throughput in vitro drug screening in NUT midline carcinoma-an aggressive and fatal disease.

NUT midline carcinoma (NMC) is a rare and aggressive cancer, with survival typically less than seven months, that can arise in people of any age. Genetically, NMC is defined by the chromosomal fusion of NUTM1 with a chromatin-binding partner, typically the bromodomain-containing protein BRD4. However, little is known about other genetic aberrations in this disease. In this study, we used a unique panel of cell lines to describe the molecular-genetic features of NMC. Next-generation sequencing identified a recurring high-impact mutation in the DNA-helicase gene RECQL5 in 75% of lines studied, and biological signals from mutation-signature and network analyses consistent with a general failure in DNA-repair. A high-throughput drug screen confirmed that microtubule inhibitors, topoisomerase inhibitors and anthracyclines are highly cytotoxic in the majority of NMC lines, and that cell lines expressing the BRD4-NUTM1 (exon11:exon2) variant are an order of magnitude more responsive to bromodomain inhibitors (iBETs) on average than those with other BRD4-NUTM1 translocation variants. We also identified a highly significant correlation between iBET and aurora kinase inhibitor efficacy in this study. Integration of exome sequencing, transcriptome, and drug sensitivity profiles suggested that aberrant activity of the nuclear receptor co-activator NCOA3 may correlate with poor response to iBETs. In conclusion, our data emphasize the heterogeneity of NMC and highlights genetic aberrations that could be explored to improve therapeutic strategies. The novel finding of a recurring RECQL5 mutation, together with recent reports of chromoplexy in this disease, suggests that DNA-repair pathways are likely to play a central role in NMC tumorigenesis.

Heterogeneity in mechanisms of emergent resistance in pediatric T-cell acute lymphoblastic leukemia.

Relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem and is thought to be associated with clonal selection during treatment. In this study we used an established pre-clinical model of induction therapy to increase our understanding of the effect of engraftment and chemotherapy on clonal selection and acquisition of drug resistance in vivo. Immune-deficient mice were engrafted with patient diagnostic specimens and exposed to a repeated combination therapy consisting of vincristine, dexamethasone, L-asparaginase and daunorubicin. Any re-emergence of disease following therapy was shown to be associated with resistance to dexamethasone, no resistance was observed to the other three drugs. Immunoglobulin/T-cell receptor gene rearrangements closely matched those in respective diagnosis and relapse patient specimens, highlighting that these clonal markers do not fully reflect the biological changes associated with drug resistance. Gene expression profiling revealed the significant underlying heterogeneity of dexamethasone-resistant xenografts. Alterations were observed in a large number of biological pathways, yet no dominant signature was common to all lines. These findings indicate that the biological changes associated with T-ALL relapse and resistance are stochastic and highly individual, and underline the importance of using sophisticated molecular techniques or single cell analyses in developing personalized approaches to therapy.

Bioenergetic modulation overcomes glucocorticoid resistance in T-lineage acute lymphoblastic leukaemia.

Drug-resistant forms of acute lymphoblastic leukaemia (ALL) are a leading cause of death from disease in children. Up to 25% of patients with T-cell ALL (T-ALL) develop resistance to chemotherapeutic agents, particularly to glucocorticoids (GCs), a class of drug to which resistance is one of the strongest indicators of poor clinical outcome. Despite their clinical importance, the molecular mechanisms that underpin GC resistance and leukaemia relapse are not well understood. Recently, we demonstrated that GC-resistance is associated with a proliferative metabolism involving the up-regulation of glycolysis, oxidative phosphorylation and cholesterol biosynthesis. Here we confirm that resistance is directly associated with a glycolytic phenotype and show that GC-resistant T-ALL cells are able to shift between glucose bioenergetic pathways. We evaluated the potential for targeting these pathways in vitro using a glycolysis inhibitor, 2-deoxyglucose (2DG), and the oxidative phosphorylation inhibitor oligomycin in combination with methylprednisolone (MPRED). We found that oligomycin synergized with MPRED to sensitize cells otherwise resistant to GCs. Similarly we observed synergy between MPRED and simvastatin, an inhibitor of cholesterol metabolism. Collectively, our findings suggest that dual targeting of bioenergetic pathways in combination with GCs may offer a promising therapeutic strategy to overcome drug resistance in ALL.

Localization of sulphonylurea receptor proteins SUR2A and SUR2B and/or SUR1 in rat kidney.

To further explore precise expression and localization of sulphonylurea receptor isoforms SUR2A and SUR2B (SUR1) in rat kidney, total RNA was isolated from the kidney tissue using the TRIzol kit. Three different primer sets designed against SUR isoforms were used in reverse transcriptase reactions. Western blotting was done on membrane fractions obtained from kidney tissues using the primary antisera for SUR2A and SUR2B (SUR1). Paraformaldehyde fixed kidney sections were immunostained with SUR2A and SUR2B (SUR1) primary antisera. Sections were developed with DAB as a chromogen. RT-PCR results demonstrated mRNA consistent with SUR1 isoform to be the only identifiable transcript. Western blotting could not identify any protein consistent with SUR2A or SUR2B (SUR1) but recognized instead a smaller 55kD protein of unknown identity. Immunohistochemistry demonstrated a differential staining pattern whereby SUR2A was localized to the mesangial cells, intra- and extrarenal blood vessels and smooth muscles. In contrast, SUR2B (SUR1) was localized only to distal nephron epithelia. Intense immunoreactivity was localized to the thick ascending limb and as well as in the outer and inner medullary collecting ducts, both. Our results demonstrate differential and highly localized expression pattern of sulphonylurea receptor proteins SUR2A and 2B (SUR1) in rat kidney with implications for drug design.

Drug-gene modeling in pediatric T-cell acute lymphoblastic leukemia highlights importance of 6-mercaptopurine for outcome.

Patients relapsing with T-cell acute lymphoblastic leukemia (T-ALL) face a dismal outcome. The aim of this study was to identify new markers of drug resistance and clinical response in T-ALL. We measured gene expression and drug sensitivity in 15 pediatric T-ALL cell lines to find signatures predictive of resistance to 10 agents used in therapy. These were used to generate a model for outcome prediction in patient cohorts using microarray data from diagnosis specimens. In three independent T-ALL cohorts, the 10-drug model was able to accurately identify patient outcome, indicating that the in vitro-derived drug-gene profiles were clinically relevant. Importantly, predictions of outcome within each cohort were linked to distinct drugs, suggesting that different mechanisms contribute to relapse. Sulfite oxidase (SUOX) expression and the drug-transporter ABCC1 (MRP1) were linked to thiopurine sensitivity, suggesting novel pathways for targeting resistance. This study advances our understanding of drug resistance in T-ALL and provides new markers for patient stratification. The results suggest potential benefit from the earlier use of 6-mercaptopurine in T-ALL therapy or the development of adjuvants that may sensitize blasts to this drug. The methodology developed in this study could be applied to other cancers to achieve patient stratification at the time of diagnosis.

FusionFinder: a software tool to identify expressed gene fusion candidates from RNA-Seq data.

The hallmarks of many haematological malignancies and solid tumours are chromosomal translocations, which may lead to gene fusions. Recently, next-generation sequencing techniques at the transcriptome level (RNA-Seq) have been used to verify known and discover novel transcribed gene fusions. We present FusionFinder, a Perl-based software designed to automate the discovery of candidate gene fusion partners from single-end (SE) or paired-end (PE) RNA-Seq read data. FusionFinder was applied to data from a previously published analysis of the K562 chronic myeloid leukaemia (CML) cell line. Using FusionFinder we successfully replicated the findings of this study and detected additional previously unreported fusion genes in their dataset, which were confirmed experimentally. These included two isoforms of a fusion involving the genes BRK1 and VHL, whose co-deletion has previously been associated with the prevalence and severity of renal-cell carcinoma. FusionFinder is made freely available for non-commercial use and can be downloaded from the project website (http://bioinformatics.childhealthresearch.org.au/software/fusionfinder/).

Influence of wild-type MLL on glucocorticoid sensitivity and response to DNA-damage in pediatric acute lymphoblastic leukemia.

BACKGROUND: Rearrangement of the mixed-lineage leukemia gene (MLL) is found in 80% of infant acute lymphoblastic leukemia (ALL) and is associated with poor prognosis and resistance to glucocorticoids (GCs). We have recently observed that GC resistance in T-ALL cell lines is associated with a proliferative metabolism and reduced expression of MLL. In this study we have further explored the relationship between MLL status and GC sensitivity. RESULTS: Negative correlation of MLL expression with GC resistance in 15 T-ALL cell lines was confirmed by quantitative RT-PCR. The absence of MLL-rearrangements suggested that this relationship represented expression of wild-type MLL. Analysis of MLL expression patterns revealed a negative relationship with cellular metabolism, proliferation and anti-apoptotic transcriptional networks. In silico analysis of published data demonstrated that reduced levels of MLL mRNA are associated with relapse and prednisolone resistance in T-ALL patients and adverse clinical outcome in children with MLL-rearranged ALL. RNAi knockdown of MLL expression in T-ALL cell lines significantly increased resistance to dexamethasone and gamma irradiation indicating an important role for wild-type MLL in the control of cellular apoptosis. CONCLUSIONS: The data suggests that reduced expression of wild-type MLL can contribute to GC resistance in ALL patients both with and without MLL-translocations.

Gene-based outcome prediction in multiple cohorts of pediatric T-cell acute lymphoblastic leukemia: a Children's Oncology Group study.

BACKGROUND: Continuous complete clinical remission in T-cell acute lymphoblastic leukemia (T-ALL) is now approaching 80% due to the implementation of aggressive chemotherapy protocols but patients that relapse continue to have a poor prognosis. Such patients could benefit from augmented therapy if their clinical outcome could be more accurately predicted at the time of diagnosis. Gene expression profiling offers the potential to identify additional prognostic markers but has had limited success in generating robust signatures that predict outcome across multiple patient cohorts. This study aimed to identify robust gene classifiers that could be used for the accurate prediction of relapse in independent cohorts and across different experimental platforms. RESULTS: Using HG-U133Plus2 microarrays we modeled a five-gene classifier (5-GC) that accurately predicted clinical outcome in a cohort of 50 T-ALL patients. The 5-GC was further tested against three independent cohorts of T-ALL patients, using either qRT-PCR or microarray gene expression, and could predict patients with significantly adverse clinical outcome in each. The 5-GC featured the interleukin-7 receptor (IL-7R), low-expression of which was independently predictive of relapse in T-ALL patients. In T-ALL cell lines, low IL-7R expression was correlated with diminished growth response to IL-7 and enhanced glucocorticoid resistance. Analysis of biological pathways identified the NF-kappaB and Wnt pathways, and the cell adhesion receptor family (particularly integrins) as being predictive of relapse. Outcome modeling using genes from these pathways identified patients with significantly worse relapse-free survival in each T-ALL cohort. CONCLUSIONS: We have used two different approaches to identify, for the first time, robust gene signatures that can successfully discriminate relapse and CCR patients at the time of diagnosis across multiple patient cohorts and platforms. Such genes and pathways represent markers for improved patient risk stratification and potential targets for novel T-ALL therapies.

Validation of a mouse xenograft model system for gene expression analysis of human acute lymphoblastic leukaemia.

BACKGROUND: Pre-clinical models that effectively recapitulate human disease are critical for expanding our knowledge of cancer biology and drug resistance mechanisms. For haematological malignancies, the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse is one of the most successful models to study paediatric acute lymphoblastic leukaemia (ALL). However, for this model to be effective for studying engraftment and therapy responses at the whole genome level, careful molecular characterisation is essential. RESULTS: Here, we sought to validate species-specific gene expression profiling in the high engraftment continuous ALL NOD/SCID xenograft. Using the human Affymetrix whole transcript platform we analysed transcriptional profiles from engrafted tissues without prior cell separation of mouse cells and found it to return highly reproducible profiles in xenografts from individual mice. The model was further tested with experimental mixtures of human and mouse cells, demonstrating that the presence of mouse cells does not significantly skew expression profiles when xenografts contain 90% or more human cells. In addition, we present a novel in silico and experimental masking approach to identify probes and transcript clusters susceptible to cross-species hybridisation. CONCLUSIONS: We demonstrate species-specific transcriptional profiles can be obtained from xenografts when high levels of engraftment are achieved or with the application of transcript cluster masks. Importantly, this masking approach can be applied and adapted to other xenograft models where human tissue infiltration is lower. This model provides a powerful platform for identifying genes and pathways associated with ALL disease progression and response to therapy in vivo.

Receptor mutation is not a common mechanism of naturally occurring glucocorticoid resistance in leukaemia cell lines.

Glucocorticoids (GCs) are among the most important drugs for the treatment of acute lymphoblastic leukaemia (ALL). Cell lines cultured in high GC concentrations typically contain mutated glucocorticoid receptor (GR), something that is rarely found in primary ALL specimens. We studied naturally occurring mechanisms of GC resistance and examined sensitivity to GC in 15 T-ALL cell lines grown without prior exposure to drugs. Resistance could not be attributed to mutations in GR or variations in levels of its expression. We conclude that this panel of cell lines provides a suitable in vitro model since it reflects GC resistance in primary ALL.

Mechanism of relapse in pediatric acute lymphoblastic leukemia.

Relapse following initial chemotherapy remains a barrier to survival in approximately 20% of children suffering from acute lymphoblastic leukemia (ALL). Recently, to investigate the mechanism of relapse, we analysed clonal populations in 27 pairs of matched diagnosis and relapse ALL samples using PCR-based detection of multiple antigen receptor gene rearrangements. These clonal markers revealed the emergence of apparently new populations at relapse in 13 patients. In those cases where the new 'relapse clone' could be detected in the diagnosis population, there was a close correlation between length of first remission and quantity of the relapse clone in the diagnosis sample. A shorter length of time to first relapse correlated with a higher quantity of the relapsing clone at diagnosis. This observation, together with demonstrated differential chemosensitivity between sub-clones at diagnosis, indicates that relapse in ALL patients may commonly involve selection of a minor intrinsically resistant sub-clone that is undetectable by routine PCR-based methods. From a clinical perspective, relapse prediction may be improved with strategies to detect minor potentially resistant sub-clones early during treatment, hence allowing intensification of therapy. Together with the availability of relevant in vivo experimental models and powerful technology for detailed analysis of patient specimens, this new information will help shape future experimentation towards targeted therapy for high-risk ALL.

Prediction of relapse in paediatric pre-B acute lymphoblastic leukaemia using a three-gene risk index.

Despite high cure rates 25% of children with acute lymphoblastic leukaemia (ALL) relapse and have dismal outcome. Crucially, many are currently stratified as standard risk (SR) and additional markers to improve patient stratification are required. Here we have used diagnostic bone marrow specimens from 101 children with pre-B ALL to examine the use of gene expression profiles (GEP) as predictors of long-term clinical outcome. Patients were divided into two cohorts for model development and validation based on availability of specimen material. Initially, GEP from 55 patients with sufficient material were analysed using HG-U133A microarrays, identifying an 18-gene classifier (GC) that was more predictive of outcome than conventional prognostic parameters. After feature selection and validation of expression levels by quantitative reverse transcription polymerase chain reaction (qRT-PCR), a three-gene qRT-PCR risk index [glutamine synthetase (GLUL), ornithine decarboxylase antizyme inhibitor (AZIN), immunoglobulin J chain (IGJ)] was developed that predicted outcome with an accuracy of 89% in the array cohort and 87% in the independent validation cohort. The data demonstrate the feasibility of using GEP to improve risk stratification in childhood ALL. This is particularly important for the identification of patients destined to relapse despite their current stratification as SR, as more intensive front-line treatment options for these individuals are already available.

High expression of connective tissue growth factor in pre-B acute lymphoblastic leukaemia.

In recent years microarrays have been used extensively to characterize gene expression in acute lymphoblastic leukaemia (ALL). Few studies, however, have analysed normal haematopoietic cell populations to identify altered gene expression in ALL. We used oligonucleotide microarrays to compare the gene expression profile of paediatric precursor-B (pre-B) ALL specimens with two control cell populations, normal CD34(+) and CD19(+)IgM(-) cells, to focus on genes linked to leukemogenesis. A set of eight genes was identified with a ninefold higher average expression in ALL specimens compared with control cells. All of these genes were significantly deregulated in an independent cohort of 101 ALL specimens. One gene, connective tissue growth factor (CTGF, also known as CCN2), had exceptionally high expression, which was confirmed in three independent leukaemia studies. Further analysis of CTGF expression in ALL revealed exclusive expression in B-lineage, not T-lineage, ALL. Within B-lineage ALL approximately 75% of specimens were consistently positive for CTGF expression, however, specimens containing the E2A-PBX1 translocation showed low or no expression. Protein studies using Western blot analysis demonstrated the presence of CTGF in ALL cell-conditioned media. These findings indicate that CTGF is secreted by pre-B ALL cells and may play a role in the pathophysiology of this disease.

Identification of novel molecular prognostic markers for paediatric T-cell acute lymphoblastic leukaemia.

In the last four decades the survival of patients with newly diagnosed childhood T-cell acute lymphoblastic leukaemia (T-ALL) has improved dramatically. In sharp contrast, relapsed T-ALL continues to confer a dismal prognosis. We sought to determine if gene expression profiling could uncover a signature of outcome for children with T-ALL. Using 12 patient specimens obtained before therapy started, we examined the gene expression profile by oligonucleotide microarrays. We identified three genes, CFLAR, NOTCH2 and BTG3, whose expression at the time of diagnosis accurately distinguished the patients according to disease outcome. These genes are involved in the regulation of apoptosis and cellular proliferation. The prognostic value of the three predictive genes was assessed in an independent cohort of 25 paediatric T-ALL patients using quantitative real-time reverse transcription polymerase chain reaction. Patients assigned to the adverse outcome group had a significantly higher cumulative incidence of relapse compared with patients assigned to the favourable outcome group (46% vs. 8%, P = 0.029). Five-year overall survival was also significantly worse in the patients assigned to the adverse outcome group (P = 0.0039). The independent influence of the 3-gene predictor was confirmed by multivariate analysis. Our study provides proof of principle that genome-wide expression profiling can detect novel molecular prognostic markers in paediatric T-ALL.

In vitro cytotoxicity of nelarabine, clofarabine and flavopiridol in paediatric acute lymphoblastic leukaemia.

The in vitro efficacies of three new drugs--clofarabine (CLOF), nelarabine (NEL) and flavopiridol (FP) - were assessed in a panel of acute lymphoblastic leukaemia (ALL) cell lines. The 50% inhibitory concentration (IC50) for CLOF across all lines was 188-fold lower than that of NEL. B-lineage, but not T-lineage lines, were >7-fold more sensitive to CLOF than cytosine arabinoside (ARAC). NEL IC50 was 25-fold and 113-fold higher than ARAC in T- and B-lineage, respectively. T-ALL cells were eightfold more sensitive to NEL than B-lineage but there was considerable overlap. FP was more potent in vitro than glucocorticoids and thiopurines and at doses that recent phase I experience predicts will translate into clinical efficacy. Potential cross-resistance of CLOF, NEL and FP was observed with many front-line ALL therapeutics but not methotrexate or thiopurines. Methotrexate sensitivity was inversely related to that of NEL and FP. Whilst NEL was particularly effective in T-ALL, a subset of patients with B-lineage ALL might also be sensitive. CLOF appeared to be marginally more effective in B-lineage than T-ALL and has a distinct resistance profile that may prove useful in combination with other compounds. FP should be widely effective in ALL if sufficient plasma levels can be achieved clinically.