Matthew J. Boyer

Blood vessels engineered from human cells

Tissue engineering has made considerable progress in the past decade, but advances have stopped short of clinical application for most tissues. We postulated that an obstacle in engineering human tissues is the limited replicative capacity of adult somatic cells. To test this hypothesis, the effectiveness of telomerase expression to extend cellular lifespan was assessed in a model of human vascular tissue engineering. Telomerase expression in vascular cells isolated from elderly patients enabled the successful culture of engineered autologous blood vessels. Engineered vessels may one day provide a source of bypass conduit for patients with atherosclerotic disease.

Human arteries engineered in vitro

There is a pressing need to develop methods to engineer small‐calibre arteries for bypass surgery. We hypothesized that the rate‐limiting step that has thwarted previous attempts to engineer such vessels from non‐neonatal tissues is the limited proliferative capacity of smooth muscle cells (SMCs), which are the main cellular component of these vessels. Ectopic expression of the human telomerase reverse transcriptase subunit (hTERT) has been shown recently to extend the lifespan of certain human cells. We therefore introduced hTERT into human SMCs and found that the resulting cells proliferated far beyond their normal lifespan but retained characteristics of normal control SMCs. Importantly, using these non‐neonatal SMCs, we were able to engineer mechanically robust human vessels, a crucial step towards creating arteries of clinical value for bypass surgery.

ADAR1 is required for hematopoietic progenitor cell survival via RNA editing

Adenosine Deaminase Acting on RNA 1 (ADAR1) is an RNA-editing enzyme that converts adenosine to inosine, following RNA transcription. ADAR1s essential role in embryonic development, especially within the hematopoietic lineage, has been demonstrated in knock-out mice. However, a specific role for ADAR1 in adult hematopoietic progenitor cells (HPCs) remains elusive. In this report, we show that ADAR1 is required for survival of differentiating HPCs as opposed to more primitive cells in adult mice by multiple strategies targeting floxed ADAR1 for deletion by Cre recombinase. As a consequence, ADAR1-deficient hematopoietic stem cells (HSCs) were incapable of reconstituting irradiated recipients although being phenotypically present in the recipient bone marrow. While an effect on HSCs cannot be completely ruled out, the preferential effect of ADAR1 absence on HPCs over more primitive hematopoietic cells was consistent with the increased expression of ADAR1 within HPCs, as well as the inability of ADAR1-deficient HPCs to form differentiated colonies and increased apoptotic fraction during ex vivo culture. Moreover, we have obtained direct evidence that ADAR1 functions in HPCs via an RNA-editing dependent mechanism. Therefore, ADAR1 plays an essential role in adult hematopoiesis through its RNA editing activity in HPCs.

Flow Cytometry-Based Cell Cycle Measurement of Mouse Hematopoietic Stem and Progenitor Cells

The balance between the quiescent hematopoietic stem cell (HSC) and the highly proliferative hematopoietic progenitor compartments maintains homeostasis in the hematopoietic system. Therefore, the entry of HSCs into the cell cycle and the rate of proliferation of hematopoietic progenitor cells are fundamental aspects in the field. This chapter describes two intracellular staining methods for DNA and RNA in conjunction with membrane staining for multiple hematopoietic cell-surface markers, and subsequent flow cytometric analysis to determine the cell cycle characteristics of primitive hematopoietic cells. First, the DNA stain Hoechst 33342 and the RNA dye Pyronin Y are used in combination with cell-surface markers to identify the proportion of cells in G(0) and G(1) in hematopoietic stem and progenitor cells. The second details the staining of bromodeoxyuridine incorporated into replicating DNA as a measure for the cycling cell fraction within a specific hematopoietic cell subset.

Hematopoietic Stem Cells Are Not the Direct Target of Spontaneous Leukemic Transformation in p18INK4C-Null Reconstituted Mice

Cell cycle inhibitors are important regulators in normal tissue regeneration and disruption of the regulators are involved in cancer development. Our recent study showed that the absence of the CDK inhibitor p18(INK4C) (p18) enhances self-renewal of normal hematopoietic stem cell (HSC) in vivo, whereas previous studies by others showed an increased incidence of leukemogenesis in older p18-null mice. Here, we have examined potential leukemogenesis during experimentally induced regeneration of HSC in the absence of p18 in order to gauge the relation between these two processes. Reconstituted mice with p18-deficient HSCs under the condition of repetitive proliferative stress (serial transplantation) were followed for >3 years. T cell leukemia from the p18-/- origin was recapitulated 24 months after secondary transplantation. However, no myeloid leukemia was found in the recipients. The T cell leukemia-initiating cells (mainly in a CD3(lo) cell subset) did not share the same immunophenotype with normal HSCs and, in fact, the function of HSCs was significantly compromised with decreased abundance in the leukemic mice. Furthermore, we found that the p15 or p16 gene promoters were frequently methylated in the leukemic cells but not in HSCs. Our present study argues against the possibility of overgrowth of p18-null HSCs leading to a leukemic phenotype. The data also support the notion that p18 has an independent role in T cell maintenance such that CD3+ CD8+ cells, unlike HSCs, are more accessible to leukemogenic transformation after the loss of p18.

Toxicity of definitive and post-operative radiation following ipilimumab in non-small cell lung cancer

To determine the feasibility and toxicity of radiation therapy, delivered either as definitive treatment or following surgery, following neo-adjuvant immune checkpoint inhibition for locally advanced NSCLC sixteen patients who received neo-adjuvant chemotherapy including ipilimumab as part of a phase II study were identified. Patients were analyzed by intent of radiation and toxicity graded based on CTCAE 4.0. There were seven patients identified who received definitive radiation and nine who received post-operative radiation. There was no grade 3 or greater toxicity in the definitive treatment group although one patient stopped treatment early due to back pain secondary to progression outside of the treatment field. In the post-operative treatment group, one patient required a one week break due to grade 2 odynophagia and no grade 3 or greater toxicity was observed. In this study of radiation as definitive or post-operative treatment following neo-adjuvant chemotherapy including ipilimumab for locally advanced NSCLC was feasible and well tolerated with limited toxicity.

Ablative Approaches for Pulmonary Metastases.

Pulmonary metastases are common in patients with cancer for which surgery is considered a standard approach in appropriately selected patients. A number of patients are not candidates for surgery due to a medical comorbidities or the extent of surgery required. For these patients, noninvasive or minimally invasive approaches to ablate pulmonary metastases are potential treatment strategies. This article summarizes the rationale and outcomes for non-surgical treatment approaches, including radiotherapy, radiofrequency and microwave ablation, for pulmonary metastases.

Improved Survival of Stage I Non–Small Cell Lung Cancer: A VA Central Cancer Registry Analysis

Introduction The combined impact of advances in diagnosis and treatment of stage I NSCLC has not been assessed comprehensively. To define the survival impact of modern staging and treatment techniques for clinical stage I NSCLC, the Veterans Administration Central Cancer Registry, a database of U.S. veterans in whom the disease was diagnosed in the Veterans Health Administration, was queried. From this database, patients who had stage I NSCLC diagnosed from 2001 to 2010 and were treated with either surgery or radiation were identified. Methods Overall survival (OS) and lung cancer‐specific survival were determined. Propensity score matching and Cox multivariate analysis were used to adjust for baseline patient characteristics. Results A total of 11,997 patients were identified. The 4‐year OS rate increased from 38.9% to 53.2% from 2001 to 2010 for all patients. Positron emission tomography and endobronchial ultrasound did not improve OS. Survival of radiated patients improved from 12.7% to 28.5%. The introduction of stereotactic body radiation therapy (SBRT) significantly improved OS (hazard ratio [HR] = 0.60, 95% confidence interval [CI]: 0.54–0.68) and lung cancer‐specific survival (HR = 0.39, 95% CI: 0.32–0.46) compared with conventionally fractionated radiation. The 4‐year OS rate also improved after surgery (from 51.5% to 66.5%). This increase was associated with use of adjuvant chemotherapy, increased use of video‐assisted thoracoscopic surgical procedures, and decreased pneumonectomy rates, with similar survival between open and video‐assisted thoracoscopic surgical procedures. OS after lobectomy was superior to that after sublobar resection (HR = 0.82, 95% CI: 0.75–0.89). In the era of available SBRT (2008–2010), 4‐year OS was not significantly different after sublobar resection or lobectomy for medically unfit patients (Charlson comorbidity index = 2) (55.4% and 58.1%, respectively; p = 0.69) but was significantly worse for fit patients (Charlson comorbidity index = 0–1) undergoing sublobar resection (55.5% and 68.0%, respectively; p < 0.001). OS (HR = 0.36, 95% CI: 0.35–0.38) and lung cancer‐specific survival (HR = 0.31, 95% CI: 0.29–0.33) were improved after surgery as compared with after radiation, with the improvement maintained on matched comparison of lobectomy and SBRT. Conclusions OS increased in veterans with a diagnosis of stage I NSCLC from 2001 to 2010; the increase was coincident with improved radiation and surgical techniques.

Esophageal Cancer: Radiation Therapy Planning

The treatment of esophageal cancer with radiation therapy presents many challenges. The propensity of esophageal cancer to invade and spread within the submucosa with resultant lymph node involvement requires particular attention to areas of elective coverage beyond the gross tumor. Moreover, the intimate association of the esophagus and gastroesophageal junction with adjacent organs in the thoracic cavity and upper abdomen dictates attention to normal tissue constraints. Although most patients receive preoperative chemoradiation, patients treated postoperatively may require larger treatment fields, further increasing treatment-related normal tissue toxicities. This chapter will discuss the rationale of radiation treatment field design and dose for esophageal cancer patients based on thoracic anatomy, imaging, and patterns of spread/failure, as well as practical considerations for patient simulation and treatment.

Esophageal Cancer: Background and Clinical Evidence

Although esophageal cancer represents only 6 % of all gastrointestinal malignancies diagnosed in the United States, it remains a highly lethal disease and the seventh most common cause of cancer death in males. Rates of squamous cell carcinoma have been declining, while the incidence of esophageal adenocarcinoma is increasing with rising rates of gastroesophageal reflux disease (GERD) and associated risk factors. Although the overall prognosis of esophageal cancer patients is poor, particularly with more advanced disease, trimodality therapy with radiation, chemotherapy, and surgery has improved outcomes. This chapter reviews the clinical data underlying the current management of esophageal cancer.