Suzanne Kamel-Reid

Recurrent genomic alterations in sequential progressive leukoplakia and oral cancer: drivers of oral tumorigenesis?

A significant proportion (up to 62%) of oral squamous cell carcinomas (OSCCs) may arise from oral potential malignant lesions (OPMLs), such as leukoplakia. Patient outcomes may thus be improved through detection of lesions at a risk for malignant transformation, by identifying and categorizing genetic changes in sequential, progressive OPMLs. We conducted array comparative genomic hybridization analysis of 25 sequential, progressive OPMLs and same-site OSCCs from five patients. Recurrent DNA copy number gains were identified on 1p in 20/25 cases (80%) with minimal, high-level amplification regions on 1p35 and 1p36. Other regions of gains were frequently observed: 11q13.4 (68%), 9q34.13 (64%), 21q22.3 (60%), 6p21 and 6q25 (56%) and 10q24, 19q13.2, 22q12, 5q31.2, 7p13, 10q24 and 14q22 (48%). DNA losses were observed in >20% of samples and mainly detected on 5q31.2 (35%), 16p13.2 (30%), 9q33.1 and 9q33.29 (25%) and 17q11.2, 3p26.2, 18q21.1, 4q34.1 and 8p23.2 (20%). Such copy number alterations (CNAs) were mapped in all grades of dysplasia that progressed, and their corresponding OSCCs, in 70% of patients, indicating that these CNAs may be associated with disease progression. Amplified genes mapping within recurrent CNAs (KHDRBS1, PARP1, RAB1A, HBEGF, PAIP2, BTBD7) were selected for validation, by quantitative real-time PCR, in an independent set of 32 progressive leukoplakia, 32 OSSCs and 21 non-progressive leukoplakia samples. Amplification of BTBD7, KHDRBS1, PARP1 and RAB1A was exclusively detected in progressive leukoplakia and corresponding OSCC. BTBD7, KHDRBS1, PARP1 and RAB1A may be associated with OSCC progression. Protein–protein interaction networks were created to identify possible pathways associated with OSCC progression.

Exon-skipping in BCR/ABL is induced by ABL exon 2.

The BCR/ABL fusion gene is pathognomonic for chronic myelogenous leukaemia (CML). We have previously reported alternative splicing of BCR/ABL, as indicated by the detection of both p190- and p210-encoding transcripts, in about 60% of CML patient samples. These exon-skipping events involved the joining of ABL exon 2 to variable upstream BCR exons. Similarly, ABL exon 2 is alternatively spliced to either of two upstream ABL exons (1a or 1b) in c-ABL. We have constructed BCR and BCR/ABL minigenes to study this phenomenon in more detail. These constructs were transfected into various cell types and splicing was assessed by reverse transcriptase PCR. Whereas the basic BCR minigene expressed exon-inclusive transcripts only, insertion of genomic DNA spanning ABL exon 2 induced exon-skipping but only when expressed in the CML cell lines K562 and EM3. In this study we localized the required sequence element to ABL exon 2 itself. These results mimic the splicing phenotype displayed by most CML patients. We propose a model where a trans-factor present in some CML cells interacts with ABL exon 2 pre-mRNA to promote skipping of upstream BCR exons.

Minimally Invasive Real-Time Detection of Actionable Mutations in Patients With Metastatic Solid Tumors Using Fine-Needle and Liquid Biopsies

PurposeFine-needle biopsy (FNB) and liquid biopsy are minimally invasive methods of tumor sampling that provide feasible means to assess tumor genotypes in real time. However, more data are needed to establish the strength of these methods by benchmarking against the current gold standard methods, core-needle biopsy (CNB) or surgical excision of the tumor.Patients and MethodsEligible patients with advanced solid tumors were prospectively recruited. We performed mutation profiling using matched tumor DNA obtained by CNB, FNB and liquid biopsy, and matrix-assisted laser desorption/ionization time-of-flight custom mass-spectrometry or targeted next-generation DNA sequencing. The actionability of detected mutations was determined using the OncoKB Web tool. Agreement between mutations detected in CNBs, FNBs, and circulating tumor DNA (ctDNA) was examined.ResultsForty-one patients underwent tumor biopsy. Thirty CNBs (73%) and 34 FNBs (83%) had sufficient tumor and DNA for mutation profiling. Median DNA yield fr...

Abstract 5010: Nucleophosmin (NPM) is univerally deregulated in acute promyelocytic leukemia

NPM is a multifunctional nucleolar phosphoprotein with roles in ribosome biogenesis, centrosome duplication, p53 response and DNA repair, and is commonly mutated in Acute myelogenous leukemia (AML). NPM is juxtaposed with retinoic acid receptor α (RARα[[Unsupported Character - [[Unsupported Character - uf029]]]]) in Acute Promyelocytic Leukemia (APL), raising the possibility that NPM functions are disrupted in this leukemia. We sought to determine the extent of NPM deregulation in the U937-NPM-RARα + cell line model, and to extend this analysis more generally to APL. Immunofluorescent microscopy (IF) demonstrated abnormal distribution of NPM in patient-derived APL cells carrying NPM-RARα or PML-RARα, as well as cell lines expressing X-RARα: NPM was distributed into abnormally large aggregates within the nucleus and/or throughout the cytoplasm. NPM distribution reverted to normal after 48 hr of treatment with 1.0 μM all-trans retinoic acid (ATRA). NPM protein levels were also significantly increased in X-RARα + cell lines. This may by due to an alteration at the protein level, as NPM mRNA expression was unaffected by X-RARα, and no mutations were detected in the NPM locus. Indeed, we found that NPM protein half-life was increased in U937 cells expressing X-RARα. These data suggested a potential disruption in nucleolar architecture in APL cells. Alterations in size and number of nucleolar organizing regions (NORs) in NB4 and U937-X-RARα cells were evident, when compared to U937 controls. Defects in nucleolar organization may lead to altered ribosome biogenesis, protein synthesis, as well as cell size and proliferation. Ribosomal RNA precursor expression was found to be increased significantly in U937-NPM-RARα cells, compared to controls. Relative cell volume, assessed by a flow cytometric assay, was moderately increased, by approximately 10% in NPM-RARα + cells, while cell proliferation rates were significantly increased, and doubling time decreased, compared to U937 controls. Analysis of protein synthesis rates in U937-NPM-RARα + cells indicated that the incorporation of a fluorescent Methionine analogue was twice that in control U937 cells. Finally, in order to determine the applicability of our in vitro results to APL patients, we analyzed pre-rRNA and 18S rRNA expression in bone marrow RNA extracted at diagnosis from 16 APL patients (10 BCR1/2 and 6 BCR3), in order to determine whether elevated nucleolar function was found in APL. 10/16 APLs had elevated 18S rRNA levels (>1.5-fold compared to normal BM); 8/16 had similarly elevated pre-rRNA levels. Overall, pre-rRNA levels were elevated an average of 2-fold compared to normal controls, while 18S rRNA levels were elevated an average 1.8-fold (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5010.

Abstract B32: Retrospective correlative study between EGFRvIII, HPV, p16, c‐MET and response to EGFR inhibitors in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN)

Background: No validated biomarkers exist to predict the response to EGFR inhibitors in SCCHN. A constitutively activated mutant, EGFRvIII, confers oncogenicity in human cancers and resistance to EGFR blockade, and is detected in 42% of 33 SCCHN tumors (Sok et al. Clin Cancer Res 2006). The aim of this study is to confirm the prevalence of EGFRvIII, HPV, p16, c‐MET in R/M SCCHN and evaluate their potential prognostic and predictive role. Materials and Methods: Archival tumor specimens of 53 patients (pts) who were treated in 4 phase I/II trials for R/M SCCHN at Princess Margaret Hospital from 2000–2005 were examined. Two of the 4 trials involved the EGFR inhibitor erlotinib (tumor specimens available in 35 of 48 pts) whereas the remaining 2 trials involved non‐EGFR targeted agents (tumor specimens available in 18 of 37 pts). EGFRvIII mutation was determined by quantitative RT‐PCR (positive result determined by decreased expression of exon 4 compared with exon 9 of the EGFR gene), presence of HPV DNA by Linear Array Genotyping, p16 and c‐MET expression by immunohistochemistry, using p16CINtec (Westborough, MA) and SP44 (Ventana, Tuczon, AZ) antibodies, respectively. Results: Demographics of the 53 pts were: median age 56 (range 15–78), F:M (%) = 23:77, ECOG 0:1:2 (%) = 28:64:8, locoregional recurrence = 85%, metastatic disease = 36%, oropharyngeal primary = 38%. Overall response rate (CR+PR) of the entire cohort to study treatment = 4/53 (7.5%), median TTP = 1.8 months, median OS = 5.9 months. Univariate analyses were significant for erlotinib‐treated pts compared to non‐erlotinib treated pts in both TTP and OS. EGFRvIII was detected in 22 pts (42%); median fold change was 6.8 (0.56–576.36). The presence of EGFRvIII mutation was associated with better disease control (PR+SD) on univariate analysis (p = 0.01), but no difference was seen between erlotinib‐treated versus non‐erlortinib treated pts. Median EGFRvIII fold changes were higher for pts with PR+SD than pts with PD (11.11 vs 3.16, p=0.04). The presence of EGFRvIII mutation was not associated with TTP or OS. HPV DNA (16, 6 or 33) was detected in 20 pts (38%), p16 immunostaining was present in 17 pts (32%) and c‐MET was highly expressed (IHC score >2) in 31 pts (58%). HPV, p16 and c‐MET were not associated with response, TTP or OS. Conclusions: This retrospective study confirms the presence of EGFRvIII mutation in about 40% of SCCHN, and it appears to be a prognostic biomarker associated with better disease control in R/M SCCHN regardless of treatment with erlotinib. Interestingly, the presence of activating mutations conferring a better prognosis has been reported with EGFR mutations in NSCLC (Eberhand et al. J Clin Oncol 2005) and with PIK3CA mutations in breast cancer (Kalinsky et al. Clin Cancer Res 2009). The positive prognostic value of EGFRvIII in R/M SCCHN is unexpected and large prospective studies are required to validate its significance. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B32.

Monitoring of Minimal Residual Hematologic Disease

In recent years, several advances have been made in predicting the outcome in hematologic malignancies, among them measurement of minimal residual disease (MRD). The term was coined to describe a scenario in which the leukemia cells are not eradicated from a patient in clinical remission but are at a level below the sensitivity of classic cytomorphologic methods. Determination of MRD is an evolving field in which the technology and result interpretation are continually being refined. Its detection relies on the presence of a leukemia-specific marker, which should be present in all leukemia cells and remain stable during disease evolution.