Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction.

Background: Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Patients and methods: Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Conclusion(s): Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival.

Phase I study of pazopanib and vorinostat: a therapeutic approach for inhibiting mutant p53-mediated angiogenesis and facilitating mutant p53 degradation.

BACKGROUND: We carried out a phase I trial of the vascular endothelial growth factor inhibitor pazopanib and the histone deacetylase inhibitor vorinostat to determine the safety and efficacy. Because these agents are known to target factors activated by TP53 mutation and facilitate mutant p53 degradation, a subgroup analysis may be interesting in patients with TP53 mutant malignancies. PATIENTS AND METHODS: Patients with advanced solid tumors (n = 78) were enrolled following a 3 + 3 design, with dose expansion for those with responsive tumors. Hotspot TP53 mutations were tested when tumor specimens were available. RESULTS: Adverse events of ≥grade 3 included thrombocytopenia, neutropenia, fatigue, hypertension, diarrhea and vomiting. Overall, the treatment produced stable disease for at least 6 months or partial response (SD ≥6 months/PR) in 19% of the patients, median progression-free survival (PFS) of 2.2 months, and median overall survival (OS) of 8.9 months. In patients with detected hotspot TP53 mutant advanced solid tumors (n = 11), the treatment led to a 45% rate of SD ≥6 months/PR (1 PR and 3 SD ≥6 months), median PFS of 3.5 months, and median OS of 12.7 months, compared favorably with the results for patients with undetected hotspot TP53 mutations (n = 25): 16% (1 PR and 3 SD ≥6 months, P = 0.096), 2.0 months (P = 0.042), and 7.4 months (P = 0.1), respectively. CONCLUSION: The recommended phase II dosage was oral pazopanib at 600 mg daily plus oral vorinostat at 300 mg daily. The preliminary evidence supports further evaluation of the combination in cancer patients with mutated TP53, especially in those with metastatic sarcoma or metastatic colorectal cancer. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov, NCT01339871.

Retreatment with anti-EGFR based therapies in metastatic colorectal cancer: impact of intervening time interval and prior anti-EGFR response.

BACKGROUND: This retrospective study aims to investigate the activity of retreatment with anti-EGFR-based therapies in order to explore the concept of clonal evolution by evaluating the impact of prior activity and intervening time interval. METHODS: Eighty-nine KRAS exon 2-wild-type metastatic colorectal patients were retreated on phase I/II clinical trials containing anti-EGFR therapies after progressing on prior cetuximab or panitumumab. Response on prior anti-EGFR therapy was defined retrospectively per physician-records as response or stable disease ≥6 months. Multivariable statistical methods included a multiple logistic regression model for response, and Cox proportional hazards model for progression-free survival. RESULTS: Retreatment anti-EGFR agents were cetuximab (n = 76) or cetuximab plus erlotinib (n = 13). The median interval time between prior and retreatment regimens was 4.57 months (range: 0.46-58.7). Patients who responded to the prior cetuximab or panitumumab were more likely to obtain clinical benefit to the retreatment compared to the non-responders in both univariate (p = 0.007) and multivariate analyses (OR: 3.38, 95 % CI: 1.27, 9.31, p = 0.019). The clinical benefit rate on retreatment also showed a marginally significant association with interval time between the two anti-EGFR based therapies (p = 0.053). Median progression-free survival on retreatment was increased in prior responders (4.9 months, 95 % CI: 3.6, 6.2) compared to prior non-responders (2.5 months, 95 % CI, 1.58, 3.42) in univariate (p = 0.064) and multivariate analysis (HR: 0.70, 95 % CI: 0.43-1.15, p = 0.156). CONCLUSION: Our data lends support to the concept of clonal evolution, though the clinical impact appears less robust than previously reported. Further work to determine which patients benefit from retreatment post progression is needed.

The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells.

The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.

A phase 1 study of intermittently administered pazopanib in combination with continuous daily dosing of lapatinib in patients with solid tumors.

PURPOSE: Preclinically, pazopanib/lapatinib combination acted synergistically to suppress the activity of multiple tyrosine kinases, including VEGFR-1, 2, 3, PDGFR and c-kit (pazopanib), HER1/EGFR and HER2 (lapatinib), and several other tyrosine kinases including c-Met through, plausibly, network inhibition effects. Clinically, continuous dosing of pazopanib/lapatinib combination was associated with a higher response rate than with lapatinib monotherapy, with poor tolerance. We explored multiple intermittent dose levels of pazopanib combined with continuous daily dosing of lapatinib in patients with solid tumors. METHODS: The present study used a phase 1, modified 3 + 3, dose-escalation design to evaluate the safety and tolerability of the combination of orally received pazopanib once every other day with continuous daily dosing of lapatinib for 28 days. In the expansion phase, tumor response was evaluated in patients with specific genetic alterations (HER2 amplification, HER2 mutation, c-Met amplification, c-Met mutation, and EGFR mutation). RESULTS: Twenty-four patients were treated. The most common drug-related adverse events were fatigue 7/24 (29%), skin rash 5/21 (21%), and diarrhea 3/24 (17%), with 4/24 (16%) patients experiencing grade ≥3 drug-related adverse events. Escalation to the FDA-approved dose (800 mg daily for pazopanib and 1500 mg every day for lapatinib) was not feasible due to toxicities. Pazopanib 200 mg every other day + lapatinib 500 mg daily was considered the maximum tolerated dose (MTD). No tumor response was observed, including in patients with the specific molecular genetic alterations tested. CONCLUSION: Every other day dosing of pazopanib combined with daily lapatinib was tolerated at the established MTD, but no complete or partial tumor responses were observed at these dose levels.

Randomized phase III study of chemoradiation with or without amifostine for patients with favorable performance status inoperable stage II-III non-small cell lung cancer: preliminary results.

A prospective randomized study was conducted to determine whether amifostine (Ethyol) reduces the rate of severe esophagitis and hematologic and pulmonary toxicity associated with chemoradiation or improves control of non-small cell lung cancer (NSCLC). Sixty patients with inoperable stage II or III NSCLC were treated with concurrent chemoradiotherapy. Both groups received thoracic radiation therapy (TRT) with 1.2 Gy/fraction, 2 fraction per day, 5 days per week for a total dose 69.6 Gy. All patients received oral etoposide (VP-16), 50 mg Bid, 30 minutes before TRT beginning day 1 for 10 days, repeated on day 29, and cisplatin 50 mg/m(2) intravenously on days 1, 8, 29, and 36. Patients in the study group received amifostine, 500 mg intravenously, twice weekly before chemoradiation (arm 1); patients in the control group received chemoradiation without amifostine (arm 2). Patient and tumor characteristics were distributed equally in both groups. Of the 60 patients enrolled, 53 were evaluable (27 in arm 1, 26 in arm 2) with a median follow-up of 6 months. Median survival times were 26 months for arm 1 and 15 months for arm 2, not statistically significantly different. Morphine intake to reduce severe esophagitis was significantly lower in arm 1 (2 of 27, 7.4%) than arm 2 (8 of 26, 31%; P =.03). Acute pneumonitis was significantly lower in arm 1 (1 of 27, 3.7%) than in arm 2 (6 of 26, 23%; P =.037). Hypotension (20 mm Hg decrease from baseline blood pressure) was significantly more frequent in arm 1 (19 of 27, 70%) than arm 2 (1 of 26, 3.8%; P =.0001). Only 1 patient discontinued treatment because of hypotension. These preliminary results showed that amifostine significantly reduced acute severe esophagitis and pneumonitis. Further observation is required to assess long-term efficacy.

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei.

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.

Predicting high-throughput screening results with scalable literature-based discovery methods.

The identification of new therapeutic uses for existing agents has been proposed as a means to mitigate the escalating cost of drug development. A common approach to such repurposing involves screening libraries of agents for activities against cell lines. In silico methods using knowledge from the biomedical literature have been proposed to constrain the costs of screening by identifying agents that are likely to be effective a priori. However, results obtained with these methods are seldom evaluated empirically. Conversely, screening experiments have been criticized for their inability to reveal the biological basis of their results. In this paper, we evaluate the ability of a scalable literature-based approach, discovery-by-analogy, to identify a small number of active agents within a large library screened for activity against prostate cancer cells. The methods used permit retrieval of the knowledge used to infer their predictions, providing a plausible biological basis for predicted activity.

[A cortico-cancellous graft with a periosteal pedicle for the treatment of deep single-walled bone pockets]. / Ein periostgestieltes, kortikospongiöses Transplantat zur Behandlung tiefer, einwandiger Knochentaschen.

The treatment of single-walled bone pockets involves a number of problems. Conventional treatment methods fail to bring about permanent success, particularly in the more advanced stages. A new method has been developed and described using a cortico-cancellous graft harvested from the apical region of the teeth with a periosteal pedicle that can be inserted into the highly infection-prone area of a bone pocket. The advantage of this procedure is that the transplant is perfused and thus not subjected to necrosis but highly resistant to infections. Indications, practical procedure and success rates are discussed and illustrated in one example.

[Treatment of one-walled periodontal osseous defect by Periost-inserted bone transplant]. / Die Behandlung der einwandigen Knochentasche mit einem periostigestielten Knochentransplantat.

A method is described to treat deep isolated periodontal osseous defects in maxillary incisor and canine region with periost-inserted bonetransplant. Indication, procedure and results are demonstrated by a case.

[Sealer-free surgical filling of root canal by means of Gutta-percha]. / Die sealerfreie chirurgische Wurzelkanalfüllung mit Guttapercha.

The author demonstrates a procedure which is aimed at the insertion of fitting, conically pressed Guttapercha pins for filling of standardized, prepared dental root canals in the apectomy. The renunciation of a sealer is the advantages of the procedure. Indication and performance are explained, the advantages of the procedure discussed.

[Organisation of the dispensary care of the maxillary and facial surgery using mini-computers]. / Organisation der Dispensairebetreuung in der Kiefer-Gesichts-Chirurgie unter Nutzung von Kleincomputern.

The presented hardware and software solution enables to realize an effective and complete patients' recall and thus the comprehensive dispensary care in an average special department for maxillary and facial surgery.

Effect of nucleotide on the binding of peptides to 70-kDa heat shock protein.

In previous work we found that bovine brain hsp70 has a single binding site for nucleotide, and that, with ATP at this site, the rates of association and dissociation of clathrin from hsp70 are fast, whereas with ADP at this site, these rates are unmeasurably slow. In the present study we show, first, that peptide C, cytochrome c peptide, and RNase S peptide bind competitively with clathrin, suggesting that they bind to the same site on hsp70, although RNase S peptide binds an order of magnitude more weakly than peptide C and cytochrome c peptide. Second, we show that, with ADP bound to hsp70, as occurs with clathrin, the rate constant for dissociation of peptide markedly decreases compared to the rate constant observed in ATP. In contrast, ADP only slightly decreases the rate of association of peptide. Based on these data we propose a model in which substrates of hsp70 bind to and dissociate from the ATP form of the enzyme, while, following ATP hydrolysis, they are locked onto the ATP form of the enzyme, unable to dissociate until ADP is released and ATP rebinds.