Annual Meeting Proceedings Part 1 - American Society of Clinical ...

678s Tumor Biology
10590 General Poster Session (Board #48G), Mon, 1:15 PM-5:15 PM
Next-generation sequencing (NGS) to identify actionable genomic changes
in common and rare solid tumors: The FMI experience with the initial 50
consecutive patients. Presenting Author: Gary A. Palmer, Foundation
Medicine, Cambridge, MA
Background: Solid tumor (ST) oncology has been transformed by the linkage
of genomic changes with targeted therapeutics. Unfortunately, most STs
still have no alteration detected with currently available assays. Thus, more
informative testing platforms are needed to analyze genomic changes in
FFPE biopsy samples often limited by tumor size, heterogeneity and ploidy.
Methods: We reviewed the genomic profiles of the first 50 clinical
specimens received by our CLIA lab (Foundation Medicine) and analyzed by
our NGS assay (Ross J. ASCO 2011). 182 genes known altered in STs and
14 genes known rearranged in STs were included. Genomic alterations
were categorized as “actionable” if linked to an approved therapy in the ST
under study or another ST (e.g. ALK rearrangement in lung or breast cancer,
respectively), a known or suspected contraindication to a given therapy
(e.g. KRAS G12D in colorectal cancer) or a clinical trial linked to the
alteration (e.g. mTOR inhibition in a tumor with STK11 loss). Results: Fifty
tumor specimens were received from 50 patients (median age 54,
F/M�30/20) representing 35 sites. Two samples failed analysis and 6 had
no detectable genomic alteration. Among 16 primary tumor types, lung
(n�15), colorectal (n�7), sarcoma (n�5), breast and esophageal/gastric
(n�4 each) were most common. We reported 135 genomic alterations
(mean 2.8, range 0-6) unequivocally involved in oncogenesis and 69
actionable alterations. At most, 29 of these (42%) would have been
detected by current assays (138% increase with NGS). Thirty seven
patients had one or more actionable alterations (range 1-4) detected by
NGS (74%, 95% CI 60-85%) versus only 21 (42%, 95% CI 28-57%) in
whom changes would have been detected by current assays. Conclusions:
Use of a broad, comprehensive NGS assay identifies an unprecedented
number of actionable genomic alterations across a variety of STs from
routine FFPE samples. This assay can serve as a paradigm for improving
access to approved therapies in STs, minimizing use of ineffective
therapies and enhancing enrollment in rationally chosen trials. Ongoing
and planned trials will study the impact on several efficacy endpoints.
10592 General Poster Session (Board #49A), Mon, 1:15 PM-5:15 PM
Genomic profiling of non-small cell lung cancer (NSCLC) for personalized
targeted therapy using CT-guided transthoracic needle biopsy (TTNB).
Presenting Author: Ritu R. Gill, Brigham and Women’s Hospital, Harvard
Medical School, Boston, MA
Background: Genomic profiling for personalized targeted therapy is emerging
for NSCLC. DFCI introduced systematic testing for mutations in BRAF,
HER2, PIK3CA and ALK translocations in addition to EGFR and KRAS in
July 2009 as part of a prospective study. We report the utility, efficacy and
safety of CT guided TTNB in this cohort. Methods: Patients with stage IV or
relapsed NSCLC seen at the DFCI were referred to BWH for CT guided TTNB
of their tumors to identify driver mutations prior to starting therapy.
Pathology specimens were dissected and analyzed by PCR-Sanger sequencing
for mutations in selected exons of EGFR, KRAS, BRAF, PIK3CA and
HER2. ALK rearrangements were detected with fluorescence in-situ hybridization
(FISH). Testing was performed after the pathologist deemed that
the tissue was adequate. Complications such as pneumothorax and
hemorrhage were recorded. Admission rates were also recorded. Results:
Between 7/1/2009 and 1/09/2011, 81 patients underwent TTNB. The
median age was 63 years. 54 (67%) were female, 66 (88%) were
former/current smokers and 58(72%) had stage IIIB/IV disease. 64(79%)
patients had sufficient tissue on core biopsies for genomic profiling, 4 (6%)
of the 64 patients failed analysis for ALK rearrangements due to less than
50 tumor cells on the hybridized slide. The number of samples obtained
ranged from 1-5 (2 cm 18-20 (G)). Lesions biopsied ranged in size from
1.2–8.9 cm. Mutations were identified in 38/81 (46.9%) patients (EGFR:
18; KRAS: 17; ALK: 2;PIK3CA: 1). 23(28.3%) had pneumothoraces
15(�10%), 5 (10-30%) and 3(�30%). 6 (7%) patients needed chest
tubes. 9 (11%) were admitted post procedure for observation (8 for (24hrs)
and 1 (72hrs). 19(23%) (18 grade1; 1 grade 2) had intra-parenchymal
hemorrhage. A higher rate of pneumothorax was observed with the 18
gauge needles (p �.05). 15 of 20 (75%) patients with EGFR, HER2, BRAF
or ALK alterations were treated with molecularly targeted therapy based on
their genetic alteration. Conclusions: CT guided TTNB is a feasible, safe and
efficacious technique for genomic profiling for targeted therapy and
enables the acquisition of sufficient tissue for gene mutation analyses.
10591 General Poster Session (Board #48H), Mon, 1:15 PM-5:15 PM
Combining semiconductor-based sequencing with amplicon-based cancer
gene panels: A rapid next-gen approach to clinical cancer genotyping.
Presenting Author: Christopher L. Corless, Knight Diagnostic Laboratories,
Oregon Health & Science University, Portland, OR
Background: Bringing next-gen sequencing into clinical (CLIA-licensed)
laboratories is an important step in the advancement of personalized
cancer care. We have validated a new sequencing approach on the Ion
Torrent (IT) PGM using the AmpliSeq Cancer Panel, which covers hotspot
regions across 46 commonly mutated cancer genes. Methods: The AmpliSeq
panel is comprised of 190 primer pairs that are co-amplified in a
single tube to generate amplicons for sequencing. In our testing only 10ng
of input DNA was used. Initial PCR was for 20 cycles, after which the
amplicons were ligated with sequencing/barcode adapters, amplified for an
additional 7 cycles, and then subjected to emulsion PCR. The resulting
nanospheres were sequenced on an IT 316 chip. Results: We sequenced 44
samples of FFPE-derived tumor DNA that were previously genotyped on a
mass spectroscopy (MS)-based panel. Samples were barcoded and sequenced
in batches of 4, yielding an average of 2034 reads per amplicon
(range: 95-5162) and an average read length of 76bp. Overall, 95.4% of
reads were on target, and 79% of reads were AQ20 or better; 95% of the
190 amplicons had over 500 reads. All 42 known point mutations were
accurately identified by the variant caller software. Seventeen in/dels from
4 to 63 bp in length were at least partially visible upon manual inspection
of the read alignments, but some were not accurately called by the
software. In addition, 22 new mutations were identified in gene regions not
covered on our MS-based panel. We demonstrated sensitivity to the level of
5% mutant allele, and the correlation between allelic ratios measured by
MS and IT sequencing was excellent (r2�0.81). Two DNA samples from
laser-captured tumor worked well with the AmpliSeq Panel. Conclusions:
Combining solid-state sequencing with a highly multiplexed PCR method
for library construction is a rapid (48 hr) approach for next-gen sequencing
of clinical cancer samples. The process is highly scalable and larger,
cancer-specific amplicon panels are in development. Automated identification
of in/dels remains a challenge in next-gen sequencing output.
10593 General Poster Session (Board #49B), Mon, 1:15 PM-5:15 PM
Prevalence of TP53 p.R337H mutation in children with cancer from a
public hospital in southern Brazil. Presenting Author: Juliana Giacomazzi,
Programa de Pós Graduação em Medicina: Ciências Médicas, Universidade
Federal do Rio Grande do Sul (UFRGS) e Laboratório de Medicina
Genômica, Centro de Pesquisa Experimental, Hospital de Clinicas de Porto
Alegre (HCPA), Porto Alegre, Brazil
Background: Childhood cancers are a common feature in Li-Fraumeni/Li
Fraumeni-Like Syndromes (LFS/LFL), associated with the inheritance of a
germline TP53 mutation. Recently, a specific germline mutation in exon 10
of the TP53 gene, p.R337H, has been reported at a high prevalence in
Southeastern Brazil. Initial studies on this mutation claimed that the main,
if not exclusive, cancer risk in carriers is childhood adrenocortical carcinoma
(ADR). However, others recent reports identified p.R377H carriers
among LFL families and among a larger spectrum of tumors, such as
choroid plexus carcinoma (CPC) and osteosarcoma. The aim of this study
was to assess the frequency of the p.R337H mutation in children
diagnosed with tumors of the LFS/LFL spectrum: ADR, sarcoma, central
nervous system tumors, leukemia, germline cell tumors and Wilm´s tumor)
at the Children´s Cancer Institute of Rio Grande do Sul (Brazil) between
1998 and 2011. Methods: Family history (FH) was recorded in pedigrees,
and DNA was extracted from peripheral blood and FFPE samples by
standard methods. Screening for the p.R337H mutation was performed by
allelic discrimination (TaqMan assay) and by TP53 exon 10 sequencing.
Results: Analysis of 295 children diagnosed with tumors of the LFS/LFL
spectrum identified the p.R337H mutation in 9/11 (81,8%) children with
ADR (including a homozygous p.R337H individual) and in 2/2 (100%)
children with CPC. All individuals had loss of heterozygosity in the tumor
and in all cases the mutant allele occurred on the same founder haplotype.
One hundred fourth-six (49,5%) patients had a cancer FH in first or second
degree relatives, 47 (16,0%) had a FH of breast cancer and 49 (16,6%)
had FH of LFL consistent with Chompret and/or Birch criteria. Conclusions:
These results confirm the strong association of p.R337H with ADR and
CPC. A FH consistent with LFL syndrome is present in a high proportion of
children diagnosed with tumors of the LFS/LFL spectrum in Southern
Brazil. Financial support: FIPE-HCPA, CAPES, FAPERGS and Glaxo Smith
Kline.
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