Li-Xuan Qin

Phase II Trial of the CDK4 Inhibitor PD0332991 in Patients With Advanced CDK4-Amplified Well-Differentiated or Dedifferentiated Liposarcoma

PURPOSE CDK4 is amplified in > 90% of well-differentiated (WDLS) and dedifferentiated liposarcomas (DDLS). The selective cyclin-dependent kinase 4 (CDK4)/CDK6 inhibitor PD0332991 inhibits growth and induces senescence in cell lines and xenografts. In a phase I trial of PD0332991, several patients with WDLS or DDLS experienced prolonged stable disease. We performed an open-label phase II study to determine the safety and efficacy of PD0332991 in patients with advanced WDLS/DDLS. PATIENTS AND METHODS Patients age ≥ 18 years experiencing disease progression while receiving systemic therapy before enrollment received PD0332991 200 mg orally once per day for 14 consecutive days in 21-day cycles. All were required to have CDK4 amplification by fluorescence in situ hybridization and retinoblastoma protein (RB) expression by immunohistochemistry (≥ 1+). The primary end point was progression-free survival (PFS) at 12 weeks, with 12-week PFS of ≥ 40% considered promising and ≤ 20% not promising. If ≥ nine of 28 patients were progression free at 12 weeks, PD0332991 would be considered active. RESULTS We screened 48 patients (44 of 48 had CDK4 amplification; 41 of 44 were RB positive). Of those, 30 were enrolled, and 29 were evaluable for the primary end point. Grade 3 to 4 events included anemia (17%), thrombocytopenia (30%), neutropenia (50%), and febrile neutropenia (3%). At 12 weeks, PFS was 66% (90% CI, 51% to 100%), significantly exceeding the primary end point. The median PFS was 18 weeks. There was one partial response. CONCLUSION Treatment with the CDK4 inhibitor PD0332991 was associated with a favorable progression-free rate in patients with CDK4-amplified and RB-expressing WDLS/DDLS who had progressive disease despite systemic therapy.

γ-Secretase Inhibitors Abrogate Oxaliplatin-Induced Activation of the Notch-1 Signaling Pathway in Colon Cancer Cells Resulting in Enhanced Chemosensitivity

Because Notch signaling is implicated in colon cancer tumorigenesis and protects cells from apoptosis by inducing prosurvival targets, it was hypothesized that inhibition of Notch signaling with gamma-secretase inhibitors (GSI) may enhance the chemosensitivity of colon cancer cells. We first show that the Notch-1 receptor, as well as its downstream target Hes-1, is up-regulated with colon cancer progression, similar to other genes involved in chemoresistance. We then report that chemotherapy induces Notch-1, as oxaliplatin, 5-fluorouracil (5-FU), or SN-38 (the active metabolite of irinotecan) induced Notch-1 intracellular domain (NICD) protein and activated Hes-1. Induction of NICD by oxaliplatin was caused by an increase in the activity and expression of gamma-secretase complex, as suppression of the protein subunit nicastrin with small interfering RNA (siRNA) prevented NICD induction after oxaliplatin. Subsequent inhibition of Notch-1 signaling with a sulfonamide GSI (GSI34) prevented the induction of NICD by chemotherapy and blunted Hes-1 activation. Blocking the activation of Notch signaling with GSI34 sensitized cells to chemotherapy and was synergistic with oxaliplatin, 5-FU, and SN-38. This chemosensitization was mediated by Notch-1, as inhibition of Notch-1 with siRNA enhanced chemosensitivity whereas overexpression of NICD increased chemoresistance. Down-regulation of Notch signaling also prevented the induction of prosurvival pathways, most notably phosphoinositide kinase-3/Akt, after oxaliplatin. In summary, colon cancer cells may up-regulate Notch-1 as a protective mechanism in response to chemotherapy. Therefore, combining GSIs with chemotherapy may represent a novel approach for treating metastatic colon cancers by mitigating the development of chemoresistance.

KDR Activating Mutations in Human Angiosarcomas Are Sensitive to Specific Kinase Inhibitors

Angiosarcomas (AS) represent a heterogeneous group of malignant vascular tumors occurring not only in different anatomic locations but also in distinct clinical settings, such as radiation or associated chronic lymphedema. Although representing only 1% to 2% of soft tissue sarcomas, vascular sarcomas provide unique insight into the general process of tumor angiogenesis. However, no molecular candidates have been identified to guide a specific therapeutic intervention. By expression profiling, AS show distinct up-regulation of vascular-specific receptor tyrosine kinases, including TIE1, KDR, SNRK, TEK, and FLT1. Full sequencing of these five candidate genes identified 10% of patients harboring KDR mutations. A KDR-positive genotype was associated with strong KDR protein expression and was restricted to the breast anatomic site with or without prior exposure to radiation. Transient transfection of KDR mutants into COS-7 cells showed ligand-independent activation of the kinase, which was inhibited by specific KDR inhibitors. These data provide a basis for the activity of vascular endothelial growth factor receptor-directed therapy in the treatment of primary and radiation-induced AS.

Do Radiation-Associated Soft Tissue Sarcomas Have the Same Prognosis As Sporadic Soft Tissue Sarcomas?

PURPOSE To determine the prognostic significance of histologic type in radiation-associated soft tissue sarcomas (RASs) and determine whether RASs are associated with an inferior prognosis compared with sporadic soft tissue sarcomas (STSs). PATIENTS AND METHODS One hundred thirty primary RASs were identified from 7,649 STS patients from 1982 to 2007. Multivariate analysis of clinicopathologic factors for disease-specific survival (DSS) was performed for RASs, and a multivariate analysis of radiation exposure was also performed for RASs and sporadic sarcomas. A matched-cohort analysis was performed for radiation-associated and sporadic malignant fibrous histiocytoma (MFH). Results Most RASs were high grade (83%), deep (87%), and truncal (61.5%). The median interval between radiation therapy and RAS development was 10 years (range, 1.3 to 74 years), which varied significantly by histologic type (P = .003). The 5-year DSS was 58%, and independent predictors were size > 5 cm, margin positivity, and histologic type. Multivariate analysis of histologic types of primary, high-grade radiation-associated and sporadic STSs showed that RAS was associated with a worse DSS (hazard ratio, 1.7; range, 1.1 to 2.4; P = .007). For pleomorphic MFH-the most common RAS type-the 5-year DSS was 44% versus 66% in a matched cohort of sporadic MFH patients (P = .07). DSS was significantly worse in primary RAS malignant peripheral nerve sheath tumors (MPNSTs) compared with unmatched sporadic MPNSTs (P = .001). CONCLUSION Histologic type, margin status, and tumor size are the most important independent predictors of DSS in patients with RASs. DSS in patients with primary RAS is significantly worse compared with sporadic STS independent of sarcoma histologic type.

A Novel Survival-Based Tissue Microarray of Pancreatic Cancer Validates MUC1 and Mesothelin as Biomarkers

Background One–fifth of patients with seemingly ‘curable’ pancreatic ductal adenocarcinoma (PDA) experience an early recurrence and death, receiving no definable benefit from a major operation. Some patients with advanced stage tumors are deemed ‘unresectable’ by conventional staging criteria (e.g. liver metastasis), yet progress slowly. Effective biomarkers that stratify PDA based on biologic behavior are needed. To help researchers sort through the maze of biomarker data, a compendium of ∼2500 published candidate biomarkers in PDA was compiled (PLoS Med, 2009. 6(4) p. e1000046). Methods and Findings Building on this compendium, we constructed a survival tissue microarray (termed s-TMA) comprised of short-term (cancer-specific death <12 months, n = 58) and long-term survivors (>30 months, n = 79) who underwent resection for PDA (total, n = 137). The s-TMA functions as a biological filter to identify bona fide prognostic markers associated with survival group extremes (at least 18 months separate survival groups). Based on a stringent selection process, 13 putative PDA biomarkers were identified from the public biomarker repository. Candidates were tested against the s-TMA by immunohistochemistry to identify the best markers of tumor biology. In a multivariate model, MUC1 (odds ratio, OR = 28.95, 3+ vs. negative expression, p = 0.004) and MSLN (OR = 12.47, 3+ vs. negative expression, p = 0.01) were highly predictive of early cancer-specific death. By comparison, pathologic factors (size, lymph node metastases, resection margin status, and grade) had ORs below three, and none reached statistical significance. ROC curves were used to compare the four pathologic prognostic features (ROC area = 0.70) to three univariate molecular predictors (MUC1, MSLN, MUC2) of survival group (ROC area = 0.80, p = 0.07). Conclusions MUC1 and MSLN were superior to pathologic features and other putative biomarkers as predicting survival group. Molecular assays comparing cancers from short and long survivors are an effective strategy to screen biomarkers and prioritize candidate cancer genes for diagnostic and therapeutic studies.

A postoperative nomogram for local recurrence risk in extremity soft tissue sarcomas after limb-sparing surgery without adjuvant radiation.

Purpose: To develop a nomogram based on clinicopathologic factors to quantify the risk of local recurrence (LR) after limb-sparing surgery without adjuvant radiation (RT). Methods: Review of our prospective sarcoma database identified 684 patients with primary, nonmetastatic, extremity STS treated with limb-sparing surgery alone between June 1982 and December 2006. No patient received adjuvant radiation or chemotherapy. Age, sex, grade, depth, size, site, margin status and histology were analyzed for prognostic significance with respect to local recurrence rates using Grays test. Variables which were significant in univariate analysis at the 0.05 level were entered into a multivariate competing risk regression model. On the basis of the multivariate analysis, a nomogram for predicting the 3- and 5-year risk of LR was developed using R libraries cmprsk and QHScrnomo. Concordance index (C-index) was calculated to evaluate the discriminatory power of the prognostic model. Results: With a median follow-up of 58 months for censored patients (73 months for all patients), the overall 3- and 5-year actuarial local recurrence rates were 11% and 13%, respectively. Factors included in the nomogram were age (⩽50 vs. >50), size (⩽5 vs. >5 cm), margin status (negative vs. positive), grade (low vs. high), and histology (atypical lipomatous tumor/well differentiated liposarcoma vs. other). The STS nomogram predicted the local recurrence rate with a C-index of 0.73. Conclusions: A nomogram for extremity STS that includes age, size, margin status, grade of tumor, and histology predicts the 3- and 5-year risk of local recurrence after limb-sparing surgery in the absence of adjuvant RT.

Prevalence of tumor-infiltrating lymphocytes and PD-L1 expression in the soft tissue sarcoma microenvironment ☆ ☆☆

The prognostic and predictive implications of programmed death-ligand 1 (PD-L1) is unknown in sarcoma. We sought to examine the immune milieu in sarcoma specimens. We evaluated PD-L1 expression by immunohistochemistry in sarcoma specimens and quantified tumor-infiltrating lymphocytes (TIL). We correlated expression with clinical parameters and outcomes. Fifty sarcoma patients treated at Memorial Sloan Kettering Cancer Center were selected. Using the DAKO PD-L1 immunohistochemistry assay and archival formalin-fixed paraffin-embedded tissue specimens; PD-L1 expression was examined. Macrophage and lymphocyte PD-L1 status was determined qualitatively. TIL was quantified. Associations between PD-L1 expression in tumor, macrophages and lymphocytes, TIL and clinical-pathological characteristics were performed. The median age was 46 years (range, 22-76), and 66% of patients were men. Tumor, lymphocyte and macrophage PD-L1 expression was noted in 12%, 30% and 58%, respectively, with the highest prevalence in gastrointestinal stromal tumors (29%). Lymphocyte and macrophage infiltration was present in 98% and 90%, respectively. There was no association between clinical features, overall survival and PD-L1 expression in tumor or immune infiltrates. Lymphocyte and macrophage infiltration is common in sarcoma, but PD-L1 tumor expression is uncommon in sarcoma with the highest frequency observed in gastrointestinal stromal tumors. There was no association between PD-L1 expression, TIL and clinicopathological features and overall survival; however, this is limited by the heterogenous patient sample and minimal death events in the studied cohort.

Histology-based Classification Predicts Pattern of Recurrence and Improves Risk Stratification in Primary Retroperitoneal Sarcoma.

Objective:To determine the prognostic significance of histologic type/subtype in a large series of patients with primary resected retroperitoneal sarcoma. Background:The histologic diversity and rarity of retroperitoneal sarcoma has hampered the ability to predict patient outcome. Methods:From a single-institution, prospective database, 675 patients treated surgically for primary, nonmetastatic retroperitoneal sarcoma during 1982 to 2010 were identified and histologic type/subtype was reviewed. Clinicopathologic variables were analyzed for association with disease-specific death (DSD), local recurrence (LR), and distant recurrence (DR). Results:Median follow-up for survivors was 7.5 years. The predominant histologies were well-differentiated liposarcoma, dedifferentiated liposarcoma, and leiomyosarcoma. Five-year cumulative incidence of DSD was 31%, and factors independently associated with DSD were R2 resection, resection of 3 or more contiguous organs, and histologic type. Five-year cumulative incidence for LR was 39% and for DR was 24%. R1 resection, age, tumor size, and histologic type were independently associated with LR; size, resection of 3 or more organs, and histologic type were independently associated with DR. Liposarcoma and leiomyosarcoma were associated with late recurrence and DSD (as long as 15 years from diagnosis). For solitary fibrous tumor, LR was uncommon (<10%), but early distant recurrence was common (36% at 5 years). Nomograms were developed to predict DSD, LR, and DR. Conclusions:Histologic type/subtype is the most important independent predictor of DSD, LR, and DR in primary retroperitoneal sarcoma. Histology predicts the pattern and incidence of LR and DR and will aid in more accurate patient counseling and selection of patients for adjuvant therapy trials.

Expression Profiling of Liposarcoma Yields a Multigene Predictor of Patient Outcome and Identifies Genes That Contribute to Liposarcomagenesis

Liposarcomas are the most common type of soft tissue sarcoma but their genetics are poorly defined. To identify genes that contribute to liposarcomagenesis and serve as prognostic candidates, we undertook expression profiling of 140 primary liposarcoma samples, which were randomly split into training set (n = 95) and test set (n = 45). A multigene predictor for distant recurrence-free survival (DRFS) was developed by the supervised principal component method. Expression levels of the 588 genes in the predictor were used to calculate a risk score for each patient. In validation of the predictor in the test set, patients with low risk score had a 3-year DRFS of 83% versus 45% for high risk score patients (P = 0.001). The HR for high versus low score, adjusted for histologic subtype, was 4.42 (95% CI, 1.26-15.55; P = 0.021). The concordance probability for risk score was 0.732. In contrast, the concordance probability for histologic subtype, which had been considered the best predictor of outcome in liposarcoma, was 0.669. Genes related to adipogenesis, DNA replication, mitosis, and spindle assembly checkpoint control were all highly represented in the multigene predictor. Three genes from the predictor, TOP2A, PTK7, and CHEK1, were found to be overexpressed in liposarcoma samples of all five subtypes and in liposarcoma cell lines. RNAi-mediated knockdown of these genes in liposarcoma cell lines reduced proliferation and invasiveness and increased apoptosis. Taken together, our findings identify genes that seem to be involved in liposarcomagenesis and have promise as therapeutic targets, and support the use of this multigene predictor to improve risk stratification for individual patients with liposarcoma.

Pancreatic Cyst Fluid Protein Expression Profiling for Discriminating Between Serous Cystadenoma and Intraductal Papillary Mucinous Neoplasm

Background:Many patients with benign serous cystadenoma (SCA) of the pancreas will undergo resection because of the inability to reliably discriminate between SCA and premalignant mucinous cysts (intraductal papillary mucinous neoplasm [IPMN], mucinous cystic neoplasm [MCN]). Methods:Cyst fluid from patients with SCA (n = 15), non main-duct and noninvasive IPMN (n = 32), and noninvasive MCN (n = 12) was aspirated at the time of operative resection and analyzed. Commercially available and custom designed multiplex assays (Luminex) were performed using a biomarker panel developed for pancreatic cancer. Differential protein expression (fluorescence intensity, FI) was compared between the 3 groups for each protein (Wilcoxon rank sum test). Unsupervised sample clustering (hierarchical clustering) and supervised sample classification (prediction analysis for microarrays [PAM]) was then performed. Results:Differential protein expression (P < 0.05) was identified between SCA and IPMN (34/51 proteins, 67%) and between SCA and MCN (13/51 proteins, 25%). The majority of proteins were down-regulated in IPMN and MCN compared with SCA. The only proteins significantly overexpressed in the cyst fluid of patients with mucinous cysts were CEA (median FI: IPMN 11.4, MCN 13.0, SCA 5.3; P < 0.001, IPMN vs. SCA) and CA72.4 (median FI: IPMN 10.4, MCN 10.5, SCA 9.9; P = 0.003, IPMN vs. SCA). Unsupervised cluster analysis demonstrated distinct clustering of SCA and IPMN with some cross-over between MCN. Supervised sample classification with 14 proteins had an overall accuracy rate of 92% between SCA and IPMN. Conclusions:In this study differential cyst fluid protein expression was observed between SCA and IPMN for the majority of proteins assessed and multimarker sample classification accurately discriminated between SCA and IPMN in 92% of patients.