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Col6A1 and Lcn2 promote the migration of PDA cells.

140 Illumina probes corresponding to138 unique genes differentially expressed in regressing (R) vs. progressing (P) cutaneous metastases based on whole genome microarray analysis (fold change magnitude {greater than or equal to}1.7, p value {less than or equal to}0.05 )

KPC and KPTC Secretomes

Histologic features of liver metastases in KPTC, KPC, KTC and KC mice.

Purpose: To explore factors associated with response and resistance to anti–PD-1 therapy, we analyzed multiple disease sites at autopsy in a patient with widely metastatic melanoma who had a heterogeneous response.Materials and Methods: Twenty-six melanoma specimens (four premortem, 22 postmortem) were subjected to whole exome sequencing. Candidate immunologic markers and gene expression were assessed in 10 cutaneous metastases showing response or progression during therapy.Results: The melanoma was driven by biallelic inactivation of NF1. All lesions had highly concordant mutational profiles and copy number alterations, indicating linear clonal evolution. Expression of candidate immunologic markers was similar in responding and progressing lesions. However, progressing cutaneous metastases were associated with overexpression of genes associated with extracellular matrix and neutrophil function.Conclusions: Although mutational and immunologic differences have been proposed as the primary determinants of heterogeneous response/resistance to targeted therapies and immunotherapies, respectively, differential lesional gene expression profiles may also dictate anti–PD-1 outcomes. Clin Cancer Res; 23(12); 3168–80. ©2017 AACR.See related commentary by Wilmott et al., p. 2921

Purpose: TP53 and the TGFβ pathway are major mediators of pancreatic cancer metastasis. The mechanisms by which they cause hematogenous metastasis have not been fully explored.Experimental Design: KPC (LSL-KRASG12D/+;LSL-Trp53R172H/+; Ptf1aCre/+) mice were generated, and the frequency and morphology of organ-specific hematogenous metastases compared with that seen in KPTC and KTC littermates (Tgfbr2+/−). Key findings were validated in primary cells from each genotype and samples of human pancreatic cancer liver metastases.Results: The frequency of hematogenous metastasis in KPTC mice was significantly lower than for KPC mice (41% vs. 68%, P < 0.05), largely due to a reduction in liver metastases. No differences were found between KPC and KPTC lung metastases, whereas liver metastases in KPTC mice showed a profound extravasation deficiency characterized by sinusoidal growth and lack of desmoplastic stroma. Analogous findings were confirmed in liver samples from patients indicating their clinical relevance. Portal vein colonization as a direct mode of access to the liver was observed in both mice and humans. Secretome analyses of KPC cells revealed an abundance of secreted prometastatic mediators including Col6A1 and Lcn2 that promoted early steps of metastatic colonization. These mediators were overexpressed in primary tumors but not metastases, suggesting that the ability to colonize is, in part, developed within the primary site, a phenomenon we refer to as the “Cinderella effect.”Conclusions: These findings establish a novel paradigm for understanding pancreatic cancer metastasis and the observed clinical latencies of liver versus lung metastases specifically. Clin Cancer Res; 23(6); 1607–20. ©2016 AACR.

Characterization of lung metastases in KPC and KPTC mice.

Supplementary methods

Patterns of Metastatic Spread by Genotype

Genotyping of Mice and Features of PanINs.

Radiographic changes of brain metastases after stereotactic radiosurgery (SRS) can signify tumor recurrence and/or radiation necrosis (RN); however, standard imaging modalities cannot easily distinguish between these two entities. We investigated whether 18F-Fluorocholine uptake in surgical samples of the resected lesions correlates with pathologic evidence of recurrent tumor and PET imaging. About 14 patients previously treated with SRS that developed radiographic changes were included. All patients underwent a preoperative 40-min dynamic PET/CT concurrent with 392 ± 11 MBq bolus injection of 18F-Fluorocholine. 18F-Fluorocholine pharmacokinetics were evaluated by standardized uptake value (SUV), graphical analysis (Patlak plot; KiP) and an irreversible two-compartment model (K1, k2, k3, and Ki). 12 out of 14 patients were administered an additional 72 ± 14 MBq injection of 18F-Fluorocholine 95 ± 26 minutes prior to surgical resection. About 113 resected samples from 12 patients were blindly reviewed by a neuropathologist to assess the viable tumor and necrotic content, microvascular proliferation, reactive gliosis, and mono- and polymorphonuclear inflammatory infiltrates. Correlation between these metrics 18F-Fluorocholine SUV was investigated with a linear mixed model. Comparison of survival distributions of two groups of patients (population median split of PET SUVmax) was performed with the log-rank test. Exactly 10 out of 12 patients for which surgical samples were acquired exhibited pathologic recurrence. Strong correlation was observed between SUVmax as measured from a surgically removed sample with highest uptake and by PET (Pearson’s r = 0.66). Patients with 18F-Fluorocholine PET SUVmax > 6 experienced poor survival. Surgical samples with viable tumor had higher 18F-fluorocholine uptake (SUV) than those without tumor (4.5 ± 3.7 and 2.6 ± 3.0; p = 0.01). 18F-fluorocholine count data from surgical samples is driven not only by the percentage viable tumor but also by the degree of inflammation and reactive gliosis (p ≤ 0.02; multivariate regression). 18F-Fluorocholine accumulation is increased in viable tumor; however, inflammation and gliosis may also lead to elevated uptake. Higher 18F-Fluorocholine PET uptake portends worse prognosis. Kinetic analysis of dynamic 18F-Fluorocholine PET imaging supports the adequacy of the simpler static SUV metric.

Purpose For patients with long bone metastases who undergo orthopedic stabilization surgery followed by radiotherapy (RT), it is unclear what extent of hardware coverage by the radiation field is needed for optimal tumor control. Methods and Materials Long bone metastases treated with surgical intervention followed by radiation between August 2011 to May 2019 from a single institution were reviewed. Local recurrence, defined as any in-bone recurrence, was identified by chart review. Accompanying demographic and treatment characteristics were recorded. Statistical analysis to evaluate factors associated with tumor recurrence included univariate analysis, multivariate analysis, and propensity score matching. Results Among 138 patients with 145 long bone metastases undergoing postoperative RT with a median follow-up of 29.5 months, 36 bone metastases experienced a local recurrence. Most patients (92%) were treated with conventional RT and the median delivered dose was 30 Gy (interquarile range, 20-30 Gy). On univariate analysis, whole hardware RT field coverage and higher dose (biologically effective dose 10 ≥39 Gy) were associated with reduced local recurrence (0.44 hazard ratio [HR]; 95% confidence interval [CI], 0.22%-0.86%; P = .017; 0.5 HR; 95% CI, 0.26%-0.96%; P = .038, respectively). Covariates of time from surgery to RT start, histology of primary tumor (categorized as resistant vs sensitive), intramedullary hardware placement, reaming procedure, and margin status did not reach statistical significance. To adjust for confounding effects, we also conducted a propensity score matched analysis which confirmed that whole hardware coverage was statistically associated with a decreased risk of recurrence on the matched dataset (0.24 HR; 95% CI, 0.07%-0.84%; P = .026). Conclusions In this analysis of mostly patients undergoing conventional radiation, coverage of the whole hardware was associated with reduced local recurrence for patients with long bone metastases, consistent with prior reports. Investigation of approaches to further reduce local recurrence, such as preoperative stereotactic radiation, may be warranted.

Pancreatic cancer expression profiles largely reflect a classical or basal-like phenotype. The extent to which these profiles vary within a patient is unknown. We integrated evolutionary analysis and expression profiling in multiregion-sampled metastatic pancreatic cancers, finding that squamous features are the histologic correlate of an RNA-seq-defined basal-like subtype. In patients with coexisting basal and squamous and classical and glandular morphology, phylogenetic studies revealed that squamous morphology represented a subclonal population in an otherwise classical and glandular tumor. Cancers with squamous features were significantly more likely to have clonal mutations in chromatin modifiers, intercellular heterogeneity for MYC amplification and entosis. These data provide a unifying paradigm for integrating basal-type expression profiles, squamous histology and somatic mutations in chromatin modifier genes in the context of clonal evolution of pancreatic cancer. Iacobuzio-Donahue and colleagues use integrated transcriptomic, histologic and mutational data to analyze squamous features of pancreatic ductal adenocarcinoma (PDAC), further refining the understanding of heterogeneity and evolution in PDAC.

Purpose: To explore factors associated with response and resistance to anti–PD-1 therapy, we analyzed multiple disease sites at autopsy in a patient with widely metastatic melanoma who had a heterogeneous response. Materials and Methods: Twenty-six melanoma specimens (four premortem, 22 postmortem) were subjected to whole exome sequencing. Candidate immunologic markers and gene expression were assessed in 10 cutaneous metastases showing response or progression during therapy. Results: The melanoma was driven by biallelic inactivation of NF1. All lesions had highly concordant mutational profiles and copy number alterations, indicating linear clonal evolution. Expression of candidate immunologic markers was similar in responding and progressing lesions. However, progressing cutaneous metastases were associated with overexpression of genes associated with extracellular matrix and neutrophil function. Conclusions: Although mutational and immunologic differences have been proposed as the primary determinants of heterogeneous response/resistance to targeted therapies and immunotherapies, respectively, differential lesional gene expression profiles may also dictate anti–PD-1 outcomes. Clin Cancer Res; 23(12); 3168–80. ©2017 AACR. See related commentary by Wilmott et al., p. 2921

Purpose: TP53 and the TGFβ pathway are major mediators of pancreatic cancer metastasis. The mechanisms by which they cause hematogenous metastasis have not been fully explored. Experimental Design: KPC (LSL-KRASG12D/+;LSL-Trp53R172H/+; Ptf1aCre/+) mice were generated, and the frequency and morphology of organ-specific hematogenous metastases compared with that seen in KPTC and KTC littermates (Tgfbr2+/−). Key findings were validated in primary cells from each genotype and samples of human pancreatic cancer liver metastases. Results: The frequency of hematogenous metastasis in KPTC mice was significantly lower than for KPC mice (41% vs. 68%, P < 0.05), largely due to a reduction in liver metastases. No differences were found between KPC and KPTC lung metastases, whereas liver metastases in KPTC mice showed a profound extravasation deficiency characterized by sinusoidal growth and lack of desmoplastic stroma. Analogous findings were confirmed in liver samples from patients indicating their clinical relevance. Portal vein colonization as a direct mode of access to the liver was observed in both mice and humans. Secretome analyses of KPC cells revealed an abundance of secreted prometastatic mediators including Col6A1 and Lcn2 that promoted early steps of metastatic colonization. These mediators were overexpressed in primary tumors but not metastases, suggesting that the ability to colonize is, in part, developed within the primary site, a phenomenon we refer to as the “Cinderella effect.” Conclusions: These findings establish a novel paradigm for understanding pancreatic cancer metastasis and the observed clinical latencies of liver versus lung metastases specifically. Clin Cancer Res; 23(6); 1607–20. ©2016 AACR.

SummaryRecent studies indicate that pancreatic cancer expression profiles are variable and largely reflect a classical or basal-type phenotype. We performed genetic sequencing, RNA-seq, and histologic review of multiregion sampled pancreatic cancers and found that squamous and squamoid features, indicators of poor prognosis, correlate with a “basal-like” expressional type. Cancers with squamous features were more likely to have truncal mutations in chromatin modifier genes and intercellular heterogeneity for MYC amplification that was associated with entosis. In most patients the basal phenotype coexisted with a glandular component, and phylogenetic studies indicated that it arose from a subclonal population in the tumor. These data provide a unifying paradigm for understanding the interrelationship of basal-type features, squamous histology, and somatic mutations in chromatin modifier genes in the context of the clonal evolution of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed after the disease has metastasized; it is among the most lethal forms of cancer. We recently described aberrant expression of an open reading frame 1 protein, ORF1p, encoded by long interspersed element-1 (LINE-1; L1) retrotransposon, in PDAC. To test whether LINE-1 expression leads to somatic insertions of this mobile DNA, we used a targeted method to sequence LINE-1 insertion sites in matched PDAC and normal samples. We found evidence of 465 somatic LINE-1 insertions in 20 PDAC genomes, which were absent from corresponding normal samples. In cases in which matched normal tissue, primary PDAC and metastatic disease sites were available, insertions were found in primary and metastatic tissues in differing proportions. Two adenocarcinomas secondarily involving the pancreas, but originating in the stomach and duodenum, acquired insertions with a similar discordance between primary and metastatic sites. Together, our findings show that LINE-1 contributes to the genetic evolution of PDAC and suggest that somatic insertions are acquired discontinuously in gastrointestinal neoplasms.

Glucose metabolic activity measured by [(18)F]-fluoro-2-deoxy-glucose positron emission tomography (FDG-PET) has shown prognostic value in multiple malignancies, but results are often confounded by the inclusion of patients with various disease stages and undergoing various therapies. This study was designed to evaluate the prognostic value of tumor FDG uptake quantified by maximum standardized uptake value (SUVmax) in a large group of early-stage non-small cell lung cancer (NSCLC) patients treated with stereotactic body radiotherapy (SBRT) using consistent treatment techniques.Two hundred nineteen lesions in 211 patients treated with definitive SBRT for stage I NSCLC were analyzed after a median follow-up of 25.2 months. Cox regression was used to determine associations between SUVmax and overall survival (OS), disease-specific survival (DSS), and freedom from local recurrence (FFLR) or distant metastasis (FFDM).SUVmax3.0 was associated with worse OS (p0.001), FFLR (p=0.003) and FFDM (p=0.003). On multivariate analysis, OS was associated with SUVmax (HR 1.89, p=0.03), gross tumor volume (GTV) (HR 1.94, p=0.005) and Karnofsky performance status (KPS) (HR 0.51, p=0.008). DSS was associated only with SUVmax (HR 2.58, p=0.04). Both LR (HR 11.47, p=0.02) and DM (HR 3.75, p=0.006) were also associated with higher SUVmax.In a large patient population, SUVmax3.0 was associated with worse survival and a greater propensity for local recurrence and distant metastasis after SBRT for NSCLC.

The extent of heterogeneity among driver gene mutations present in naturally occurring metastases-that is, treatment-naive metastatic disease-is largely unknown. To address this issue, we carried out 60× whole-genome sequencing of 26 metastases from four patients with pancreatic cancer. We found that identical mutations in known driver genes were present in every metastatic lesion for each patient studied. Passenger gene mutations, which do not have known or predicted functional consequences, accounted for all intratumoral heterogeneity. Even with respect to these passenger mutations, our analysis suggests that the genetic similarity among the founding cells of metastases was higher than that expected for any two cells randomly taken from a normal tissue. The uniformity of known driver gene mutations among metastases in the same patient has critical and encouraging implications for the success of future targeted therapies in advanced-stage disease.

We describe an 85-yr-old male of Ashkenazi Jewish descent with biopsy-proven locally advanced pancreatic ductal adenocarcinoma (PDA). The patient underwent a modified course of gemcitabine and stereotactic body radiation therapy and survived for 42 mo with a stable pancreatic head mass and no evidence of metastatic disease before death due to complications from a stroke. Whole-exome sequencing of his tumor revealed a simple genome landscape with no evidence of mutations, copy-number changes, or structural alterations in genes most commonly associated with PDA (i.e., KRAS, CDKN2A, TP53, or SMAD4). An analysis of his germline DNA revealed no pathogenic variants of significance. Whole-exome and whole-genome sequencing identified a somatic mutation of RNF213 and an inversion/deletion of CTNNA2 as the genetic basis of his PDA. Although PDA is classically characterized by a predictable set of mutations, these data suggest that alternate genetic paths to PDA may exist, which can be associated with a more indolent clinical course.

To characterize the impact of androgen-deprivation therapy (ADT) on the incidence of cardiovascular events (CE) in prostate cancer patients treated with radiotherapy (RT).There were 2211 patients with localized prostate cancer treated with RT from 1988 to 2008 at our institution. There were 991 patients (44.8%) who received ADT at the time of RT for a median of 6.1 months. Salvage ADT was initiated prior to CE in 365 men (16.5%) at a median of 5.5 years (range: 0.6 to 18.4 years) after RT and continued for a median of 4.3 years. A nomogram was constructed to predict the 10-year risk of CE "post-RT" (i.e., after RT).Patients receiving ADT at the time of RT exhibited significantly higher 10-year incidence of CE (19.6%, 95% CI 17.0%-22.6%) than those not receiving ADT (14.3%, 95% CI 12.2%-16.7%, P = .005). On multivariate analysis, both ADT at the time of RT (P = .007) and the time of salvage (P = .0004) were associated with increased CE risk, as were advanced age (P = .02), smoking (P = .0007), history of diabetes (P = .0007), and history of CE before RT (P .0001). A nomogram using patient age, smoking status, history of pre-RT CE, history of diabetes, and ADT use at the time of RT predicted the rate of 10-year CE with a C-index of 0.81 (95% CI, 0.72-0.88).While ADT is often an essential part of prostate cancer treatment, patients should be counseled regarding increased risks of CE and prophylactic efforts should be considered to mitigate that risk.

10122 Background: Bone metastases impair function and decrease quality of life due to acute or chronic pain. The standard approach for patients with multiple bone metastases is systemic therapy and palliative radiation therapy (RT) when the metastases become symptomatic. This study aims to understand the characteristics and outcomes for inpatients admitted for painful bone metastases. Methods: An inpatient radiation oncology consult registry was created in 2015 to evaluate patterns of care for patients receiving RT in the inpatient setting. Of the 1151 consults requested between 7/2015 and 6/2016, 28% (n = 323) were for evaluation of symptomatic bone metastases in patients who were hospitalized for acute or chronic pain. Among this cohort, 64% (n = 208) went on to receive RT for 225 bone metastases. Sixty percent of RT courses were initiated while the patient was hospitalized. Clinical characteristics correlated with overall survival (OS) were evaluated through Cox regression analysis. Results: The median follow up for the 208 patients who received RT was 4 months (0.1-9 months). Patient median age was 61 (10-92 years), and the median KPS was 70 (20-90). The most common sites treated were spine (50%), joints such as hip and shoulder (11%), long bones including femur and humerus (11%), and pelvis (10%). Sixty-one percent (n = 138) of the treated metastases were diagnosed ≥4 months prior to RT. The median survival after receiving palliative RT was 4 months (0-19 months). Among the 141 patients who had died at the time of analysis, 92 (65%) died within 2 months, and 128 (91%) within 6 months. Eighteen patients (9%) discontinued RT to transition to hospice care. OS after RT is significantly correlated with KPS (p < 0.0001) at the time of consult but not with patient age or site of treated disease. Conclusions: In this select group of inpatients who were evaluated for palliation of symptomatic bone metastases, we found a short OS after RT. The majority of metastases were present for ≥4 months prior to RT. This study suggests that earlier RT for high-risk metastases should be considered to prevent development of symptomatic disease that requires hospitalization. Risk factors for development of painful bone metastases are being studied prospectively at our institution.

The oral cavity is the most anterior head and neck site. It is separated from the oropharynx by an imaginary line extending from the anterior tonsillar pillars to the junction of hard and soft palate to the circumvallate papillae.The oral cavity is divided into several subsites including the oral tongue, floor of the mouth, retromolar trigone, alveolar ridge, hard palate, buccal mucosa, gingiva and lips (Fig. 1).

MMPs (matrix metalloproteinases) and the related “a disintegrin and metalloproteinases” (ADAMs) promote tumorigenesis by cleaving extracellular matrix and protein substrates, including N-cadherin. Although N-cadherin is thought to regulate cell adhesion, migration, and invasion, its role has not been characterized in glioblastomas (GBMs). In this study, we investigated the expression and function of posttranslational N-cadherin cleavage in GBM cells as well as its regulation by protein kinase C (PKC). N-Cadherin cleavage occurred at a higher level in glioblastoma cells than in non-neoplastic astrocytes. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA) increased N-cadherin cleavage, which was reduced by pharmacological inhibitors and short interfering RNA (siRNA) specific for ADAM-10 or PKC-α. Furthermore, treatment of GBM cells with PMA induced the translocation of ADAM-10 to the cell membrane, the site at which N-cadherin was cleaved, and this translocation was significantly reduced by the PKC-α inhibitor Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole] or PKC-α short hairpin RNA. In functional studies, N-cadherin cleavage was required for GBM cell migration, as depletion of N-cadherin cleavage by N-cadherin siRNA, ADAM-10 siRNA, or a cleavage-site mutant N-cadherin, decreased GBM cell migration. Together, these results suggest that N-cadherin cleavage is regulated by a PKC-α–ADAM-10 cascade in GBM cells and may be involved in mediating GBM cell migration.

Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor–related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-α (PKC-α) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-α small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate–induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-α/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-α and PI3K could provide a new treatment paradigm for glioblastomas. [Cancer Res 2007;67(21):10241–51]