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1.  CTLA-4 Blockade Synergizes Therapeutically with PARP Inhibition in BRCA1-Deficient Ovarian Cancer 
Cancer immunology research  2015;3(11):1257-1268.
Immune checkpoint blockade has shown significant therapeutic efficacy in melanoma and other solid tumors, but results in ovarian cancer have been limited. With evidence that tumor immunogenicity modulates the response to checkpoint blockade, and data indicating that BRCA-deficient ovarian cancers express higher levels of immune response genes, we hypothesized that BRCA ovarian tumors would be vulnerable to checkpoint blockade. To test this hypothesis, we used an immunocompetent BRCA1-deficient murine ovarian cancer model to compare treatment with CTLA-4 or PD-1/PD-L1 antibodies alone or combined with targeted cytotoxic therapy using a PARP inhibitor. Correlative studies were performed in vitro using human BRCA1 cells. We found that CTLA-4 antibody, but not PD-1/PD-L1 blockade, synergized therapeutically with the PARP inhibitor, resulting in immune-mediated tumor clearance and long-term survival in a majority of animals (P < 0.0001). The survival benefit of this combination was T-cell mediated and dependent on increases in local IFNγ production in the peritoneal tumor environment. Evidence of protective immune memory was observed more than 60 days after completion of therapy. Similar increases in the cytotoxic effect of PARP inhibition in the presence of elevated levels of IFNγ in human BRCA1 cancer cells support the translational potential of this treatment protocol. These results demonstrate that CTLA-4 blockade combined with PARP inhibition induces protective antitumor immunity and significant survival benefit in the BRCA1 tumor model, and support clinical testing of this regimen to improve outcomes for women with hereditary ovarian cancer.
doi:10.1158/2326-6066.CIR-15-0044
PMCID: PMC4984269  PMID: 26138335
2.  Delays in Post-Remission Chemotherapy for Philadelphia Chromosome Negative Acute Lymphoblastic Leukemia are Associated with Inferior Outcomes in Patients who Undergo Allogeneic Transplant: an Analysis from ECOG 2993/MRC UK ALLXII 
American journal of hematology  2016;91(11):1107-1112.
Adults with acute lymphoblastic leukemia (ALL) have a poorer prognosis than children due to a high risk of relapse. One explanation may be variable adherence to dose-intense chemotherapy. However, little is known about risk factors for delays in therapy and their impact on survival.
We conducted an analysis of ECOG 2993/UKALLXII trial to study delays in post-remission chemotherapy in adults with newly-diagnosed ALL. Logistic regression was used to identify risk factors for a very long delay (>4 weeks, VLD) in start of intensification therapy. Cox regression was used to evaluate the impact of delays on overall and event-free survival (OS, EFS).
We evaluated 1076 Philadelphia chromosome negative (Ph-) patients who completed induction chemotherapy, achieved complete remission, and started intensification. Factors independently associated with VLD included: duration of hospitalization (Odds Ratio (OR)=1.2, p<0.001) during Phase I; thrombocytopenia during Phase I (OR=1.16, p=0.004) or Phase II (OR 1.13, p=0.001); chemotherapy dose reductions during induction Phase I (OR=1.72, p<0.014); female sex (OR=1.53, p=0.010); Black (OR=3.24, p=0.003) and Asian (OR=2.26, p=0.021) race; and increasing age (OR=1.31, p<0.001). In multivariate Cox regression, patients who underwent allogeneic stem cell transplant (alloHCT) had significantly worse OS (HR 1.4, p=0.03) and EFS (HR 1.4, p=0.02) after experiencing a VLD compared to alloHCT patients who experienced <=4 weeks delay.
Specific populations (female, older, Black, and Asian patients) were more likely to experience delays in chemotherapy, as were those with significant toxicity during induction. Very long delays in therapy negatively affected outcomes in patients undergoing allografting.
doi:10.1002/ajh.24497
PMCID: PMC5073003  PMID: 27468137
acute lymphoblastic leukemia; chemotherapy; allogeneic stem cell transplant
3.  AMERICAN JOINT COMMITTEE ON CANCER ACCEPTANCE CRITERIA FOR INCLUSION OF RISK MODELS FOR INDIVIDUALIZED PROGNOSIS IN THE PRACTICE OF PRECISION MEDICINE 
The American Joint Committee on Cancer (AJCC) has increasingly recognized the need for more personalized probabilistic predictions than those delivered by ordinal staging systems, particularly through the use of accurate risk models or calculators. However, judging the quality and acceptability of a risk model is complex.
The AJCC Precision Medicine Core conducted a two-day meeting to discuss characteristics necessary for a quality risk model in cancer patients. More specifically, the committee established inclusion and exclusion criteria necessary for a risk model to potentially be endorsed by the AJCC. This committee reviewed and discussed relevant literature before creating a checklist unique to this need of AJCC risk model endorsement.
The committee identified 13 inclusion and 3 exclusion criteria for AJCC risk model endorsement in cancer. The emphasis centered on performance metrics, implementation clarity, and clinical relevance.
The facilitation of personalized probabilistic predictions for cancer patients holds tremendous promise, and these criteria will hopefully greatly accelerate this process. Moreover, these criteria might be useful for a general audience when trying to judge the potential applicability of a published risk model in any clinical domain.
doi:10.3322/caac.21339
PMCID: PMC4955656  PMID: 26784705
4.  Radiofrequency Ablation is Associated with Decreased Neoplastic Progression in Patients with Barrett’s Esophagus and Confirmed Low-Grade Dysplasia 
Gastroenterology  2015;149(3):567-576.e3.
Background & Aims
Barrett’s esophagus (BE) with low-grade dysplasia (LGD) can progress to high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). Radiofrequency ablation (RFA) has been shown to be an effective treatment for LGD in clinical trials but its effectiveness in clinical practice is unclear. We compared the rate of progression of LGD following RFA to that with endoscopic surveillance alone in routine clinical practice.
Methods
We performed a retrospective study of patients who either underwent RFA (n=45) or surveillance endoscopy (n=125) for LGD, confirmed by at least 1 expert pathologist, from October 1992 through December 2013 at 3 medical centers in the US. Cox regression analysis was used to assess the association between progression and RFA.
Results
Data were collected over median follow-up periods of 889 days (inter-quartile range, 264–1623 days) after RFA and 848 days (inter-quartile range, 322–2355 days) after surveillance endoscopy (P=.32). The annual rates of progression to HGD or EAC was 6.6% in the surveillance group and 0.77% in the RFA group. The risk of progression to HGD or EAC was significantly lower among patients who underwent RFA than those who underwent surveillance (adjusted hazard ratio, 0.06; 95% confidence interval, 0.008–0.48).
Conclusions
Among patients with BE and confirmed LGD, rates of progression to a combined endpoint of HGD and EAC were lower among those treated with RFA than among untreated patients. Although selection bias cannot be excluded, these findings provide additional evidence for the use of endoscopic ablation therapy for LGD.
doi:10.1053/j.gastro.2015.04.013
PMCID: PMC4550488  PMID: 25917785
eradication; clinical setting; prevention; esophageal cancer
5.  A clinical measure of DNA methylation predicts outcome in de novo acute myeloid leukemia 
JCI insight  2016;1(9):e87323.
BACKGROUND
Variable response to chemotherapy in acute myeloid leukemia (AML) represents a major treatment challenge. Clinical and genetic features incompletely predict outcome. The value of clinical epigenetic assays for risk classification has not been extensively explored. We assess the prognostic implications of a clinical assay for multilocus DNA methylation on adult patients with de novo AML.
METHODS
We performed multilocus DNA methylation assessment using xMELP on samples and calculated a methylation statistic (M-score) for 166 patients from UPENN with de novo AML who received induction chemotherapy. The association of M-score with complete remission (CR) and overall survival (OS) was evaluated. The optimal M-score cut-point for identifying groups with differing survival was used to define a binary M-score classifier. This classifier was validated in an independent cohort of 383 patients from the Eastern Cooperative Oncology Group Trial 1900 (E1900; NCT00049517).
RESULTS
A higher mean M-score was associated with death and failure to achieve CR. Multivariable analysis confirmed that a higher M-score was associated with death (P = 0.011) and failure to achieve CR (P = 0.034). Median survival was 26.6 months versus 10.6 months for low and high M-score groups. The ability of the M-score to perform as a classifier was confirmed in patients ≤ 60 years with intermediate cytogenetics and patients who achieved CR, as well as in the E1900 validation cohort.
CONCLUSION
The M-score represents a valid binary prognostic classifier for patients with de novo AML. The xMELP assay and associated M-score can be used for prognosis and should be further investigated for clinical decision making in AML patients.
doi:10.1172/jci.insight.87323
PMCID: PMC4951094  PMID: 27446991
6.  Lymphatic Invasion as a Prognostic Biomarker in Primary Cutaneous Melanoma 
Melanoma has a propensity for lymph node metastasis. However, the incidence of lymphatic invasion detected by histology alone in primary melanoma is disproportionately low in comparison to the incidence of positive sentinel lymph nodes (SLN). With the discovery of lymphatic endothelial cell markers, such as podoplanin and LYVE-1, lymphatic vessels can be reliably detected in formalin-fixed paraffin-embedded (FFPE) tissues. There is a now consensus that lymphatic invasion detected by immunohistochemical stains in primary melanoma is much more common than previously reported by histological examination alone. Immunohistochemical stains show that lymphangiogenesis and lymphatic invasion in primary melanoma may occur intratumorally or peritumorally, and lymphatic invasion is common across the range of tumor thicknesses in primary vertical growth phase (VGP) melanomas. A number of studies have shown that lymphatic invasion in primary melanoma is associated with a positive sentinel lymph node biopsy and a worse clinical outcome. Although not currently a part of the standard of care for staging of melanoma, the detection of lymphatic invasion in primary melanoma using immunohistochemical markers may be helpful in planning of therapy in some cases and may find a routine role in primary melanoma microscopic attributes in future.
doi:10.1007/978-1-62703-727-3_15
PMCID: PMC4918087  PMID: 24258984
Lymphangiogenesis; Lymphatic invasion; Melanoma; Podoplanin; D2–40; LYVE-1; Prognosis
7.  A clinical measure of DNA methylation predicts outcome in de novo acute myeloid leukemia 
JCI Insight  null;1(9):e87323.
BACKGROUND. Variable response to chemotherapy in acute myeloid leukemia (AML) represents a major treatment challenge. Clinical and genetic features incompletely predict outcome. The value of clinical epigenetic assays for risk classification has not been extensively explored. We assess the prognostic implications of a clinical assay for multilocus DNA methylation on adult patients with de novo AML.
METHODS. We performed multilocus DNA methylation assessment using xMELP on samples and calculated a methylation statistic (M-score) for 166 patients from UPENN with de novo AML who received induction chemotherapy. The association of M-score with complete remission (CR) and overall survival (OS) was evaluated. The optimal M-score cut-point for identifying groups with differing survival was used to define a binary M-score classifier. This classifier was validated in an independent cohort of 383 patients from the Eastern Cooperative Oncology Group Trial 1900 (E1900; NCT00049517).
RESULTS. A higher mean M-score was associated with death and failure to achieve CR. Multivariable analysis confirmed that a higher M-score was associated with death (P = 0.011) and failure to achieve CR (P = 0.034). Median survival was 26.6 months versus 10.6 months for low and high M-score groups. The ability of the M-score to perform as a classifier was confirmed in patients ≤ 60 years with intermediate cytogenetics and patients who achieved CR, as well as in the E1900 validation cohort.
CONCLUSION. The M-score represents a valid binary prognostic classifier for patients with de novo AML. The xMELP assay and associated M-score can be used for prognosis and should be further investigated for clinical decision making in AML patients.
The M-score is a DNA-methylation–based biomarker developed for clinical use that predicts survival in patients with de novo AML treated with induction chemotherapy.
doi:10.1172/jci.insight.87323
PMCID: PMC4951094  PMID: 27446991
8.  A Modified Integrated Genetic Model for Risk Prediction in Younger Patients with Acute Myeloid Leukemia 
PLoS ONE  2016;11(4):e0153016.
Background
Although cytogenetics-based prognostication systems are well described in acute myeloid leukemia (AML), overall survival (OS) remains highly variable within risk groups. An integrated genetic prognostic (IGP) model using cytogenetics plus mutations in nine genes was recently proposed for patients ≤60 years to improve classification. This model has not been validated in clinical practice.
Methods and Findings
We retrospectively studied 197 patients with newly diagnosed de novo AML. We compared OS curves among the mutational profiles defined by the IGP model. The IGP model assigned patients with intermediate cytogenetics as having favorable, intermediate or unfavorable mutational profiles. The IGP model reassigned 50 of 137 patients with intermediate cytogenetics to favorable or unfavorable mutational profiles. Median OS was 2.8 years among 14 patients with intermediate cytogenetics and favorable mutational profiles (mutant NPM1 and mutant IDH1 or IDH2) and 1.3 years among patients with intermediate mutational profiles. Among patients with intermediate cytogenetics labeled as having unfavorable mutational profiles, median OS was 0.8 years among 24 patients with FLT3-ITD positive AML and high-risk genetic changes (trisomy 8, TET2 and/or DNMT3A) and 1.7 years among 12 patients with FLT3-ITD negative AML and high-risk mutations (TET2, ASXL1 and/or PHF6). OS for patients with intermediate cytogenetics and favorable mutational profiles was similar to OS for patients with favorable cytogenetics (p = 0.697) and different from patients with intermediate cytogenetics and intermediate mutational profiles (p = 0.028). OS among patients with FLT3-ITD positive AML and high-risk genetic changes was similar to patients with unfavorable cytogenetics (p = 0.793) and different from patients with intermediate IGP profile (p = 0.022). Patients with FLT3-ITD negative AML and high-risk mutations, defined as ‘unfavorable’ in the IGP model, had OS similar to patients with intermediate IGP profile (p = 0.919).
Conclusions
The IGP model was not completely validated in our cohort. However, mutations in six out of the nine genes can be used to characterize survival (NPMI, IDH1, IDH2, FLT3-ITD, TET2, DNMT3A) and allow for more robust prognostication in the patients who are re-categorized by the IGP model. These mutations should be incorporated into clinical testing for younger patients outside of clinical trials, in order to guide therapy.
doi:10.1371/journal.pone.0153016
PMCID: PMC4822876  PMID: 27050425
9.  DNMT3A mutational status affects the results of dose-escalated induction therapy in acute myelogenous leukemia 
Purpose
DNA methyltransferase 3A ( DNMT3A) is one of the commonly mutated genes in acute myelogenous leukemia (AML). Reports on the prognostic significance of DNMT3A mutations have been inconsistent, and most of the data is available only for patients 60 years of age or younger. We hypothesized that this inconsistency is due to an interaction between the dose of anthracycline used in induction therapy and DNMT3A status. We studied whether patients with DNMT3A-mutated AML treated with standard dose anthracyclines had an inferior survival compared to patients with other mutation profiles or those who received high dose therapy.
Experimental design
152 patients in this retrospective cohort study (median age, 54 years) with de-novo AML underwent induction therapy and next-generation sequencing of 33 commonly mutated genes in hematologic malignancies, including DNMT3A, FLT3-ITD, NPM1, and IDH1/2. Cox regression was used to if those with DNMT3A mutations who were treated with standard dose anthracycline had inferior survival.
Results
DNMT3A mutations, found in 32% of patients, were not associated with an inferior survival. Dose escalation of anthracycline in the induction regimen was associated with improved survival in those with DNMT3A mutations but not those with wild-type DNMT3A. Patients with DNMT3A mutations who received standard dose induction had shorter survival time than other patient groups (10.1 months vs. 19.8 months, p=0.0129). This relationship remained significant (HR: 1.90, p=0.006) controlling for multiple variables.
Conclusions
Patients with DNMT3A-mutated AML have an inferior survival when treated with standard-dose anthracycline induction therapy. This group should be considered for high-dose induction therapy.
doi:10.1158/1078-0432.CCR-14-0327
PMCID: PMC4383675  PMID: 25609058
Acute myeloid leukemia; DNMT3A; prognosis; induction chemotherapy
10.  Identification of secreted proteins that reflect autophagy dynamics within tumor cells 
Autophagy  2014;11(1):60-74.
Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, β), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when ATG7 (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.
doi:10.4161/15548627.2014.984273
PMCID: PMC4502670  PMID: 25484078
autophagy; biomarker; cancer; melanoma; secretome; ATG5, autophagy-related 5; ATG7, autophagy-related 7; BECN1, Beclin 1, autophagy-related; AV, autophagic vacuole; CXCL8, chemokine (C-X-C motif) ligand 8; DKK3, dickkopf WNT signaling pathway inhibitor 3; EGF, epidermal growth factor; IF, interstitial fluid; IL1B, interleukin 1, β; LC3/MAP1LC3, microtubule-associated protein 1 light chain 3; LIF, leukemia inhibitory factor; M, media; PtdIns3K, phosphatidylinositol 3-kinase; SAM, significance analysis of microarrays
11.  Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities 
Oncogene  2014;34(18):2347-2359.
Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference (RNAi) experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16INK4A-Rb pathway. Loss of p16INK4A or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence, but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as TGF-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.
doi:10.1038/onc.2014.169
PMCID: PMC4268095  PMID: 24931169
Notch; Rb; p16; HPV; E7; senescence; squamous cell carcinoma
12.  MEK inhibition affects STAT3 signaling and invasion in human melanoma cell lines 
Oncogene  2013;33(14):1850-1861.
Elevated activity of the MAPK signaling cascade is found in the majority of human melanomas and is known to regulate proliferation, survival, and invasion. Current targeted therapies focus on decreasing the activity of this pathway; however, we do not fully understand how these therapies impact tumor biology, especially given that melanoma is a heterogeneous disease. Using a three-dimensional (3D), collagen-embedded spheroid melanoma model, we observed that MEK and BRAF inhibitors can increase the invasive potential of approximately 20% of human melanoma cell lines. The invasive cell lines displayed increased receptor tyrosine kinase (RTK) activity and activation of the Src/FAK/STAT3 signaling axis, also associated with increased cell-to-cell adhesion and cadherin engagement following MEK inhibition. Targeting various RTKs, Src, FAK, and STAT3 with small molecule inhibitors in combination with a MEK inhibitor prevented the invasive phenotype, but only STAT3 inhibition caused cell death in the 3D context. We further show that STAT3 signaling is induced in BRAF-inhibitor resistant cells. Our findings suggest that MEK and BRAF inhibitors can induce STAT3 signaling, causing potential adverse effects such as increased invasion. We also provide the rationale for the combined targeting of the MAPK pathway along with inhibitors of RTKs, SRC, or STAT3 to counteract STAT3-mediated resistance phenotypes.
doi:10.1038/onc.2013.131
PMCID: PMC3769503  PMID: 23624919
melanoma; MEK; STAT3; targeted therapy; invasion; resistance
13.  Prognosis of Patients with Melanoma and Microsatellitosis Undergoing Sentinel Lymph Node Biopsy 
Annals of surgical oncology  2013;21(3):1016-1023.
Introduction
Melanoma microsatellitosis is classified as stage IIIB/C disease and is associated with a poor prognosis. Prognostic factors within this group, however, have not been well characterized.
Methods
We performed a retrospective analysis of 1621 patients undergoing sentinel lymph node (SLN) biopsy at our institution (1996–2011) to compare patients with (n=98) and patients without (n=1523) microsatellites. Univariate and multivariate logistic and Cox regression analyses were used to identify factors associated with SLN positivity and melanoma-specific survival (MSS) in patients with microsatellites.
Results
Patients with microsatellites were older and had lesions with higher Clark level and greater thickness that more frequently had mitoses, ulceration, and lymphovascular invasion (LVI) (all p<0.0001). In microsatellite patients, the SLN positivity rate was 43%. Lesional ulceration (OR=2.9, 95% CI: 1.5–8.6), absent tumor infiltrating lymphocytes (OR=2.8, 95% CI: 1.1–7.1), and LVI (OR=3.3, 95% CI: 1.7–10.0) were significantly associated with SLN positivity by multivariate analysis. With a median follow up of 4.5 years in survivors, ulceration (HR=3.4, 95% CI: 1.5–7.8) and >1 metastatic LN (HR=2.7, 95% CI: 1.1–6.6) were significantly associated with decreased MSS by multivariate analysis. In patients without these prognostic factors, the 5-year MSS was 90% (n=49), compared to 50% (n=23) among patients with ulceration only, 51% (n=12) in those with >1 metastatic LN only, or 25% in those with both (n=14, p<0.01).
Discussion
Microsatellitosis was frequently associated with multiple adverse pathologic features. In the absence of ulceration and >1 metastatic LN, however, the outcome for patients with microsatellites compared favorably to stage IIIB patients overall.
doi:10.1245/s10434-013-3388-5
PMCID: PMC4001246  PMID: 24258854
14.  EGFR Inhibition Promotes an Aggressive Invasion Pattern Mediated by Mesenchymal-like Tumor Cells within Squamous Cell Carcinomas 
Molecular cancer therapeutics  2013;12(10):2176-2186.
Squamous cell carcinomas (SCCs) with an infiltrative invasion pattern carry a higher risk of treatment failure. Such infiltrative invasion may be mediated by a mesenchymal-like subpopulation of malignant cells that we have previously shown to arise from epithelial to mesenchymal transition (EMT) and resist epidermal growth factor receptor (EGFR) targeting. Here we demonstrate that SCCs with infiltrative, high risk invasion patterns contain abundant mesenchymal-like cells, which are rare in tumors with low risk patterns. This cellular heterogeneity was modeled accurately in three dimensional culture using collagen-embedded SCC spheroids, which revealed distinct invasive fronts created by collective migration of E-cadherin-positive cells versus infiltrative migration of individual mesenchymal-like cells. Because EGFR expression by mesenchymal-like cells was diminished in the spheroid model and in human SCCs, we hypothesized that SCCs shift toward infiltrative invasion mediated by this subpopulation during anti-EGFR therapy. Anti-EGFR treatment of spheroids using erlotinib or cetuximab enhanced infiltrative invasion by targeting collective migration by E-cadherin-positive cells while sparing mesenchymal-like cells; by contrast, spheroid invasion in absence of mesenchymal-like cells was abrogated by erlotinib. Similarly, cetuximab treatment of xenografts containing mesenchymal-like cells created an infiltrative invasive front comprised of this subpopulation, whereas no such shift was observed upon treating xenografts lacking these cells. These results implicate mesenchymal-like SCC cells as key mediators of the infiltrative invasion seen in tumors with locally aggressive behavior. They further demonstrate that EGFR inhibition can promote an infiltrative invasion front comprised of mesenchymal-like cells preferentially in tumors where they are abundant prior to therapy.
doi:10.1158/1535-7163.MCT-12-1210
PMCID: PMC3796003  PMID: 23939378
pattern of invasion; EGFR inhibition; squamous cell carcinoma; EMT; tumor heterogeneity
15.  Age as a predictor of sentinel node metastasis among patients with localized melanoma 
Annals of surgical oncology  2014;21(4):1075-1081.
Purpose
We have previously reported that older patients with clinical Stage I and II primary cutaneous. melanoma had lower survival rates compared to younger patients We postulated that the incidence of nodal metastasis would therefore be higher among older melanoma patients.
Materials and Methods
The expanded AJCC Melanoma Staging Database contains a cohort of 7756 melanoma patients who presented without clinical evidence of regional lymph node or distant metastasis and who underwent a sentinel node biopsy procedure as a component of their staging workup
Results
Although older patients had primary melanoma features associated with more aggressive biology, we observed paradoxically a significant decrease in the incidence of sentinel node metastasis as patient age increased. Overall, the highest incidence of sentinel node metastasis was 25.8% in patients under 20 years of age, compared to 15.5% in patients 80 years and older (p< 0.001). In contrast, five year mortality rates for Clinical Stage II patients ranged from a low of 20% for those 20–40 years of age up to 38% for those over 70 years of age. Patient age was an independent predictor of sentinel node metastasis in a multifactorial analysis (p<0.001)
Conclusions
Patients with clinical Stage I and II melanoma under 20 years of age had a higher incidence of sentinel lymph node metastasis but, paradoxically, a more favorable survival outcome compared to all other age groups. In contrast, patients >70 years had the most aggressive primary melanoma features and a higher mortality rate compared to all other age groups, but a lower incidence of sentinel lymph node metastasis.
Synopsis
We analyzed the importance of patient age as a component of melanoma staging and in the design and interpretation of clinical trials for patients with localized disease or with regional node metastases.
doi:10.1245/s10434-013-3464-x
PMCID: PMC4121329  PMID: 24531700
16.  Age as a Prognostic Factor in Patients with Localized Melanoma and Regional Metastases 
Annals of surgical oncology  2013;20(12):3961-3968.
Purpose
We postulated that the worse prognosis of melanoma with advancing age reflected more aggressive tumor biology and that in younger patients the prognosis would be more favorable.
Materials and Methods
The expanded AJCC melanoma staging database contained 11,088 patients with complete data for analysis, including mitotic rate.
Results
With increasing age by decade, primary melanomas were thicker, exhibited higher mitotic rates, and were more likely to be ulcerated. In a multivariate analysis of patients with localized melanoma, thickness and ulceration were highly significant predictors of outcome at all decades of life (except for patients less than 20 years). Mitotic rate was significantly predictive in all age groups except patients < 20 years and >80 years. For patients with Stage III melanoma, there were four independent variables associated with patient survival: number of nodal metastases, patient age, ulceration, and mitotic rate.
Patients under 20 years of age had primary tumors with slightly more aggressive features, a higher incidence of sentinel lymph node metastasis, but, paradoxically, more favorable survival than all other age groups. In contrast, patients >70 years old had primary melanomas with the most aggressive prognostic features, were more likely to be head and neck primaries, and were associated with a higher mortality rate than the other age groups. Surprisingly, however, these patients had a lower rate of sentinel lymph node metastasis per T stage. Among patients between the two age extremes, clinicopathologic features and survival tended to be more homogeneous.
Conclusions
Melanomas in patients at the extremes of age have a distinct natural history.
doi:10.1245/s10434-013-3100-9
PMCID: PMC4121330  PMID: 23838920
17.  Overcoming intrinsic multi-drug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1Bhigh cells 
Cancer cell  2013;23(6):10.1016/j.ccr.2013.05.003.
Summary
Despite success with BRAFV600E–inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multi-drug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (OXPHOS) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1Bhigh subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anti-cancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.
doi:10.1016/j.ccr.2013.05.003
PMCID: PMC3810180  PMID: 23764003
18.  Effect of Guidelines on Primary Care Physician Use of PSA Screening: Results from the Community Tracking Study Physician Survey 
Background
Little is known about the effect of guidelines that recommend shared decision making on physician practice patterns. The objective of this study was to determine the association between physicians’ perceived effect of guidelines on clinical practice and self-reported prostate-specific antigen (PSA) screening patterns.
Methods
This was a cross-sectional study using a nationally representative sample of 3914 primary care physicians participating in the 1998–1999 Community Tracking Study Physician Survey. Responses to a case vignette that asked physicians what proportion of asymptomatic 60-year-old white men they would screen with a PSA were divided into 3 distinct groups: consistent PSA screeners (screen all), variable screeners (screen 1%–99%), and consistent nonscreeners (screen none). Logistic regression was used to determine the association between PSA screening patterns and physician-reported effect of guidelines (no effect v. any magnitude effect).
Results
Only 27% of physicians were variable PSA screeners; the rest were consistent screeners (60%) and consistent nonscreeners (13%). Only 8% of physicians perceived guidelines to have no effect on their practice. After adjustment for demographic and practice characteristics, variable screeners were more likely to report any magnitude effect of guidelines on their practice when compared with physicians in the other 2 groups (adjusted odds ratio 1.73; 95% confidence interval = 1.25–2.38; P = 0.001).
Conclusions
Physicians who perceive an effect of guidelines on their practice are almost twice as likely to exhibit screening PSA practice variability, whereas physicians who do not perceive an effect of guidelines on their practice are more likely to be consistent PSA screeners or consistent PSA nonscreeners.
doi:10.1177/0272989X08315243
PMCID: PMC3991564  PMID: 18556635
prostate-specific antigen; mass screening; guidelines; physicians’ practice patterns
19.  Optical Imaging of Periostin Enables Early Endoscopic Detection and Characterization of Esophageal Cancer in Mice 
Gastroenterology  2012;144(2):294-297.
Imaging strategies that detect early-stage esophageal squamous cell carcinoma (ESCC) could improve clinical outcomes, combined with endoscopic approaches. Periostin is an integrin-binding protein that is important in the tumor microenvironment. We created a fluorescent-labeled antibody that recognizes periostin and binds specifically to ESCC xenograft tumors in mice. In L2-cre;p120ctnLoxP/LoxP mice, which develop squamous cell cancers that resemble human ESCC, we visualized the probe in preneoplastic and neoplastic esophageal lesions using near-infrared fluorescent imaging with upper gastrointestinal endoscopy. Periostin might be a biomarker of the esophageal tumor microenvironment that can be used to detect preneoplastic lesions.
doi:10.1053/j.gastro.2012.10.030
PMCID: PMC3624041  PMID: 23085486
mouse model; neoplasm; extracellular matrix; POSTN
20.  IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment 
Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
PMCID: PMC3902230  PMID: 24482736
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
21.  Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation 
Oncogene  2012;31(47):4898-4911.
There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4 induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a subpopulation of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4 mediated tumor cell dedifferentiation may play an important role during tumor progression.
doi:10.1038/onc.2011.656
PMCID: PMC3343184  PMID: 22286766
Oct4; Cancer stem cell; Melanoma; Dedifferentiation; hypoxia
22.  Direct reprogramming of melanocytes to neural crest stem-like cells by one defined factor 
Stem cells (Dayton, Ohio)  2011;29(11):1752-1762.
Mouse and human somatic cells can either be reprogrammed to a pluripotent state or converted to another lineage with a combination of transcription factors suggesting that lineage commitment is a reversible process. Here we show that only one factor, the active intracellular form of Notch1, is sufficient to convert mature pigmented epidermal-derived melanocytes into functional multipotent neural crest stem-like cells. These induced neural crest stem cells (iNCSCs) proliferate as spheres under stem cell media conditions, re-express neural crest-related genes and differentiate into multiple neural crest derived mesenchymal and neuronal lineages. Moreover, iNCSCs are highly migratory and functional in ovo. These results demonstrate that mature melanocytes can be reprogrammed toward their primitive neural crest cell precursors through the activation of a single stem cell-related pathway. Reprogramming of melanocytes to iNCSCs may provide an alternate source of NCSCs for neuroregenerative applications.
doi:10.1002/stem.740
PMCID: PMC3615703  PMID: 21948558
Notch; melanocytes; neural crest stem cells; reprogramming; dedifferentiation
23.  A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer 
Journal of Proteome Research  2011;11(2):678-691.
Proteomics discovery of novel cancer serum biomarkers is hindered by the great complexity of serum, patient-to-patient variability, and triggering by the tumor of an acute-phase inflammatory reaction. This host response alters many serum protein levels in cancer patients, but these changes have low specificity as they can be triggered by diverse causes. We addressed these hurdles by utilizing a xenograft mouse model coupled with an in-depth 4-D protein profiling method to identify human proteins in the mouse serum. This strategy ensures identified putative biomarkers are shed by the tumor, and detection of low-abundance proteins shed by the tumor is enhanced because the mouse blood volume is more than a thousand times smaller than that of a human. Using TOV-112D ovarian tumors, more than 200 human proteins were identified in the mouse serum, including novel candidate biomarkers and proteins previously reported to be elevated in either ovarian tumors or the blood of ovarian cancer patients. Subsequent quantitation of selected putative biomarkers in human sera using label-free multiple reaction monitoring (MRM) mass spectrometry (MS) showed that chloride intracellular channel 1, the mature form of cathepsin D, and peroxiredoxin 6 were elevated significantly in sera from ovarian carcinoma patients.
doi:10.1021/pr200603h
PMCID: PMC3272104  PMID: 22032327
Biomarker discovery; ovarian cancer; cancer biomarkers; xenograft mouse model; proteomics; multidimensional protein profiling
24.  Lymphatic Invasion is Independently Prognostic of Metastasis in Primary Cutaneous Melanoma 
Clinical Cancer Research  2011;18(1):229-237.
Purpose
Lymphatic invasion (LI) in primary cutaneous melanomas was recently found to be common. In this study, we evaluated LI as an independent prognostic factor.
Experimental Design
This study included 251 patients with vertical growth phase (VGP) primary cutaneous melanomas who had paraffin-fixed lesional tissue and were in a prospective cohort seen between 1972 and 1991, had no clinical evidence of regional nodal disease at diagnosis and had at least ten years of follow-up. Dual immunohistochemistry (IHC) staining was used to detect lymphatic endothelium (podoplanin) and melanoma cells (S-100). Multivariate logistic regression for ten-year metastasis was used to define independent prognostic factors and a prognostic tree was developed to characterize and discriminate risk groups. Kaplan-Meier disese-free survival curves for those with and without LI within current AJCC stages were compared using the log-rank statistic.
Results
LI was observed in 43% (108 of 251) of the study melanomas. The multivariate model for ten-year metastasis identified 4 independent prognostic factors: tumor thickness, mitotic rate (MR), LI, and anatomic site. The prognostic tree identified a group of patients with thin (≤1 mm thick) melanomas and poor prognosis: stage IB melanomas with LI. Survival curves for time to first metastasis demonstrated significantly poorer prognosis for patients with LI compared to those without it for both stages IB and IIA.
Conclusions
LI is common across the range of tumor thicknesses in primary VGP melanomas. It is an independent prognostic factor and significantly increases the risk of metastasis in patients in clinical stages IB and IIA.
doi:10.1158/1078-0432.CCR-11-0490
PMCID: PMC3262994  PMID: 22096024
25.  A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT competent cells that express the ZEB transcription factors 
Cancer research  2011;71(21):6836-6847.
Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated senescence, epithelial to mesenchymal transition (EMT) and cancer stem cell function. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1 enriched EMT competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage independent growth and tumor formation in nude mice. RNA interference (RNAi) experiments suggested the involvement of ZEBs in anchorage independent colony formation, invasion and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition by DNMAML1 in organotypic 3D culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of squamous esophageal cancers.
doi:10.1158/0008-5472.CAN-11-0846
PMCID: PMC3206139  PMID: 21890822
Notch; EMT; squamous cell differentiation; ZEB1; miR-200

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