Integrative multi-region molecular profiling of primary prostate cancer in men with synchronous lymph node metastasis.

Localized prostate cancer is frequently composed of multiple spatially distinct tumors with significant inter- and intra-tumoral molecular heterogeneity. This genomic diversity gives rise to many competing clones that may drive the biological trajectory of the disease. Previous large-scale sequencing efforts have focused on the evolutionary process in metastatic prostate cancer, revealing a potential clonal progression to castration resistance. However, the clonal origin of synchronous lymph node (LN) metastases in primary disease is still unknown. Here, we perform multi-region, targeted next generation sequencing and construct phylogenetic trees in men with prostate cancer with synchronous LN metastasis to better define the pathologic and molecular features of primary disease most likely to spread to the LNs. Collectively, we demonstrate that a combination of histopathologic and molecular factors, including tumor grade, presence of extra-prostatic extension, cellular morphology, and oncogenic genomic alterations are associated with synchronous LN metastasis.

Endothelial knockdown of the tumor suppressor, WWOX, increases inflammation in ventilator-induced lung injury.

Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX-silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX-silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1ß, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin.NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model.

Overlapping Features of Primary Cutaneous Marginal Zone Lymphoproliferative Disorder and Primary Cutaneous CD4 + Small/Medium T-Cell Lymphoproliferative Disorder : A Diagnostic Challenge Examined by Genomic Analysis.

Primary cutaneous marginal zone lymphoproliferative disorder (PCMZL) and primary cutaneous CD4 + small/medium T-cell lymphoproliferative disorder (CD4 + TLPD) are indolent lymphoproliferative disorders. However, cases with overlapping features can be challenging. We identified 56 CD4 + TLPD and 38 PCMZL cases from our pathology archives. Clinical, morphologic, and immunophenotypic features were reviewed. Polymerase chain reaction for immunoglobulin (IG) and T-cell receptor gamma (TRG) gene rearrangements were analyzed. Next-generation sequencing studies were performed on 26 cases with adequate material, 19 with CD4 + TLPD, and 7 with PCMZL. CD4 + TLPD presented mostly (91%) as solitary lesions, located in the head and neck area (64%), while PCMZL occurred mostly in the upper extremity (47%) and trunk (34%). Lesions were sometimes multiple (40%) and recurrences (67%) were more common. Cases of PCMZL had an increase in reactive CD3 + T cells, with frequent programmed cell death protein 1 expression, whereas cases of CD4 + TLPD often contained abundant reactive B cells. Twenty-five cases were identified as having overlapping features: 6 cases of PCMZL were clonal for both IG and TRG; 11 cases of CD4 + TLPD were clonal for IG and TRG and 6 cases of CD4 + TLPD had light chain-restricted plasma cells. By next-generation sequencing, 23 variants were detected in 15 genes, with PCMZL more likely to show alterations, most commonly affecting TNFAIP3 and FAS, altered in 5 cases. Both entities have an indolent clinical course with response to conservative therapy and management, and warrant interpretation as a lymphoproliferative disorder rather than overt lymphoma.

Comparative effects of crystalline, poorly crystalline and freshly formed iron oxides on the colloidal properties of polystyrene microplastics.

Colloid-sized microplastics (MPs) are ubiquitous in aquatic environments and can share the same transport route together with various crystalline, poorly crystalline and freshly formed iron oxides. However, the colloidal interactions between these colloid constituents are not fully understood. This study was designed to investigate the colloidal properties of polystyrene microplastics (PSMPs) under the influence of haematite, goethite, ferrihydrite and freshly formed Fe oxide (FFFO). Dynamic light scattering was coupled with a test tube method to observe changes in the surface charge and colloidal dynamics of suspensions of PSMPs and Fe oxides. The overall effects on the aggregation of PSMPs are found to decrease in the following order: FFFO > ferrihydrite > goethite > haematite. The effects of these Fe oxides are found to strongly depend on pH. While the crystalline oxides play a dominant role in the acidic environment, poorly crystalline oxides show greater effects on PSMP aggregation in an alkaline environment. Heteroaggregation due to decreasing electrostatic interactions is the major mechanism that governs the colloidal dynamics of PSMPs and Fe oxides. It can be inferred that the copresence of Fe oxides and MPs can delay the transport of MPs or even change the destination for MPs.

Development of a Whole-urine, Multiplexed, Next-generation RNA-sequencing Assay for Early Detection of Aggressive Prostate Cancer.

BACKGROUND: Despite biomarker development advances, early detection of aggressive prostate cancer (PCa) remains challenging. We previously developed a clinical-grade urine test (Michigan Prostate Score [MiPS]) for individualized aggressive PCa risk prediction. MiPS combines serum prostate-specific antigen (PSA), the TMPRSS2:ERG (T2:ERG) gene fusion, and PCA3 lncRNA in whole urine after digital rectal examination (DRE). OBJECTIVE: To improve on MiPS with a novel next-generation sequencing (NGS) multibiomarker urine assay for early detection of aggressive PCa. DESIGN, SETTING, AND PARTICIPANTS: Preclinical development and validation of a post-DRE urine RNA NGS assay (Urine Prostate Seq [UPSeq]) assessing 84 PCa transcriptomic biomarkers, including T2:ERG, PCA3, additional PCa fusions/isoforms, mRNAs, lncRNAs, and expressed mutations. Our UPSeq model was trained on 73 patients and validated on a held-out set of 36 patients representing the spectrum of disease (benign to grade group [GG] 5 PCa). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The area under the receiver operating characteristic curve (AUC) of UPSeq was compared with PSA, MiPS, and other existing models/biomarkers for predicting GG ≥3 PCa. RESULTS AND LIMITATIONS: UPSeq demonstrated high analytical accuracy and concordance with MiPS, and was able to detect expressed germline HOXB13 and somatic SPOP mutations. In an extreme design cohort (n = 109; benign/GG 1 vs GG ≥3 PCa, stratified to exclude GG 2 cancer in order to capture signal difference between extreme ends of disease), UPSeq showed differential expression for T2:ERG.T1E4 (1.2 vs 78.8 median normalized reads, p < 0.00001) and PCA3 (1024 vs 2521, p = 0.02), additional T2:ERG splice isoforms, and other candidate biomarkers. Using machine learning, we developed a 15-transcript model on the training set (n = 73) that outperformed serum PSA and sequencing-derived MiPS in predicting GG ≥3 PCa in the held-out validation set (n = 36; AUC 0.82 vs 0.69 and 0.69, respectively). CONCLUSIONS: These results support the potential utility of our novel urine-based RNA NGS assay to supplement PSA for improved early detection of aggressive PCa. PATIENT SUMMARY: We have developed a new urine-based test for the detection of aggressive prostate cancer, which promises improvement upon current biomarker tests.

Molecular Characterization of Clear Cell Renal Cell Carcinoma Reveals Prognostic Significance of Epithelial-mesenchymal Transition Gene Expression Signature.

BACKGROUND: There is an ongoing need to develop prognostic biomarkers to improve the management of clear cell renal cell carcinoma (ccRCC). OBJECTIVE: To leverage enriched pathways in ccRCC to improve risk-stratification. DESIGN, SETTING, AND PARTICIPANTS: We retrospectively identified two complementary discovery cohorts of patients with ccRCC who underwent (1) radical nephrectomy (RNx) with inferior vena cava tumor thrombectomy (patients = 5, samples = 24) and (2) RNx for localized disease and developed recurrence versus no recurrence (n = 36). Patients with localized ccRCC (M0) in The Cancer Genome Atlas (TCGA, n = 386) were used for validation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A differential expression gene (DEG) analysis was performed on targeted RNA next-generation sequencing data from both discovery cohorts. Using TCGA for validation, Kaplan-Meier survival analysis and multivariable Cox proportional hazard testing were utilized to investigate the prognostic impact of DEGs, cell cycle proliferation (CCP), and a novel epithelial-mesenchymal transition (EMT) score on progression-free (PFS) and disease-specific (DSS) survival. RESULTS AND LIMITATIONS: In the discovery cohorts, we observed overexpression of WT1 and CCP genes in the tumor thrombus versus the primary tumor, as well as in patients with recurrence versus those without recurrence. A hallmark pathway analysis demonstrated enrichment of the EMT- and CCP-related pathways in patients with high WT1 expression in the TCGA (validation) ccRCC cohort. CCP and EMT scores were derived in the validation cohort, which was stratified into four risk groups using Youden Index cut points: CCPlow/EMTlow, CCPlow/EMThigh, CCPhigh/EMTlow, and CCPhigh/EMThigh. The CCPhigh/EMThigh risk group was associated with the worst PFS and DSS (both p < 0.001). In a multivariable analysis, CCPhigh/EMThigh was independently associated with poor PFS and DSS (hazard ratio = 4.6 and 10.3, respectively; p < 0.001). CONCLUSIONS: We demonstrate the synergistic prognostic impact of EMT in tumors with a high CCP score. Our novel EMT score has the potential to improve risk stratification and provide potential novel therapeutic targets. PATIENT SUMMARY: Genes involved in epithelial-mesenchymal transition provides important prognostic information for patients with clear cell renal cell carcinoma.

Longitudinal Circulating Tumor DNA Analysis in Blood and Saliva for Prediction of Response to Osimertinib and Disease Progression in EGFR-Mutant Lung Adenocarcinoma.

Background: We assessed whether serial ctDNA monitoring of plasma and saliva predicts response and resistance to osimertinib in EGFR-mutant lung adenocarcinoma. Three ctDNA technologies-blood-based droplet-digital PCR (ddPCR), next-generation sequencing (NGS), and saliva-based EFIRM liquid biopsy (eLB)-were employed to investigate their complementary roles. Methods: Plasma and saliva samples were collected from patients enrolled in a prospective clinical trial of osimertinib and local ablative therapy upon progression (NCT02759835). Plasma was analyzed by ddPCR and NGS. Saliva was analyzed by eLB. Results: A total of 25 patients were included. We analyzed 534 samples by ddPCR (n = 25), 256 samples by NGS (n = 24) and 371 samples by eLB (n = 22). Among 20 patients who progressed, ctDNA progression predated RECIST 1.1 progression by a median of 118 days (range: 61-272 days) in 11 (55%) patients. Of nine patients without ctDNA progression by ddPCR, two patients had an increase in mutant EGFR by eLB and two patients were found to have ctDNA progression by NGS. Levels of ctDNA measured by ddPCR and NGS at early time points, but not volumetric tumor burden, were associated with PFS. EGFR/ERBB2/MET/KRAS amplifications, EGFR C797S, PIK3CA E545K, PTEN V9del, and CTNNB1 S45P were key resistance mechanisms identified by NGS. Conclusion: Serial assessment of ctDNA in plasma and saliva predicts response and resistance to osimertinib, with each assay having supplementary roles.

Abiraterone Alone or in Combination With Enzalutamide in Metastatic Castration-Resistant Prostate Cancer With Rising Prostate-Specific Antigen During Enzalutamide Treatment.

Purpose Enzalutamide resistance could result from raised androgens and be overcome by combination with abiraterone acetate. PLATO ( ClinicalTrials.gov identifier: NCT01995513) interrogated this hypothesis using a randomized, double-blind, placebo-controlled design. Patients and Methods In period one, men with chemotherapy-naïve metastatic castration-resistant prostate cancer received open-label enzalutamide 160 mg daily. Men with no prostate-specific antigen (PSA) increase at weeks 13 and 21 were treated until PSA progression (≥ 25% increase and ≥ 2 ng/mL above nadir), then randomly assigned at a one-to-one ratio in period two to abiraterone acetate 1,000 mg daily and prednisone 5 mg twice daily with either enzalutamide or placebo (combination or control group, respectively) until disease progression as defined by the primary end point: progression-free survival (radiographic or unequivocal clinical progression or death during study). Secondary end points included time to PSA progression and PSA response in period two. Results Of 509 patients enrolled in period one, 251 were randomly assigned in period two. Median progression-free survival was 5.7 months in the combination group and 5.6 months in the control group (hazard ratio, 0.83; 95% CI, 0.61 to 1.12; P = .22). There was no difference in the secondary end points. Grade 3 hypertension (10% v 2%) and increased ALT (6% v 2%) or AST (2% v 0%) were more frequent in the combination than the control group. Conclusion Combining enzalutamide with abiraterone acetate and prednisone is not indicated after PSA progression during treatment with enzalutamide alone; hypertension and elevated liver enzymes are more frequent with combination therapy.

Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study.

BACKGROUND: Uveal melanoma is a rare tumour with no established treatments once metastases develop. Although a variety of immune-based therapies have shown efficacy in metastatic cutaneous melanoma, their use in ocular variants has been disappointing. Recently, adoptive T-cell therapy has shown salvage responses in multiple refractory solid tumours. Thus, we sought to determine if adoptive transfer of autologous tumour-infiltrating lymphocytes (TILs) could mediate regression of metastatic uveal melanoma. METHODS: In this ongoing single-centre, two-stage, phase 2, single-arm trial, patients (aged ≥16 years) with histologically confirmed metastatic ocular melanoma were enrolled. Key eligibility criteria were an Eastern Cooperative Oncology Group performance status of 0 or 1, progressive metastatic disease, and adequate haematological, renal, and hepatic function. Metastasectomies were done to procure tumour tissue to generate autologous TIL cultures, which then underwent large scale ex-vivo expansion. Patients were treated with lymphodepleting conditioning chemotherapy (intravenous cyclophosphamide [60 mg/kg] daily for 2 days followed by fludarabine [25 mg/m2] daily for 5 days, followed by a single intravenous infusion of autologous TILs and high-dose interleukin-2 [720 000 IU/kg] every 8 h). The primary endpoint was objective tumour response in evaluable patients per protocol using Response to Evaluation Criteria in Solid Tumors, version 1.0. An interim analysis of this trial is reported here. The trial is registered at ClinicalTrials.gov, number NCT01814046. FINDINGS: From the completed first stage and ongoing expansion stage of this trial, a total of 21 consecutive patients with metastatic uveal melanoma were enrolled between June 7, 2013, and Sept 9, 2016, and received TIL therapy. Seven (35%, 95% CI 16-59) of 20 evaluable patients had objective tumour regression. Among the responders, six patients achieved a partial response, two of which are ongoing and have not reached maximum response. One patient achieved complete response of numerous hepatic metastases, currently ongoing at 21 months post therapy. Three of the responders were refractory to previous immune checkpoint blockade. Common grade 3 or worse toxic effects were related to the lymphodepleting chemotherapy regimen and included lymphopenia, neutropenia, and thrombocytopenia (21 [100%] patients for each toxicity); anaemia (14 [67%] patients); and infection (six [29%] patients). There was one treatment-related death secondary to sepsis-induced multiorgan failure. INTERPRETATION: To our knowledge, this is the first report describing adoptive transfer of autologous TILs to mediate objective tumour regression in patients with metastatic uveal melanoma. These initial results challenge the belief that metastatic uveal melanoma is immunotherapy resistant and support the further investigation of immune-based therapies for this cancer. Refinement of this T-cell therapy is crucial to improve the frequency of clinical responses and the general applicability of this treatment modality. FUNDING: Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

EBV-negative Aggressive NK-cell Leukemia/Lymphoma: Clinical, Pathologic, and Genetic Features.

Aggressive natural killer cell leukemia (ANKL) is a systemic NK-cell neoplasm, almost always associated with Epstein-Barr virus (EBV). Rare cases of EBV-negative ANKL have been described, and some reports suggested more indolent behavior. We report the clinicopathologic, immunophenotypic, and molecular characteristics of 7 EBV-negative ANKL. All patients were adults, with a median age of 63 years (range 22 to 83 y) and an M:F ratio of 2.5:1. Five patients were White, 1 Black, and 1 Asian. All patients presented acutely, with fever (6/7), cytopenias (6/7), and splenomegaly (4/7). Four patients had lymphadenopathy, 4 had extranodal disease. Bone marrow involvement was present in 5, with hemophagocytosis in 3. Peripheral blood was involved in 5 with the neoplastic cells containing prominent azurophilic granules. By immunohistochemistry and/or flow cytometry, the tumor cells lacked surface CD3 and were positive for CD56 (7/7), CD2 (5/5), CD8 (3/7), CD30 (4/5), and granzyme-B (6/6). They were negative for CD4, CD5, ßF1, TCRγ, LMP1, and EBV-encoded RNA. Polymerase chain reaction for TCRG clonality was polyclonal. Mutational analysis revealed missense mutations in the STAT3 gene in both cases studied. Median survival was 8 weeks from the onset of disease. One patient received allogeneic bone marrow transplant and is alive with no disease (follow-up 15 mo). EBV-negative ANKL exists but is rare. It tends to occur in older patients and is indistinguishable clinically and pathologically from EBV-positive ANKL, with a similar fulminant clinical course. The high prevalence of Asian patients seen with EBV-positive disease seems less evident with EBV-negative cases.

Circulating Tumor DNA as an Early Indicator of Response to T-cell Transfer Immunotherapy in Metastatic Melanoma.

PURPOSE: Adoptive transfer of activated autologous tumor-infiltrating lymphocytes (TIL) can mediate complete, durable regressions in patients with metastatic melanoma. Responding patients generally do not have significant changes in noncutaneous RECIST targets before 30 to 60 days following TIL infusion, and complete responses are often not confirmed for 1 to 2 years. There is a critical need for a biomarker that can provide early information regarding the likelihood and duration of a response to enable rational decisions about altering therapy. We wished to evaluate the role of circulating tumor DNA (ctDNA) in separating responding from nonresponding patients. EXPERIMENTAL DESIGN: We studied BRAF V600E ctDNA levels by a sensitive allele-specific PCR assay in 388 serum samples from 48 patients who received TIL immunotherapy at the NCI and correlated differences in the dynamic patterns of their ctDNA measurements with response outcomes. RESULTS: A strong correlation was found between the presence or absence of an early serum peak of V600E ctDNA, and the likelihood of an objective response. Furthermore, patients that developed an early ctDNA peak and cleared their serum of V600E ctDNA were highly likely to achieve a complete response over the next 1 to 2 years. Patients that showed no peak of V600E ctDNA failed to achieve an objective response, with one exception. CONCLUSIONS: We show that the dynamic changes occurring in BRAF V600E ctDNA levels within the first month following T-cell transfer immunotherapy in metastatic melanoma can be used to rapidly identify responding from nonresponding patients, potentially allowing clinicians to make critical treatment-related decisions in a more timely manner. These data also suggest that the majority of tumor killing by TIL occurs very early after the initiation of therapy. Clin Cancer Res; 22(22); 5480-6. ©2016 AACR.

Identification of an Immunogenic Subset of Metastatic Uveal Melanoma.

PURPOSE: Uveal melanoma is a rare melanoma variant with no effective therapies once metastases develop. Although durable cancer regression can be achieved in metastatic cutaneous melanoma with immunotherapies that augment naturally existing antitumor T-cell responses, the role of these treatments for metastatic uveal melanoma remains unclear. We sought to define the relative immunogenicity of these two melanoma variants and determine whether endogenous antitumor immune responses exist against uveal melanoma. EXPERIMENTAL DESIGN: We surgically procured liver metastases from uveal melanoma (n = 16) and cutaneous melanoma (n = 35) patients and compared the attributes of their respective tumor cell populations and their infiltrating T cells (TIL) using clinical radiology, histopathology, immune assays, and whole-exomic sequencing. RESULTS: Despite having common melanocytic lineage, uveal melanoma and cutaneous melanoma metastases differed in their melanin content, tumor differentiation antigen expression, and somatic mutational profile. Immunologic analysis of TIL cultures expanded from these divergent forms of melanoma revealed cutaneous melanoma TIL were predominantly composed of CD8(+) T cells, whereas uveal melanoma TIL were CD4(+) dominant. Reactivity against autologous tumor was significantly greater in cutaneous melanoma TIL compared with uveal melanoma TIL. However, we identified TIL from a subset of uveal melanoma patients which had robust antitumor reactivity comparable in magnitude with cutaneous melanoma TIL. Interestingly, the absence of melanin pigmentation in the parental tumor strongly correlated with the generation of highly reactive uveal melanoma TIL. CONCLUSIONS: The discovery of this immunogenic group of uveal melanoma metastases should prompt clinical efforts to determine whether patients who harbor these unique tumors can benefit from immunotherapies that exploit endogenous antitumor T-cell populations. Clin Cancer Res; 22(9); 2237-49. ©2015 AACR.

Use of arsenic trioxide in a hemodialysis-dependent patient with relapsed acute promyelocytic leukemia.

Arsenic trioxide has been established for use in both relapsed and front-line treatment of acute promyelocytic leukemia. Dose adjustments are recommended to be considered in severe renal impairment although dosage reduction guidelines are not provided. In addition, toxicities of arsenic are significant. The use of arsenic trioxide has not been well studied in dialysis patients and there is a paucity of data in the literature to support the use in such a situation. We describe an 81-year-old relapsed acute promyelocytic leukemia hemodialysis-dependent patient with a pre-existing cardiac condition who was treated with 10 mg arsenic trioxide three times weekly after dialysis. These findings provide support along with the marginal amount of currently published data for an arsenic trioxide dosing regimen in hemodialysis patients.

Sensitive capture of circulating tumour cells by functionalized graphene oxide nanosheets.

The spread of cancer throughout the body is driven by circulating tumour cells (CTCs). These cells detach from the primary tumour and move from the bloodstream to a new site of subsequent tumour growth. They also carry information about the primary tumour and have the potential to be valuable biomarkers for disease diagnosis and progression, and for the molecular characterization of certain biological properties of the tumour. However, the limited sensitivity and specificity of current methods for measuring and studying these cells in patient blood samples prevents the realization of their full clinical potential. The use of microfluidic devices is a promising method for isolating CTCs. However, the devices are reliant on three-dimensional structures, which limits further characterization and expansion of cells on the chip. Here we demonstrate an effective approach to isolating CTCs from blood samples of pancreatic, breast and lung cancer patients, by using functionalized graphene oxide nanosheets on a patterned gold surface. CTCs were captured with high sensitivity at a low concentration of target cells (73 ± 32.4% at 3-5 cells per ml blood).

Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease.

In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.

EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection.

The discovery of activating mutations in EGFR and KRAS in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Fine-needle aspirates and other cytologic procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used laser capture microdissection to isolate small numbers of tumor cells to assess for EGFR and KRAS mutations from cell block sections of 19 cytology samples from patients with known lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient's initial clinical work-up. Not only were we able to detect the identical EGFR or KRAS mutation that was present in the patient's prior molecular assay in every case, but we were also able to consistently detect the mutation from as few as 50 microdissected tumor cells. Furthermore, isolating a more pure population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from laser capture microdissection-enriched cases where the tumor load was low and traditional methods of whole slide scraping failed. Therefore, this method can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR and KRAS mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.

Panitumumab: human monoclonal antibody against epidermal growth factor receptors for the treatment of metastatic colorectal cancer.

BACKGROUND: Panitumumab, formerly known as ABX-EGF, was the first recombinant human immunoglobulin G2 monoclonal antibody approved by the US Food and Drug Administration for the treatment of patients with epidermal growth factor receptor-expressing metastatic colorectal cancer (mCRC) refractory to fluoropyrimidine-, oxaliplatin-, and/or irinotecan-containing chemotherapeutic regimens. OBJECTIVE: The purpose of this review was to evaluate the pharmacokinetic and pharmacodynamic properties, clinical efficacy, and safety profile of panitumumab in the treatment of metastatic colorectal cancer. METHODS: Computerized searches of MEDLINE and International Pharmaceutical Abstracts from 1985 to August 15, 2007, were performed with the search terms panitumumab, ABX-EGF, EGFr, and colorectal cancer. All available clinical trials and ongoing trials were included in this review. Relevant abstracts presented at the annual meetings of the American Society of Clinical Oncology, American Association for Cancer Research, and Gastrointestinal Cancer Symposium (1999-2007) also were reviewed and included. RESULTS: Preclinical and clinical studies have established a role for panitumumab in mCRC refractory to multiple chemotherapeutic regimens. In a Phase III trial of panitumumab plus best supportive care (BSC) versus BSC alone in patients with refractory mCRC, panitumumab was found to have efficacy in time-related end points, such as progression-free survival. In the panitumumab group, a significant (46%) reduction in tumor progression rate was reported compared with BSC (hazard ratio, 0.54; 95% CI, 0.44-0.66; P < 0.001). At the present time, the use of panitumumab as first-line treatment for mCRC with standard chemotherapy and bevacizumab is not indicated due to increased toxicity with no advantage in efficacy. The efficacy of panitumumab is being evaluated in other solid tumors, such as lung, breast, ovarian, bladder, and head and neck cancers. CONCLUSION: Panitumumab appears to have relatively acceptable tolerability and is now available as an additional option for patients with mCRC refractory to multiple chemotherapeutic regimens.

Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma.

To determine whether the PI3K/Akt signaling pathway is involved in the pathogenesis of mantle cell lymphoma (MCL), we investigated the phosphorylation status of Akt and multiple downstream targets in primary MCL cases and cell lines. Akt was phosphorylated in 12 of 12 aggressive blastoid MCL variants and in 4 of 4 MCL cell lines. In contrast, phosphorylated Akt was present in only 5 of 16 typical MCL, 3 at comparable levels to the blastoid cases, and 2 at low levels. The presence of p-Akt was accompanied by the phosphorylation of p27(kip1), FRKHL-1, MDM2, Bad, mTOR, and p70S6K. Inhibition of the PI3K/Akt pathway in the MCL cell lines abrogated or reduced the phosphorylation of Akt, p27(kip1), FRKHL-1, MDM2, Bad, mTOR, GSK-3beta, IkappaB, and led to cell-cycle arrest and apoptosis. Six MCL cases (5 with activated Akt and 1 with inactive Akt) and 3 of 4 cell lines showed loss of PTEN expression. PIK3CA mutations were not detected. We conclude that constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis of MCL and preferentially occurs in blastoid variants. One possible mechanism of activation is loss of PTEN expression. These data suggest that PI3K/Akt inhibitors may be effective in the treatment of Akt-activated MCL.

Thalidomide use in pediatric patients.

OBJECTIVE: To provide a detailed overview of thalidomide use in pediatric patients. DATA SOURCES: English-language articles were identified through a MEDLINE search (1966-February 2001); key terms included thalidomide, child, graft-versus-host disease, cancer, HIV, Crohn's disease, Behçet's disease, and lupus erythematosus. References cited in those articles were also evaluated. DATA SYNTHESIS: Thalidomide appears to be effective in patients with chronic, not acute, graft-versus-host disease (GVHD) and in healing aphthous ulcers in patients with HIV infection. Limited case reports suggest efficacy of thalidomide in the treatment of cutaneous manifestations of Behçet's disease, Crohn's disease, and lupus in children; however, the recurrence of disease is almost universal on drug discontinuation. CONCLUSIONS: Thalidomide should be used as a last resort when all other therapies fail, preferably in male or prepubescent female patients.