Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.

Purpose

Serious adverse drug event (sADE) reporting to Institutional Review Boards (IRB) is essential to ensure pharmaceutical safety. However, the quality of these reports has not been studied. Safety reports are especially important for cancer drugs that receive accelerated Food and Drug Administration approval, like imatinib, as preapproval experience with these drugs is limited. We evaluated the quality, accuracy, and completeness of sADE reports submitted to an IRB.

Experimental Design

sADE reports submitted to an IRB from 14 clinical trials with imatinib were reviewed. Structured case report forms, containing detailed clinical data fields and a validated causality assessment instrument, were developed. Two forms were generated for each ADE, the first populated with data abstracted from the IRB reports, and the second populated with data from the corresponding clinical record. Completeness and causality assessments were evaluated for each of the two sources, and then compared. Accuracy (concordance between sources) was also assessed.

Results

Of 115 sADEs reported for 177 cancer patients to the IRB, overall completeness of adverse event descriptions was 2.4-fold greater for structured case report forms populated with information from the clinical record versus the corresponding forms from IRB reports (95.0% versus 40.3%, P < 0.05). Information supporting causality assessments was recorded 3.5-fold more often in primary data sources versus IRB adverse event descriptions (93% versus 26%, P < 0.05). Some key clinical information was discrepant between the two sources.

Conclusions

The use of structured syndrome-specific case report forms could enhance the quality of reporting to IRBs, thereby improving the safety of pharmaceuticals administered to cancer patients.

Purpose

This study was conducted to identify novel genes with importance to the biology of adult acute myelogenous leukemia (AML).

Experimental design

We analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for recurrent genomic microdeletions using ultra-high density Affymetrix SNP 6.0 array-based genomic profiling.

Results

Through fine mapping of microdeletions on 17q, we derived a minimal deleted region of ~0.9Mb length that harbors 11 known genes; this region includes Neurofibromin 1 (NF1). Sequence analysis of all NF1 coding exons in the 11 AML cases with NF1 copy number changes identified acquired truncating frameshift mutations in 2 patients. These NF1 mutations were already present in the hematopoetic stem cell compartment. Subsequent expression analysis of NF1 mRNA in the entire AML cohort using FACS sorted blasts as a source of RNA identified 6 patients (one with a NF1 mutation) with absent NF1 expression. The NF1 null states were associated with increased Ras-bound GTP, and shRNA-mediated NF1 suppression in primary AML blasts with wild type NF1 facilitated colony formation in methylcellulose. Primary AML blasts without functional NF1, unlike blasts with functional NF1, displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mTOR signaling for survival. Finally, colony formation in methylcellulose ex vivo of NF1 null CD34+/CD38− cells sorted from AML bone marrow samples was inhibited by low dose rapamycin.

Conclusions

NF1 null states are present in 7/95=7% of adult AML and delineate a disease subset that could be preferentially targeted by Ras or mTOR-directed therapeutics.

Background

Interferon-α (IFN) induces complete cytogenetic remission (CCR) in 20–25% CML patients and in a small minority of patients; CCR persists after IFN is stopped. IFN induces CCR in part by increasing cytotoxic T lymphocytes (CTL) specific for PR1, the HLA-A2-restricted 9-mer peptide from proteinase 3 and neutrophil elastase, but it is unknown how CCR persists after IFN is stopped.

Principal Findings

We reasoned that PR1-CTL persist and mediate CML-specific immunity in patients that maintain CCR after IFN withdrawal. We found that PR1-CTL were increased in peripheral blood of 7/7 HLA-A2+ patients during unmaintained CCR from 3 to 88 months after IFN withdrawal, as compared to no detectable PR1-CTL in 2/2 IFN-treated CML patients not in CCR. Unprimed PR1-CTL secreted IFNγ and were predominantly CD45RA±CD28+CCR7+CD57-, consistent with functional naïve and central memory (CM) T cells. Similarly, following stimulation, proliferation occurred predominantly in CM PR1-CTL, consistent with long-term immunity sustained by self-renewing CM T cells. PR1-CTL were functionally anergic in one patient 6 months prior to cytogenetic relapse at 26 months after IFN withdrawal, and in three relapsed patients PR1-CTL were undetectable but re-emerged 3–6 months after starting imatinib.

Conclusion

These data support the hypothesis that IFN elicits CML-specific CM CTL that may contribute to continuous CCR after IFN withdrawal and suggest a role for T cell immune therapy with or without tyrosine kinase inhibitors as a strategy to prolong CR in CML.

Background

Imatinib is a tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). BCR-ABL mutations are associated with failure of imatinib treatment in many CML patients. LYN kinase regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase, and differences in LYN regulation have been found between imatinib-sensitive and -resistant CML cell lines.

Methods

We evaluated cells from 12 imatinib-resistant CML patients with mutation-negative BCR-ABL and from six imatinib-sensitive patients who discontinued therapy because of imatinib intolerance. Phosphorylation of BCR-ABL and LYN was assessed in patient cells and cell lines by immunoblotting with activation state–specific antibodies, co-immunoprecipitation studies, and mass spectroscopy analysis of phosphopeptides. Cell viability, caspase activation, and apoptosis were also measured. Mutations were analyzed by sequencing. The effect of silencing LYN with short interfering RNAs (siRNAs) or reducing activation by treatment with tyrosine kinase inhibitors was evaluated in cell lines and patient cells.

Results

Imatinib treatment suppressed LYN phosphorylation in cells from imatinib-sensitive CML patients and imatinib-sensitive cell lines. Imatinib treatment blocked BCR-ABL signaling but did not suppress LYN phosphorylation in cells from imatinib-resistant patients, and persistent activation of LYN kinase was not associated with mutations in LYN kinase or its carboxyl-terminal regulatory domains. Unique LYN phosphorylation sites (tyrosine-193 and tyrosine-459) and associated proteins (c-Cbl and p80) were identified in cells from imatinib-resistant patients. Reducing LYN expression (siRNA) or activation (dasatinib) was associated with loss of cell survival and cytogenetic or complete hematologic responses in imatinib-resistant disease.

Conclusions

LYN activation was independent of BCR-ABL in cells from imatinib-resistant patients. Thus, LYN kinase may be involved in imatinib resistance in CML patients with mutation-negative BCR-ABL and its direct inhibition is consistent with clinical responses in these patients.