Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Case Reports
. 2016 Oct 31;8(1):116.
doi: 10.1186/s13073-016-0366-0.

A Case Study of an Integrative Genomic and Experimental Therapeutic Approach for Rare Tumors: Identification of Vulnerabilities in a Pediatric Poorly Differentiated Carcinoma

Affiliations
Free PMC article
Case Reports

A Case Study of an Integrative Genomic and Experimental Therapeutic Approach for Rare Tumors: Identification of Vulnerabilities in a Pediatric Poorly Differentiated Carcinoma

Filemon S Dela Cruz et al. Genome Med. .
Free PMC article

Abstract

Background: Precision medicine approaches are ideally suited for rare tumors where comprehensive characterization may have diagnostic, prognostic, and therapeutic value. We describe the clinical case and molecular characterization of an adolescent with metastatic poorly differentiated carcinoma (PDC). Given the rarity and poor prognosis associated with PDC in children, we utilized genomic analysis and preclinical models to validate oncogenic drivers and identify molecular vulnerabilities.

Methods: We utilized whole exome sequencing (WES) and transcriptome analysis to identify germline and somatic alterations in the patient's tumor. In silico and in vitro studies were used to determine the functional consequences of genomic alterations. Primary tumor was used to generate a patient-derived xenograft (PDX) model, which was used for in vivo assessment of predicted therapeutic options.

Results: WES revealed a novel germline frameshift variant (p.E1554fs) in APC, establishing a diagnosis of Gardner syndrome, along with a somatic nonsense (p.R790*) APC mutation in the tumor. Somatic mutations in TP53, MAX, BRAF, ROS1, and RPTOR were also identified and transcriptome and immunohistochemical analyses suggested hyperactivation of the Wnt/ß-catenin and AKT/mTOR pathways. In silico and biochemical assays demonstrated that the MAX p.R60Q and BRAF p.K483E mutations were activating mutations, whereas the ROS1 and RPTOR mutations were of lower utility for therapeutic targeting. Utilizing a patient-specific PDX model, we demonstrated in vivo activity of mTOR inhibition with temsirolimus and partial response to inhibition of MEK.

Conclusions: This clinical case illustrates the depth of investigation necessary to fully characterize the functional significance of the breadth of alterations identified through genomic analysis.

Keywords: BRAF; MAX; Patient-derived xenograft (PDX) models; Poorly differentiated carcinoma (PDC); Precision medicine; Temsirolimus; Whole exome sequencing (WES); mTOR.

Figures

Fig. 1
Fig. 1
Clinical presentation of metastatic PDC. a Representative scalp nodule. b, c Diagnostic imaging demonstrating the presence of multiple lytic lesions of the calvarium (b) as well as heterogeneous lesions within the liver with associated hepatosplenomegaly (c). di Immunohistochemical staining consistent with diagnosis of a PDC with high proliferative index: (d) H&E (200X), (e) cytokeratin 5 (200X), (f) cytokeratin 10 (100X), (g) EpCAM (400X), (h) ß-catenin (400X), (i) Ki67 (200X). Scale bar = 100 μm
Fig. 2
Fig. 2
WES and transcriptome sequencing of a primary tumor. a Circos plot summarizing WES and transcriptome analysis of primary tumor. Inner circle represents structural variants and gene fusions; second tier, copy number variations (blue, loss; red, gain); third tier, mRNA expression outlier analysis of cancer related genes within the top and bottom 10th percentile (green, under-expressed; orange, over-expressed); fourth tier (outer circle), somatic mutations localized to respective chromosomes. b Scatter-plot showing the t-SNE 2D projection for 3167 samples, including at least 100 samples (indicated in the figure) for each of the 34 tissue types represented in our pan-cancer database. Tissue ID is indicated by different colors and the carcinoid sample is indicated by a bold black dot and arrow. c Relative gene expression rank of outlier genes after z-normalization across a compendium of expression profiles from the GTEx database. A z-distribution is superimposed as reference. ACC adrenocortical carcinoma, BLCA bladder urothelial carcinoma, BRCA breast carcinoma, CESC cervical carcinoma, CHOL cholangiocarcinoma, COAD colon adenocarcinoma, DLBC diffuse large B-cell lymphoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme, HNSC head and neck carcinoma, KICH kidney chromophobe, KIRC clear cell carcinoma of the kidney, KIRP renal papillary cell carcinoma, LAML acute myeloid leukemia, LGG low grade glioma, LIHC hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, MESO mesothelioma, NET gastrointestinal neuroendocrine tumor, OV ovarian carcinoma, PAAD pancreatic adenocarcinoma, PCPG pheochromocytoma and paraganglioma, PRAD prostate adenocarcinoma, READ rectal adenocarcinoma, SARC sarcoma, SKCM cutaneous melanoma, STAD gastric adenocarcinoma, TGCT testicular germ cell tumor, THCA thyroid carcinoma, THYM thymoma, UCEC uterine corpus endometrial carcinoma, UCS uterine carcinosarcoma, UVM uveal melanoma
Fig. 3
Fig. 3
Structural and functional analyses of Variants of Unknown Significance (VUS). ac Structures of MAX homodimer and C-MYC-MAX and MXD1-MAX heterodimers in complex with DNA. a MAX-MAX homodimer crystal structure (PDB id: 1AN2) in which the subunit A (yellow for carbon atoms) and B (cyan for carbon atoms) are represented and the side chains of several invariant residues are depicted with stick models and labeled. b Crystal structure of C-MYC/-MAX heterodimer in complex with DNA (PDB id: 1NKP). MAX and C-MYC carbon atoms are represented in yellow and purple, respectively. c Crystal structure of MAX-MXD1 heterodimer in complex with DNA (PDB id: 1NLW). MAX and MXD1 carbon atoms are represented in yellow and green, respectively. In all structures presented, the MAX p.R60Q mutation is shown in magenta. Dashed lines (black and magenta) represent hydrogen bonds. The sugar-phosphate backbone of DNA is shown in orange with two selected nucleotides from each subunit shown as stick models. d MAXR60Q mutant heterodimerizes with C-MYC and MXD1 and binds to DNA. The indicated proteins were transcribed and translated in vitro and incubated with an E-box containing probe. Specific proteins/DNA complex bands are indicated on the left. Non-specific (ns) binding products present in the probe-only and vector control lanes are indicated on the left. e, f Structures of wild-type BRAF and BRAF p.K483E mutant. e Model of the BRAF kinase domain in complex with ATP (black for carbon atoms) and a Mg2+ ion (dark green), in which the side chains of five essential residues in BRAF, are shown, and labeled. The helix αC in its active conformation (dark violet) (PDB id: 4MNE) and in inactive conformation (light gray) (PDB id: 4WO5) is represented as cartoon and the side chain of the invariant E501 is depicted with stick models in two orientations. f Model of the BRAF kinase domain in which K483 is replaced by E (magenta for carbon atoms). g Proteins levels and phosphorylation level of ERK1/2 upon transient transfection of the indicated BRAF proteins in HEK 293 T cells
Fig. 4
Fig. 4
a Sensitivity of PDX tumors to the mTOR inhibitor, temsirolimus. Chemoresistance to carboplatin and JQ1 were observed following a transient period of response. Mean and standard error of the mean (SEM) are shown. b Phosphorylation level of RPS6 upon temsirolimus treatment. c C-MYC and N-MYC protein levels upon JQ1 treatment. d Temsirolimus treatment results in decreased Ki-67 staining with concomitant increase in cleaved caspase 3 (Cl. CASP 3) following short-term (3 days) and long-term (50 days) treatments. * p < 0.05, ** p < 0.01. e Tumor growth after temsirolimus treatment withdrawal. Mean and SEM are shown. f Temsirolimus treatment can successfully rescue and induce tumor regression in carboplatin-resistant tumors. Mean and SEM are shown. g Combination therapy (temsirolimus and irinotecan) does not result in increased anti-tumor activity. Tumor regrowth is observed with withdrawal of treatment. Mean and SEM are shown

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

References

    1. Greco FA. Molecular diagnosis of the tissue of origin in cancer of unknown primary site: useful in patient management. Curr Treat Options Oncol. 2013;14(4):634–42. doi: 10.1007/s11864-013-0257-1. - DOI - PubMed
    1. Nystrom SJ, Hornberger JC, Varadhachary GR, Hornberger RJ, Gutierrez HR, Henner DW, et al. Clinical utility of gene-expression profiling for tumor-site origin in patients with metastatic or poorly differentiated cancer: impact on diagnosis, treatment, and survival. Oncotarget. 2012;3(6):620–8. doi: 10.18632/oncotarget.521. - DOI - PMC - PubMed
    1. Varadhachary GR, Karanth S, Qiao W, Carlson HR, Raber MN, Hainsworth JD, et al. Carcinoma of unknown primary with gastrointestinal profile: immunohistochemistry and survival data for this favorable subset. Int J Clin Oncol. 2014;19(3):479–84. doi: 10.1007/s10147-013-0583-0. - DOI - PubMed
    1. Hainsworth JD, Rubin MS, Spigel DR, Boccia RV, Raby S, Quinn R, et al. Molecular gene expression profiling to predict the tissue of origin and direct site-specific therapy in patients with carcinoma of unknown primary site: a prospective trial of the Sarah Cannon research institute. J Clin Oncol. 2013;31(2):217–23. doi: 10.1200/JCO.2012.43.3755. - DOI - PubMed
    1. Dienstmann R, Rodon J, Tabernero J. Optimal design of trials to demonstrate the utility of genomically-guided therapy: Putting Precision Cancer Medicine to the test. Mol Oncol. 2015;9(5):940–50. doi: 10.1016/j.molonc.2014.06.014. - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback