OHSU Knight Cancer Institute

BACKGROUND: The molecular causes of many hematologic cancers remain unclear. Among these cancers are chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL1-negative) chronic myeloid leukemia (CML), both of which are diagnosed on the basis of neoplastic expansion of granulocytic cells and exclusion of genetic drivers that are known to occur in other myeloproliferative neoplasms and myeloproliferative-myelodysplastic overlap neoplasms. METHODS: To identify potential genetic drivers in these disorders, we used an integrated approach of deep sequencing coupled with the screening of primary leukemia cells obtained from patients with CNL or atypical CML against panels of tyrosine kinase-specific small interfering RNAs or small-molecule kinase inhibitors. We validated candidate oncogenes using in vitro transformation assays, and drug sensitivities were validated with the use of assays of primary-cell colonies. RESULTS: We identified activating mutations in the gene encoding the receptor for colony-stimulating factor 3 (CSF3R) in 16 of 27 patients (59%) with CNL or atypical CML. These mutations segregate within two distinct regions of CSF3R and lead to preferential downstream kinase signaling through SRC family-TNK2 or JAK kinases and differential sensitivity to kinase inhibitors. A patient with CNL carrying a JAK-activating CSF3R mutation had marked clinical improvement after the administration of the JAK1/2 inhibitor ruxolitinib. CONCLUSIONS: Mutations in CSF3R are common in patients with CNL or atypical CML and represent a potentially useful criterion for diagnosing these neoplasms. (Funded by the Leukemia and Lymphoma Society and others.).

The diagnosis of gastrointestinal stromal tumor (GIST) is currently based on morphologic features and immunohistochemical demonstration of KIT (CD117). However, some tumors (in our estimation approximately 4%) have clinicopathologic features of GIST but do not express KIT. To determine if these lesions are truly GISTs, we evaluated 25 tumors with clinical and histologic features typical of GIST, but with negative KIT immunohistochemistry, for KIT and PDGFRA mutations using DNA extracted from paraffin-embedded tissue. Most tumors originated in the stomach (N = 14) or omentum/mesentery (N = 5). The neoplasms were composed of epithelioid cells (13 cases), admixed epithelioid and spindle cells (8 cases), or spindle cells (4 cases). Absence of KIT expression was confirmed by immunoblotting in 5 cases. Tumor karyotypes performed in 4 cases were noncomplex with monosomy 14 or 14q deletion, typical of GIST. Mutational analysis revealed PDGFRA and KIT mutations in 18 and 4 tumors, respectively, whereas 3 tumors did not have apparent KIT or PDGFRA mutations. The PDGFRA mutations primarily involved exon 18 (N = 15) and included 11 tumors with missense mutation in codon 842 (PDGFRA D842V or D842Y). In conclusion, a small subset of GISTs with otherwise typical clinicopathologic and cytogenetic features do not express detectable KIT protein. When compared with KIT-positive GISTs, these KIT-negative GISTs are more likely to have epithelioid cell morphology, contain PDGFRA oncogenic mutations, and arise in the omentum/peritoneal surface. Notably, some KIT-negative GISTs contain imatinib-sensitive KIT or PDGFRA mutations; therefore, patients with KIT-negative GISTs should not, a priori, be denied imatinib therapy.

The particularly interdisciplinary nature of human microbiome research makes the organization and reporting of results spanning epidemiology, biology, bioinformatics, translational medicine and statistics a challenge. Commonly used reporting guidelines for observational or genetic epidemiology studies lack key features specific to microbiome studies. Therefore, a multidisciplinary group of microbiome epidemiology researchers adapted guidelines for observational and genetic studies to culture-independent human microbiome studies, and also developed new reporting elements for laboratory, bioinformatics and statistical analyses tailored to microbiome studies. The resulting tool, called 'Strengthening The Organization and Reporting of Microbiome Studies' (STORMS), is composed of a 17-item checklist organized into six sections that correspond to the typical sections of a scientific publication, presented as an editable table for inclusion in supplementary materials. The STORMS checklist provides guidance for concise and complete reporting of microbiome studies that will facilitate manuscript preparation, peer review, and reader comprehension of publications and comparative analysis of published results.

Increased numbers of tumor-infiltrating macrophages correlate with poor disease outcome in patients affected by several types of cancer, including breast and prostate carcinomas. The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment of tumor-associated macrophages (TAMs) to the neoplastic microenvironment and promotes the differentiation of TAMs toward a pro-tumorigenic phenotype. Twelve clinical trials are currently evaluating agents that target the CSF1/CSF1R signaling pathway as a treatment against multiple malignancies, including breast carcinoma, leukemia, and glioblastoma. The blockade of CSF1R signaling has been shown to greatly decrease the number of macrophages in a tissue-specific manner. However, additional mechanistic insights are needed in order to understand how macrophages are depleted and the global effects of CSF1R inhibition on other tumor-infiltrating immune cells. Using BLZ945, a highly selective small molecule inhibitor of CSF1R, we show that CSF1R inhibition attenuates the turnover rate of TAMs while increasing the number of CD8+ T cells that infiltrate cervical and breast carcinomas. Specifically, we find that BLZ945 decreased the growth of malignant cells in the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis. Furthermore, we show that BLZ945 prevents tumor progression in the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis. Our results demonstrate that TAMs undergo a constant turnover in a CSF1R-dependent manner, and suggest that continuous inhibition of the CSF1R pathway may be essential to maintain efficacious macrophage depletion as an anticancer therapy.

The majority of renal neoplasms can be distinguished on the basis of histologic examination alone; however, there are morphologic similarities between clear cell renal carcinoma and chromophobe cell carcinoma, as well as between the granular/eosinophilic variants of these tumors and renal oncocytoma. Only a limited number of histochemical markers are available to aid in the differential diagnosis of these neoplasms. Hale's colloidal iron usually yields strong, diffuse cytoplasmic staining of chromophobe cell carcinomas whereas clear cell carcinomas are generally negative; however, interpretation of this stain is not always straightforward. By immunohistochemistry, vimentin is detectable in most clear cell carcinomas and is absent from most chromophobe cell tumors and oncocytomas, but reliance on a single antibody can be misleading. In this report we examine the use of commercially available monoclonal antibodies to RCC and CD10 in the differential diagnosis of common renal tumors. Eighty-five percent of clear cell carcinomas (53 of 62) had detectable surface membrane staining for RCC, and 94% (58 of 62) were positive for CD10. Papillary carcinomas were likewise strongly positive for RCC and CD10 in nearly all cases (13 of 14 each). In contrast, all 19 chromophobe cell carcinomas examined were completely negative for surface membrane staining with both of these markers. Oncocytomas were also negative for RCC (0 of 9), but CD10 was detectable in some cases (3 of 9). These results suggest that the presence of surface membrane staining for RCC and CD10 may be used to confirm a diagnosis of suspected clear cell or papillary renal carcinoma. Chromophobe cell carcinomas should be negative for both markers. The absence of RCC staining may also be helpful in the diagnosis of renal oncocytoma.

The impact of intrinsic aging upon human peripheral blood T cell subsets remains incompletely quantified and understood. This impact must be distinguished from the influence of latent persistent microorganisms, particularly CMV, which has been associated with age-related changes in the T cell pool. In a cross-sectional cohort of 152 CMV-negative individuals, aged 21-101 y, we found that aging correlated strictly to an absolute loss of naive CD8, but not CD4, T cells but, contrary to many reports, did not lead to an increase in memory T cell numbers. The loss of naive CD8 T cells was not altered by CMV in 239 subjects (range 21-96 y), but the decline in CD4(+) naive cells showed significance in CMV(+) individuals. These individuals also exhibited an absolute increase in the effector/effector memory CD4(+) and CD8(+) cells with age. That increase was seen mainly, if not exclusively, in older subjects with elevated anti-CMV Ab titers, suggesting that efficacy of viral control over time may determine the magnitude of CMV impact upon T cell memory, and perhaps upon immune defense. These findings provide important new insights into the age-related changes in the peripheral blood pool of older adults, demonstrating that aging and CMV exert both distinct and joint influence upon blood T cell homeostasis in humans.

Epigenetic modifications, including changes in DNA methylation, lead to altered gene expression and thus may underlie epileptogenesis via induction of permanent changes in neuronal excitability. Therapies that could inhibit or reverse these changes may be highly effective in halting disease progression. Here we identify an epigenetic function of the brain's endogenous anticonvulsant adenosine, showing that this compound induces hypomethylation of DNA via biochemical interference with the transmethylation pathway. We show that inhibition of DNA methylation inhibited epileptogenesis in multiple seizure models. Using a rat model of temporal lobe epilepsy, we identified an increase in hippocampal DNA methylation, which correlates with increased DNA methyltransferase activity, disruption of adenosine homeostasis, and spontaneous recurrent seizures. Finally, we used bioengineered silk implants to deliver a defined dose of adenosine over 10 days to the brains of epileptic rats. This transient therapeutic intervention reversed the DNA hypermethylation seen in the epileptic brain, inhibited sprouting of mossy fibers in the hippocampus, and prevented the progression of epilepsy for at least 3 months. These data demonstrate that pathological changes in DNA methylation homeostasis may underlie epileptogenesis and reversal of these epigenetic changes with adenosine augmentation therapy may halt disease progression.

The Cbl protooncogene product has emerged as a negative regulator of receptor and nonreceptor tyrosine kinases. We recently demonstrated that oncogenic Cbl mutants upregulate the endogenous tyrosine kinase signaling machinery when expressed in the NIH 3T3 cells, and identified the platelet-derived growth factor receptor-alpha (PDGFRalpha) as one of the tyrosine kinases targeted by these oncogenes. These findings suggested a role for the normal Cbl protein in negative regulation of the PDGFRalpha. However, the mechanism of such negative regulation remained to be determined. Here we show that overexpression of the wild-type Cbl enhances the ligand-induced ubiquitination of the PDGFRalpha. Concomitantly, the PDGFRalpha in Cbl-overexpressing cells undergoes a faster ligand-induced degradation compared with that in the control cells. These results identify a role for Cbl in the regulation of ligand-induced ubiquitination and degradation of receptor tyrosine kinases and suggest one potential mechanism for evolutionarily conserved negative regulatory influence of Cbl on tyrosine kinases.

BACKGROUND: Donor-site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells. Hypothesis/ PURPOSE: The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts. STUDY DESIGN: Controlled laboratory study. METHODS: Cartilage samples were from juvenile (<13 years old) and adult (>13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity. RESULTS: Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNA in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (P = .002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (P < .001). Juvenile chondrocytes did not stimulate lymphocyte proliferation. CONCLUSION: These results document a dramatic age-related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo. CLINICAL RELEVANCE: Juvenile human chondrocytes have greater potential to restore articular cartilage than adult cells, and may be transplanted without the fear of rejection, suggesting a new allogeneic approach to restoring articular cartilage in older individuals.

PURPOSE: To determine the potential of crenolanib, a potent inhibitor of PDGFRA, to treat malignancies driven by mutant PDGFRA. EXPERIMENTAL DESIGN: The biochemical activity of crenolanib was compared with imatinib using a panel of PDGFRA-mutant kinases expressed in several different cell line models, including primary gastrointestinal stromal tumors (GIST) cells. The antiproliferative activity of crenolanib was also studied in several cell lines with PDGFRA-dependent growth. RESULTS: Crenolanib was significantly more potent than imatinib in inhibiting the kinase activity of imatinib-resistant PDGFRA kinases (D842I, D842V, D842Y, DI842-843IM, and deletion I843). For example, crenolanib was 135-fold more potent than imatinib against D842V in our isogenic model system, with an IC(50) of approximately 10 nmol/L. The relative potency of crenolanib was further confirmed in BaF3 and primary GIST cells expressing PDGFRA D842V. In contrast, imatinib was at least 10-fold more potent than crenolanib in inhibiting the V561D mutation. For all other tested PDGFRA mutations, crenolanib and imatinib had comparable potency. CONCLUSIONS: Crenolanib is a potent inhibitor of imatinib-resistant PDGFRA kinases associated with GIST, including the PDGFRA D842V mutation found in approximately 5% of GISTs. The spectrum of activity of crenolanib suggests that this drug is a type I inhibitor (inhibitor of activated conformation of kinase). Based in part on these results, a phase II clinical study of this agent to treat GIST with the PDGFRA D842V mutation has been initiated.

Abstract Pre-existing serum antibodies play an important role in vaccine-mediated protection against infection but the underlying mechanisms of immune memory are unclear. Clinical studies indicate that antigen-specific antibody responses can be maintained for many years, leading to theories that reactivation/differentiation of memory B cells into plasma cells is required to sustain long-term antibody production. Here, we present a decade-long study in which we demonstrate site-specific survival of bone marrow-derived plasma cells and durable antibody responses to multiple virus and vaccine antigens in rhesus macaques for years after sustained memory B cell depletion. Moreover, BrdU + cells with plasma cell morphology can be detected for 10 years after vaccination/BrdU administration, indicating that plasma cells may persist for a prolonged period of time in the absence of cell division. On the basis of these results, long-lived plasma cells represent a key cell population responsible for long-term antibody production and serological memory.

IMPORTANCE: Clinical trials are essential to cancer care, and data about the current state of research in oncology are needed to develop benchmarks and set the stage for improvement. OBJECTIVE: To perform a comprehensive analysis of the national oncology clinical research portfolio. DESIGN: All interventional clinical studies registered on ClinicalTrials.gov between October 2007 and September 2010 were identified using Medical Subject Heading terms and submitted conditions. They were reviewed to validate classification, subcategorized by cancer type, and stratified by design characteristics to facilitate comparison across cancer types and with other specialties. RESULTS: Of 40 970 interventional studies registered between October 2007 and September 2010, a total of 8942 (21.8%) focused on oncology. Compared with other specialties, oncology trials were more likely to be single arm (62.3% vs 23.8%; P < .001), open label (87.8% vs 47.3%; P < .001), and nonrandomized (63.9% vs 22.7%; P < .001). There was moderate but significant correlation between number of trials conducted by cancer type and associated incidence and mortality (Spearman rank correlation coefficient, 0.56 [P = .04] and 0.77 [P = .001], respectively). More than one-third of all oncology trials were conducted solely outside North America. CONCLUSIONS AND RELEVANCE: There are significant variations between clinical trials in oncology and other diseases, as well as among trials within oncology. The differences must be better understood to improve both the impact of cancer research on clinical practice and the use of constrained resources.

Although activation of tyrosine kinase pathways is a shared theme among myeloproliferative neoplasms, the pathogenetic basis of chronic neutrophilic leukemia (CNL) has remained elusive. Recently, we identified high-frequency oncogenic mutations in the granulocyte-colony stimulating factor receptor (CSF3R) in CNL and in some patients with atypical chronic myeloid leukemia. Inhibition of Janus kinase 2 or SRC kinase signaling downstream of mutated CSF3R is feasible and should be explored therapeutically. Herein, we discuss the potential impact of these findings for the classification and treatment of these disorders.

Identification of targeted therapies for TNBC is an urgent medical need. Using a drug combination screen reliant on synthetic lethal interactions, we identified clinically relevant combination therapies for different TNBC subtypes. Two drug combinations targeting the BET family were further explored. The first, targeting BET and CXCR2, is specific for mesenchymal TNBC and induces apoptosis, whereas the second, targeting BET and the proteasome, is effective for major TNBC subtypes and triggers ferroptosis. Ferroptosis was induced at low drug doses and was associated with increased cellular iron and decreased glutathione levels, concomitant with reduced levels of GPX4 and key glutathione biosynthesis genes. Further functional studies, analysis of clinical datasets and breast cancer specimens revealed a unique vulnerability of TNBC to ferroptosis inducers, enrichment of ferroptosis gene signature, and differential expression of key proteins that increase labile iron and decrease glutathione levels. This study identified potent combination therapies for TNBC and unveiled ferroptosis as a promising therapeutic strategy.

BACKGROUND: Most patients with KIT-mutant gastrointestinal stromal tumours (GISTs) benefit from imatinib, but treatment resistance results from outgrowth of heterogeneous subclones with KIT secondary mutations. Once resistance emerges, targeting KIT with tyrosine kinase inhibitors (TKIs) sunitinib and regorafenib provides clinical benefit, albeit of limited duration. METHODS: We systematically explored GIST resistance mechanisms to KIT-inhibitor TKIs that are either approved or under investigation in clinical trials: the studies draw upon GIST models and clinical trial correlative science. We subsequently modelled in vitro a rapid TKI alternation approach against subclonal heterogeneity. RESULTS: Each of the KIT-inhibitor TKIs targets effectively only a subset of KIT secondary mutations in GIST. Regorafenib and sunitinib have complementary activity in that regorafenib primarily inhibits imatinib-resistance mutations in the activation loop, whereas sunitinib inhibits imatinib-resistance mutations in the ATP-binding pocket. We find that rapid alternation of sunitinib and regorafenib suppresses growth of polyclonal imatinib-resistant GIST more effectively than either agent as monotherapy. CONCLUSIONS: Our data highlight that heterogeneity of KIT secondary mutations is the main mechanism of tumour progression to KIT inhibitors in imatinib-resistant GIST patients. Therapeutic combinations of TKIs with complementary activity against resistant mutations may be useful to suppress growth of polyclonal imatinib-resistance in GIST.

Kinases are dysregulated in most cancers, but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here, we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days. Screening of 151 leukemia patient samples revealed a wide diversity of drug sensitivities, with 70% of the clinical specimens exhibiting hypersensitivity to one or more drugs. From this data set, we developed an algorithm to predict kinase pathway dependence based on analysis of inhibitor sensitivity patterns. Applying this algorithm correctly identified pathway dependence in proof-of-principle specimens with known oncogenes, including a rare FLT3 mutation outside regions covered by standard molecular diagnostic tests. Interrogation of all 151 patient specimens with this algorithm identified a diversity of kinase targets and signaling pathways that could aid prioritization of deep sequencing data sets, permitting a cumulative analysis to understand kinase pathway dependence within leukemia subsets. In a proof-of-principle case, we showed that in vitro drug sensitivity could predict both a clinical response and the development of drug resistance. Taken together, our results suggested that drug target scores derived from a comprehensive kinase inhibitor panel could predict pathway dependence in cancer cells while simultaneously identifying potential therapeutic options.

In the multidisciplinary management of gastrointestinal stromal tumours (GISTs), there is a need to coordinate the efforts of pathology, radiology, surgery and oncology. Surgery is the mainstay for resectable nonmetastatic GISTs, but virtually all GISTs are associated with a risk of metastasis. Imatinib 400 mg/day with or without surgery is the recommended first-line treatment for recurrent or metastatic GIST; a higher dose may be considered in patients who progress, develop secondary resistance or present with specific genotypic characteristics. Adjuvant or neoadjuvant imatinib is not advised for resectable nonmetastatic GISTs. Neoadjuvant imatinib may be considered when surgery would result in significant morbidity or loss of organ function. Follow-up computed tomography imaging is recommended every three to six months for at least five years. Patients with metastatic disease should be continued on imatinib due to the high risk of recurrence on discontinuation of therapy. Treatment should be continued until there is progression or intolerable adverse effects. If dose escalation with imatinib fails, a clinical trial with novel agents alone or in combination may be considered. The present recommendations were developed at a surgical subcommittee meeting and a subsequent full Advisory Committee meeting held in Toronto, Ontario, in April 2005, under the sponsorship of Novartis Pharmaceuticals Canada Inc.

Pharmacogenomic testing is viewed as an integral part of precision medicine. To achieve this, we originated The 1,200 Patients Project which offers broad, preemptive pharmacogenomic testing to patients at our institution. We analyzed enrollment, genotype, and encounter-level data from the first year of implementation to assess utility of providing pharmacogenomic results. Results were delivered via a genomic prescribing system (GPS) in the form of traffic lights: green (favorable), yellow (caution), and red (high risk). Additional supporting information was provided as a virtual pharmacogenomic consult, including citation to relevant publications. Currently, 812 patients have participated, representing 90% of those approached; 608 have been successfully genotyped across a custom array. A total of 268 clinic encounters have occurred at which results were accessible via the GPS. At 86% of visits, physicians accessed the GPS, receiving 367 result signals for medications patients were taking: 57% green lights, 41% yellow lights, and 1.4% red lights. Physician click frequencies to obtain clinical details about alerts varied according to color severity (100% of red were clicked, 72% yellow, 20% green). For 85% of visits, clinical pharmacogenomic information was available for at least one drug the patient was taking, suggesting relevance of the delivered information. We successfully implemented an individualized health care model of preemptive pharmacogenomic testing, delivering results along with pharmacogenomic decision support. Patient interest was robust, physician adoption of information was high, and results were routinely utilized. Ongoing examination of a larger number of clinic encounters and inclusion of more physicians and patients is warranted.

Abstract Bone is an active organ that continuously undergoes an orchestrated process of remodeling throughout life. Bone tissue is uniquely capable of adapting to loading, hormonal, and other changes happening in the body, as well as repairing bone that becomes damaged to maintain tissue integrity. On the other hand, diseases such as osteoporosis and metastatic cancers disrupt normal bone homeostasis leading to compromised function. Historically, the ability to investigate processes related to either physiologic or diseased bone tissue is limited by traditional models that fail to emulate the complexity of native bone. Organ‐on‐a‐chip models are based on technological advances in tissue engineering and microfluidics, enabling the reproduction of key features specific to tissue microenvironments within a microfabricated device. Compared to conventional in vitro and in vivo bone models, microfluidic models, and especially organ‐on‐a‐chip platforms, provide more biomimetic tissue culture conditions, with increased predictive power for clinical assays. In this review, microfluidic and organ‐on‐a‐chip technologies designed for understanding the biology of bone as well as bone‐related diseases and treatments are reported. Finally, the authors discuss the limitations of the current models and point toward future directions for microfluidics and organ‐on‐a‐chip technologies in bone research.

Ménétrier disease and gastrointestinal stromal tumors (GISTs) are hyperproliferative disorders of the stomach caused by dysregulated receptor tyrosine kinases (RTKs). In Ménétrier disease, overexpression of TGF-alpha, a ligand for the RTK EGFR, results in selective expansion of surface mucous cells in the body and fundus of the stomach. In GISTs, somatic mutations of the genes encoding the RTK KIT (or PDGFRA in a minority of cases) result in constitutive kinase activity and neoplastic transformation of gut pacemaker cells (interstitial cells of Cajal). On the basis of the involvement of these RTKs in the pathogenesis of these disorders, Ménétrier disease patients have been effectively treated with a blocking monoclonal antibody specific for EGFR and GIST patients with KIT and PDGFRA tyrosine kinase inhibitors.