High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult, because contaminating stromal cells overgrow the malignant cells.
We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts.
Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified seventy-seven FDA approved drugs with activity, including two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs.
Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice.
personalized chemotherapy; hprt; mice; cancer cell lines; drug screening
Pancreatic cancer (PC) is the fourth cause of death from cancer in the western world. Majority of patients present with advanced unresectable disease responding poorly to most chemotherapeutic agents. Chemotherapy for PC might be improved by adjusting it to individual genetic profiles. We attempt to identify genetic predictors of chemosensitivity to broad classes of anticancer drugs.
Using a panel of genetically defined human PC cell lines, we tested gemcitabine (anti-metabolite), docetaxel (anti-microtubule), mitomycin C (alkylating), irinotecan (topoisomerase I inhibitor), cisplatin (crosslinking), KU0058948 (Parp1 inhibitor), triptolide (terpenoid drug) and artemisinin (control).
All PC cell lines were sensitive to triptolide and docetaxel. Most PC cells were also sensitive to gemcitabine and MMC. The vast majority of PC cell lines were insensitive to cisplatin, irinotecan, and a Parp1 inhibitor. However, individual cell lines were often sensitive to these compounds in unique ways. We found that DPC4/SMAD4 inactivation sensitized PC cells to cisplatin and irinotecan by 2–4 fold, but they were modestly less sensitive to gemcitabine. PC cells were all sensitive to triptolide and 18% were sensitive to the Parp1 inhibitor. P16/CDKN2A inactivated PC cells were 3–4 fold less sensitive to gemcitabine and MMC.
Chemosensitivity of PC cells correlated with some specific genetic profiles. These results support the hypothesis that genetic subsets of pancreatic cancer exist, and these genetic backgrounds may permit one to personalize the chemotherapy of PC in the future. Further work will need to confirm these responses and determine their magnitude in vivo.
exome wide analysis; cytotoxicity screening; chemotherapeutic drugs; pancreatic cancer; personalizing chemotherapy
To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth.
We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA).
We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations.
Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts.
Next-generation sequencing; Validation; KRAS; BRAF; EGFR; Read depth; Deamination
The incidence of intrahepatic cholangiocarcinoma (ICC) is increasing worldwide. The prognosis of ICC is poor and a better understanding of ICC tumor biology is needed to more accurately predict clinical outcome and to suggest potential targets for more effective therapies. v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and BRAF are frequently mutated oncogenes that promote carcinogenesis in a variety of tumor types. In this study, we analyze a large set of ICC tumors (N = 54) for mutations in these genes and compare the clinical outcomes of wild type versus KRAS and BRAF mutant cases. Out of 54 cases, 7.4% were mutant for KRAS, 7.4% were mutant for BRAF and these were mutually exclusive. These mutant cases were associated with a higher tumor stage at time of resection and a greater likelihood of lymph node involvement. These cases were also associated with a worse long-term overall survival. Therefore, testing for KRAS and BRAF mutations could be a valuable adjunct in improving both prognosis and outcome stratification among patients with ICC.
While gene-directed enzyme prodrug therapy has shown potential as a cancer therapeutic in animal and clinical trials, concerns over the efficacy, selectivity, and safety of gene delivery vehicles have restricted its advance. In an attempt to relieve some of the demands on targeted gene delivery vehicles and achieve the full potential of enzyme prodrug therapy, cancer-targeted activity can be engineered into the enzyme itself. We previously engineered a switchable prodrug-activating enzyme that selectively kills human cancer cells accumulating the cancer marker hypoxia-inducible factor-1α (HIF-1α). This HIF-1α-activated protein switch (Haps59) is designed to increase its ability to convert the prodrug 5-fluorocytosine into the chemotherapeutic 5-fluorouracil in a HIF-1α-dependent manner. However, in cancer cell lines expressing Haps59 the 5FC sensitivity difference between the presence and absence of HIF-1α was not as large as desired. In this work, we aimed to improve the cancer specificity of this switch via a directed evolution approach utilizing random mutagenesis, linker mutagenesis, and random insertion and circular permutation. We identified improved HIF-1α-activated protein switches that confer E. coli with modest increases in HIF-1α-dependent 5FC toxicity. Additionally, the current bottleneck in the development of improved HIF-1α-activated protein switches is screening switch candidates in mammalian cells. To accommodate higher throughput and reduce experimental variability, we explored the use of Flp recombinase-mediated isogenic integration in 293 cells. These experiments raised the possibility that Haps59 can be activated by other interactors of the CH1 domain, and experiments in E. coli indicated that CITED2 can also activate Haps59. Although many CH1 binding partners are also oncogenes, CH1's promiscuous binding and subsequent off-target activation of Haps59 needs to be examined under normal physiological conditions to identify off-target activators. With aberrant activating molecules identified, further directed evolution can be performed to improve the cancer specificity of HIF-1α-activated protein switches.
Esophageal adenocarcinoma currently has one of the most rapidly
increasing tumor incidences in the United States, with the vast majority of
cases occurring on the backdrop of metaplastic epithelium (Barrett esophagus).
The availability of appropriate cell line models is essential for maintaining
the pace of esophageal cancer research and for pre-clinical validation of new
therapeutic modalities. The identity of several of the widely utilized
esophageal adenocarcinoma cell lines (BIC-1, SEG-1 and TE-7) have recently been
called into question. Here we describe the establishment and characterization of
a bona fide esophageal cancer cell line, JH-EsoAd1, from a patient with
Barrett-associated adenocarcinoma. The rapid dissemination of this cancer cell
line to the esophageal cancer research community should help ameliorate the
current scarcity of preclinical models in this disease.
Barrett esophagus; adenocarcinoma; cell line; JH-EsoAd1; genotyping
Intraductal papillary mucinous neoplasms (IPMNs) are the most common cystic precursor lesions of invasive pancreatic cancer. The recent identification of activating GNAS mutations at codon 201 in IPMNs is a promising target for early detection and therapy. The purpose of this study was to explore clinicopathological correlates of GNAS mutational status in resected IPMNs.
Clinical and pathologic characteristics were retrieved on 54 patients in whom GNAS codon 201 mutational status was previously reported (“historical group”, Wu et al. Sci Transl Med 3:92ra66, 2011). In addition, a separate cohort of 32 patients (validation group) was included. After microdissection and DNA extraction, GNAS status was determined in the validation group by pyrosequencing.
GNAS activating mutations were found in 64 % of the 32 IPMNs included in the validation group, compared with a previously reported prevalence of 57 % in the historical group. Overall, 52 of 86 (61 %) of IPMNs demonstrated GNAS mutations in the two studies combined. Analysis of both groups confirmed that demographic characteristics, tumor location, ductal system involvement, focality, size, grade of dysplasia, presence of an associated cancer, and overall survival were not correlated with GNAS mutational status. Stratified by histological subtype, 100 % of intestinal type IPMNs demonstrated GNAS mutations compared to 51 % of gastric IPMN, 71 % of pancreatobiliary IPMNs, and 0 % of oncocytic IPMNs.
GNAS activating mutations can be reliably detected in IPMNs by pyrosequencing. In terms of clinicopathological parameters, only histological subtype was correlated with mutational frequency, with the intestinal phenotype always associated with GNAS mutations.
Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with a poor prognosis in acute myeloid leukemia (AML). Detection of ITD-positive minor clones at the initial diagnosis and during the minimal residual disease (MRD) stage may be essential. We previously designed a delta-PCR strategy to improve the sensitivity to 0.1% ITD-positive leukemia cells and showed that minor mutants with an allele burden of less than 1% can be clinically significant. In this study, we report on tandem duplication PCR (TD-PCR), a modified inverse PCR assay, and demonstrate a limit of detection of a few molecules of ITD mutants. The TD-PCR was initially designed to confirm ITD mutation of an amplicon which was undetectable by capillary electrophoresis and was incidentally isolated by a molecular fraction collecting tool. Subsequently, TD-PCR detected ITD mutation in 2 of 77 patients previously reported as negative for ITD mutation by a standard PCR assay. TD-PCR can also potentially be applied to monitor MRD with high analytic sensitivity in a portion of ITD-positive AML patients. Further studies using TD-PCR to detect ITD mutants at diagnosis may clarify the clinical significance of those ITD mutants with extremely low allele burden.
FLT3; Internal Tandem Duplication; acute myeloid leukemia; minimal residual disease; Tandem duplication PCR
Pancreatic carcinomas with acinar differentiation, including acinar cell carcinoma, pancreatoblastoma, and carcinomas with mixed differentiation, are distinct pancreatic neoplasms with poor prognosis. Although recent whole exome sequencing analyses have defined the somatic mutations that characterize the other major neoplasms of the pancreas, the molecular alterations underlying pancreatic carcinomas with acinar differentiation remain largely unknown. In the current study, we sequenced the exomes of 23 surgically resected pancreatic carcinomas with acinar differentiation. These analyses revealed a relatively large number of genetic alterations at both the individual base pair and chromosomal levels. There was an average of 119 somatic mutations per carcinoma. When three outliers were excluded, there was an average of 64 somatic mutations per tumor (range 12–189). The mean fractional allelic loss (FAL) was 0.27 (range 0–0.89) and heterogeneity at the chromosome level was confirmed in selected cases using fluorescent in situ hybridization (FISH). No gene was mutated in >30% of the cancers. Genes altered in other neoplasms of the pancreas were occasionally targeted in carcinomas with acinar differentiation; SMAD4 was mutated in six tumors (26%), TP53 in three (13%), GNAS in two (9%), RNF43 in one (4%) and MEN1 in one tumor (4%). Somatic mutations were identified in genes in which constitutional alterations are associated with familial pancreatic ductal adenocarcinoma, such as ATM, BRCA2, and PALB2 (one tumor each), as well as in genes altered in extra-pancreatic neoplasms, such as JAK1 in four tumors (17%) BRAF in three (13%), RB1 in three (13%), APC in two (9%), PTEN in two (9%), ARID1A in two (9%), MLL3 in two (9%), and BAP1 in one (4%). Perhaps most importantly, we found that more than a third of these carcinomas have potentially targetable genetic alterations including mutations in BRCA2, PALB2, ATM, BAP1, BRAF and JAK1.
pancreas; carcinoma; acinar cell carcinoma; genetics; sequencing; pancreatoblastoma
Intraductal papillary mucinous neoplasms (IPMNs) are one of the 3 known curable precursor lesions of invasive pancreatic ductal adenocarcinoma, an almost uniformly fatal disease. Cell lines from IPMNs and their invasive counterparts should be valuable to identify gene mutations critical to IPMN carcinogenesis, and permit high-throughput screening to identify drugs that cause regression of these lesions.
To advance the study of the biological features of IPMNs, we attempted in vivo and in vitro growth of selected IPMNs based on the hypothesis that IPMNs could be grown in the most severely immunodeficient mice. We examined fourteen cases by implanting them into nude, severe combined immunodeficient (SCID), and NOD/SCID/IL2Rγnull (NOG) mice, in addition to direct culture, to generate tumor xenografts and cell lines. One sample was directly cultured only.
Thirteen tumors were implanted into the 3 types of mice, including 10 tumors implanted into the triple immunodeficient NOG mice, where the majority (8 of 10) grew. This included 5 IPMNs lacking an invasive component. One of the explanted IPMNs, with an associated invasive carcinoma, was successfully established as a cell line. Tumorigenicity was confirmed by growth in soft agar, growth in immunodeficient mice, and the homozygous deletion of p16/cdkn2a. Epithelial differentiation of the cell line was documented by cytokeratin expression. Patient origin was confirmed using DNA fingerprinting.
Most non-invasive IPMNs grow in NOG mice. We successfully established one IPMN cell line, and plan to use it to clarify the molecular pathogenesis of IPMNs.
Cell lines; Immunodeficient mice; Intraductal papillary mucinous neoplasm (IPMN); pancreatic cancer; precursor lesions
Pancreatic intraepithelial neoplasia (PanIN) is a precursor to invasive ductal adenocarcinoma of the pancreas. Observations made in genetically engineered mouse models suggest that the acinar/centroacinar compartment can give rise to ductal neoplasia. In order to integrate findings in mice and men, we examined human acinar cells, acinar-ductal metaplasia (ADM) lesions and PanINs for KRAS2 gene mutations.
Surgically resected pancreata were screened for foci of ADM with or without an associated PanIN lesion. Stromal cells, acinar cells, ADMs, and PanINs, were separately isolated using laser capture microdissection. KRAS2 status was analyzed using genomic DNA isolated from the microdissected tissue.
Twelve of these 31 foci of ADM occurred in isolation, while 19 were in the same lobules as a PanIN lesion. All 31 microdissected foci of acinar cells were KRAS2 gene wild-type, as were all 12 isolated ADM lesions lacking an associated PanIN. KRAS2 gene mutations were present in 14 of 19 (74%) PanIN lesions, and in 12 of the 19 (63%) foci of ADM associated with these PanINs. All ADM lesions with a KRAS2 gene mutation harbored the identical KRAS2 gene mutation found in their associated PanIN lesions.
Ductal neoplasms of the human pancreas, as defined by KRAS2 gene mutations, do not appear to arise from acinar cells. Isolated AMD lesions are genetically distinct from those associated with PanINs, and the latter may represent retrograde extension of the neoplastic PanIN cells, or less likely are PanIN precursor lesions.
Papanicolaou (Pap) smears have revolutionized the management of patients with cervical cancers by permitting the detection of early, surgically curable tumors and their precursors. In recent years, the traditional Pap smear has been replaced by a liquid-based method, which allows not only cytologic evaluation but also collection of DNA for detection of human papillomavirus, the causative agent of cervical cancer. We reasoned that this routinely collected DNA could be exploited to detect somatic mutations present in rare tumor cells that accumulate in the cervix once shed from endometrial or ovarian cancers. A panel of genes that are commonly mutated in endometrial and ovarian cancers was assembled with new whole-exome sequencing data from 22 endometrial cancers and previously published data on other tumor types. We used this panel to search for mutations in 24 endometrial and 22 ovarian cancers and identified mutations in all 46 samples. With a sensitive massively parallel sequencing method, we were able to identify the same mutations in the DNA from liquid Pap smear specimens in 100% of endometrial cancers (24 of 24) and in 41% of ovarian cancers (9 of 22). Prompted by these findings, we developed a sequence-based method to query mutations in 12 genes in a single liquid Pap smear specimen without previous knowledge of the tumor’s genotype. When applied to 14 samples selected from the positive cases described above, the expected tumor-specific mutations were identified. These results demonstrate that DNA from most endometrial and a fraction of ovarian cancers can be detected in a standard liquid-based Pap smear specimen obtained during routine pelvic examination. Although improvements need to be made before applying this test in a routine clinical manner, it represents a promising step toward a broadly applicable screening methodology for the early detection of gynecologic malignancies.
Genome-wide sequencing identified heterozygous, constitutional, Ataxia telangiectaisa mutated (ATM) gene mutations in two kindreds with familial pancreatic cancer. Mutations segregated with disease in both kindreds and tumor analysis demonstrated LOH of the wildtype allele. Sequence analysis of an additional 166 familial pancreatic cancer probands indentified four additional patients with deleterious mutations in the ATM gene, while no deleterious mutations were identified in 190 spouse controls (p=0.046). These results indicate that ATM mutations play an important role in familial pancreatic cancer predisposition.
ATM; predisposition; familial; pancreas; cancer
A better molecular characterization of Intraductal Papillary Mucinous Neoplasm (IPMN), the most frequent cystic precursor lesion of pancreatic adenocarcinoma, may have a pivotal role in its early detection and in the development of effective therapeutic strategies. BRG1, a central component of the chromatin remodeling complex SWI/SNF regulating transcription, is inactive in several malignancies. In this study we evaluate the Brg1 expression in IPMN in order to better understand its role in the pancreatic carcinogenesis. Tissue microarrays (TMAs) of 66 surgically resected IPMNs were immunolabeled for the Brg1 protein. Expression patterns were then correlated with clinicopathologic parameters. Normal pancreatic epithelium strongly immunolabeled for Brg1. Reduced Brg1 expression was observed in 32 (53.3%) of the 60 evaluable IPMN lesions and occurred more frequently in high-grade IPMNs (13 of 17 showed loss; 76%) compared to intermediate-grade (15 of 29 showed loss; 52%) and low-grade IPMNs (4 of 14 showed loss; 28%) (p=0.03). A complete loss of Brg1 expression was observed in 5 of the 60 (8.3%) lesions. Finally, a decrease in Brg1 protein expression was furthermore found in a low-passage non-invasive IPMN cell line by Western blot analysis. We did not observe correlation between Brg1 expression and IPMN subtype or with location of the cyst. We provide first evidence that Brg1 expression is lost in noninvasive cystic precursor lesions of pancreatic adenocarcinoma.
BRG1; IPMN; pancreatic cancer
To examine the clinicopathologic features and clonal relationship of multifocal intraductal papillary mucinous neoplasms (IPMNs) of the pancreas.
Intraductal papillary mucinous neoplasms are increasingly diagnosed cystic precursor lesions of pancreatic cancer. Intraductal papillary mucinous neoplasms can be multifocal and a potential cause of recurrence after partial pancreatectomy.
Thirty four patients with histologically documented multifocal IPMNs were collected and their clinicopathologic features catalogued. In addition, thirty multifocal IPMNs arising in 13 patients from 3 hospitals were subjected to laser microdissection followed by KRAS pyrosequencing and loss of heterozygosity (LOH) analysis on chromosomes 6q and 17p. Finally, we sought to assess the clonal relationships among multifocal IPMNs.
We identified 34 patients with histologically documented multifocal IPMNs. Synchronous IPMNs were present in 29 patients (85%), whereas 5 (15%) developed clinically significant metachronous IPMNs. Six patients (18%) had a history of familial pancreatic cancer. A majority of multifocal IPMNs (86% synchronous, 100% metachronous) were composed of branch duct lesions, and typically demonstrated a gastric-foveolar subtype epithelium with low or intermediate grades of dysplasia. Three synchronous IPMNs (10%) had an associated invasive cancer. Molecular analysis of multiple IPMNs from 13 patients demonstrated nonoverlapping KRAS gene mutations in 8 patients (62%) and discordant LOH profiles in 7 patients (54%); independent genetic alterations were established in 9 of the 13 patients (69%).
The majority of multifocal IPMNs arise independently and exhibit a gastric-foveolar subtype, with low to intermediate dysplasia. These findings underscore the importance of life-long follow-up after resection for an IPMN.
Mutations in the chromatin remodeling gene ARID1A have recently been identified in the majority of ovarian clear cell carcinomas. To determine the prevalence of mutations in other tumor types, we evaluated 759 malignant neoplasms including those of the pancreas, breast, colon, stomach, lung, prostate, brain and blood (leukemias). We identified truncating mutations in 6% of the neoplasms studied; non-truncating somatic mutations were identified in an additional 0.4% of neoplasms. Mutations were most commonly found in gastrointestinal samples with 12 of 119 (10%) colorectal and 10 of 100 (10%) gastric neoplasms, respectively, harboring changes. More than half of the mutated colorectal and gastric cancers displayed microsatellite instability and the mutations in these tumors were out-of-frame insertions or deletions at mononucleotide repeats. Mutations were also identified in 2% to 8% of tumors of the pancreas, breast, brain (medulloblastomas), prostate, and lung, and none of these tumors displayed microsatellite instability. These findings suggest that the aberrant chromatin remodeling consequent to ARID1A inactivation contributes to a variety of different types of neoplasms.
ARID1A; cancer; chromatin remodeling
Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.
Polymorphic short tandem repeat (STR), or microsatellite, loci have been widely used to analyze chimerism status following allogeneic hematopoietic stem cell transplantation (HSCT). The presence of a patient’s DNA, as identified by STR analysis, may indicate residual or recurrent malignant disease or may represent normal hematopoiesis of patient origin. The ratio of patient-derived to donor-derived alleles is used to calculate the relative amount of patient cells (both benign and malignant) to donor cells. STRs on chromosomes known to be gained or lost in a patient’s tumor are generally ignored because it is difficult to perform meaningful calculations of mixed chimerism. However, in this report, we present evidence that STR loci on gained or lost chromosomes are useful in distinguishing the benign or malignant nature of chimeric DNA. In the peripheral blood or bone marrow of four HSCT patients with leukemia or lymphoma, we identified tumor DNA on the basis of STR loci showing copy number alteration. We propose that a targeted evaluation of STR loci showing altered copy number in post-transplant chimerism analysis can provide evidence of residual cancer cells.
short tandem repeat; microsatellite; allogeneic hematopoietic stem cell transplantation; chimerism; leukemia relapse
Sarcina organisms were first observed and recorded in the stomach contents of a patient with vomiting by John Goodsir in 1842. Since that time, the fine structure, phylogenetic classification, and biochemical characteristics have been described. While numerous cases of fatal disease have been attributed to this organism in the veterinary literature, only a few human cases have been documented. As a result, whether this organism causes disease in humans has not been definitively established. We report the clinicopathologic findings in a series of 5 patients with Sarcina-like organisms identified in upper gastrointestinal endoscopic biopsies with molecular confirmation. Based on our findings, the organism is most commonly found in patients with a history of gastric outlet obstruction or delayed gastric emptying. While many of the patients do not demonstrate direct mucosal injury from the organism, the presence of a concurrent gastric ulcer puts the patient at increased risk for complications such as emphysematous gastritis or perforation. The finding of Sarcina organisms should prompt further investigation for functional causes of gastric outlet obstruction and delayed gastric emptying, such as occult malignancy.
Sarcina; gastrointestinal tract; gastroparesis; emphysematous gastritis; gastritis
5-fluorouracil (5FU), a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms). Prior studies implicated a VNTR (variable numbers of tandem repeats) polymorphism in the 5′-untranslated region (5′-UTR) of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T) repeats and novel single nucleotide polymorphisms (SNPs) flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt), polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing.
Metastasis and drug resistance are the major causes of mortality in patients with pancreatic cancer. Once developed, the progression of pancreatic cancer metastasis is virtually unstoppable with current therapies. Here we report the remarkable clinical outcome of a patient with advanced, gemcitabine-resistant, pancreatic cancer who was later treated with DNA damaging agents, based on the observation of significant activity of this class of drugs against a personalized xenograft generated from the patient’s surgically resected tumor. Mitomycin C treatment, selected based on its robust preclinical activity in a personalized xenograft generated from the patient’s tumor, resulted in long lasting (36+ months) tumor response. Global geneomic sequencing revealed biallelic inactivation of the gene encoding PalB2 protein in this patient’s cancer, the mutation is predicted to disrupt BRCA1 and BRCA2 interactions critical to DNA double strand break repair. This work suggests that inactivation of the PALB2 gene is a determinant of response to DNA damage in pancreatic cancer and a new target for personalizing cancer treatment. Integrating personalized xenografts with unbiased exomic sequencing led to customize therapy, tailored to the genetic environment of patient’s tumor and identification of a new biomarker of drug response in a lethal cancer.
PALB2; pancreatic cancer; personalized xenograft; mitomycin C
Esophageal adenocarcinoma (EAC) arises in the backdrop of reflux-induced metaplastic phenomenon known as Barrett esophagus. The prognosis of advanced EAC is dismal, and there is an urgent need for identifying molecular targets for therapy. Serial Analysis of Gene Expression (SAGE) was performed on metachronous mucosal biopsies from a patient who underwent progression to EAC during endoscopic surveillance. SAGE confirmed significant upregulation of Axl “tags” during the multistep progression of Barrett esophagus to EAC. In a cohort of 92 surgically resected EACs, Axl overexpression was associated with shortened median survival on both univariate (p < 0.004) and multivariate (p < 0.036) analysis. Genetic knockdown of Axl receptor tyrosine kinase (RTK) function was enabled in two EAC lines (OE33 and JH-EsoAd1) using lentiviral short hairpin RNA (shRNA). Genetic knockdown of Axl in EAC cell lines inhibited invasion, migration and in vivo engraftment, which was accompanied by downregulation in the activity of the Ral GTPase proteins (RalA and RalB). Restoration of Ral activation rescued the transformed phenotype of EAC cell lines, suggesting a novel effector mechanism for Axl in cancer cells. Pharmacological inhibition of Axl was enabled using a small molecule antagonist, R428 (Rigel Pharmaceuticals). Pharmacological inhibition of Axl with R428 in EAC cell lines significantly reduced anchorage-independent growth, invasion and migration. Blockade of Axl function abrogated phosphorylation of ERBB2 (Her-2/neu) at the Tyr877 residue, indicative of receptor crosstalk. Axl RTK is an adverse prognostic factor in EAC. The availability of small molecule inhibitors of Axl function provides a tractable strategy for molecular therapy of established EAC.
Barrett esophagus; Axl; Ral GTP; SAGE
Telomeres protect against chromosomal breakage, fusion, and interchromosome bridges during cell division. Shortened telomeres have been observed in the lowest grade of pancreatic intraepithelial neoplasia. Genetically engineered mouse models of pancreatic neoplasia develop acinar-to-ductal metaplasia prior to the development of pancreatic intraepithelial neoplasia suggesting that acinar-to-ductal metaplasias can be an early precursor lesion to pancreatic cancer. Some human pancreatic intraepithelial neoplasias are associated with acinar-to-ductal metaplasias, and it has been suggested that these acinar-to-ductal metaplasias arise as a consequence of growth of adjacent pancreatic intraepithelial neoplasias. Since the earliest known genetic lesions of pancreatic intraepithelial neoplasias is shortened telomeres we compared the telomere lengths of acinar-to-ductal metaplasia lesions, pancreatic intraepithelial neoplasias and adjacent normal cells of human pancreata to determine if acinar-to-ductal metaplasias could be precursors to pancreatic intraepithelial neoplasia. We used quantitative fluorescent in situ hybridization to measure the telomere length of cells from pancreatic lesions and adjacent normal pancreata from 22 patients, including 20 isolated acinar-to-ductal metaplasias, 13 pancreatic intraepithelial neoplasias associated with acinar-to-ductal metaplasias, and 12 pancreatic intraepithelial neoplasias. Normalized mean telomere fluorescence was significantly different among the cell types analyzed; 12.6±10.2 units in normal acinar cells, 10.2±6.4 in ductal cells, 8.4±5.9 in fibroblasts, 9.4±7.3 in isolated acinar-to-ductal metaplasias, 4.1±2.9 in pancreatic intraepithelial neoplasia-associated acinar-to-ductal metaplasias, and 1.6±1.9 in pancreatic intraepithelial neoplasias, respectively (p<0.001, ANOVA with randomized block design). Telomeres were significantly shorter in pancreatic intraepithelial neoplasia-associated acinar-to-ductal metaplasias (p<0.05, post-hoc Duncan test) and in pancreatic intraepithelial neoplasias (p<0.05), than in normal cells, or isolated acinar-to-ductal metaplasias. Thus, shortened telomeres are found in pancreatic intraepithelial neoplasia-associated acinar-to-ductal metaplasias, but not in isolated acinar-to-ductal metaplasia lesions. These results indicate that isolated acinar-to-ductal metaplasias are not a precursor to pancreatic intraepithelial neoplasia, and support the hypothesis that pancreatic intraepithelial neoplasia-associated acinar-to-ductal metaplasias arise secondary to pancreatic intraepithelial neoplasia lesions.
More than 2% of the adult U.S. population harbors a pancreatic cyst. These often pose a difficult management problem because conventional criteria cannot always distinguish cysts with malignant potential from those that are innocuous. One of the most common cystic neoplasms of the pancreas, and a bona fide precursor to invasive adenocarcinoma, is called intraductal papillary mucinous neoplasm (IPMN). To help reveal the pathogenesis of these lesions, we purified the DNA from IPMN cyst fluids from 19 patients and searched for mutations in 169 genes commonly altered in human cancers. In addition to the expected KRAS mutations, we identified recurrent mutations at codon 201 of GNAS. A larger number (113) of additional IPMNs were then analyzed to determine the prevalence of KRAS and GNAS mutations. In total, we found that GNAS mutations were present in 66% of IPMNs and that either KRAS or GNAS mutations could be identified in 96%. In eight cases, we could investigate invasive adenocarcinomas that developed in association with IPMNs containing GNAS mutations. In seven of these eight cases, the GNAS mutations present in the IPMNs were also found in the invasive lesion. GNAS mutations were not found in other types of cystic neoplasms of the pancreas or in invasive adenocarcinomas not associated with IPMNs. In addition to defining a new pathway for pancreatic neoplasia, these data suggest that GNAS mutations can inform the diagnosis and management of patients with cystic pancreatic lesions.
Metastasis, the dissemination and growth of neoplastic cells in an organ distinct from that in which they originated 12, is the most common cause of death in cancer patients. This is particularly true for pancreatic cancers, where most patients are diagnosed with metastatic disease and few show a sustained response to chemo- or radiation therapy 3. Whether the dismal prognosis of patients with pancreatic cancer compared to patients with other types of cancer is a result of late diagnosis or early dissemination of disease to distant organs is not known. Here we rely on data generated by sequencing the genomes of seven pancreatic cancer metastases to evaluate the clonal relationships among primary and metastatic cancers. We find that clonal populations that give rise to distant metastases are represented within the primary carcinoma, but these clones are genetically evolved from the original parental, non-metastatic clone. Thus, genetic heterogeneity of metastases reflects that within the primary carcinoma. A quantitative analysis of the timing of the genetic evolution of pancreatic cancer was performed, indicating at least a decade between the occurrence of the initiating mutation and the birth of the parental, non-metastatic founder cell. At least five more years are required for the acquisition of metastatic ability and patients die an average of two years thereafter. These data provide novel insights into the genetic features underlying pancreatic cancer progression and define a broad time window of opportunity for early detection to prevent deaths from metastatic disease.