Jianling Yuan

Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells

ABSTRACT The hypoxic tumor microenvironment has been shown to contribute to genetic instability. As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level. However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation. In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression. Functional MMR deficiency under hypoxia was detected as induced instability of a (CA)29 dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene. These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins. In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria.

Mutagenesis induced by the tumor microenvironment

Genomic instability is a commonly observed feature of tumors. Most investigations addressing the mechanism of tumor progression have focused on the genetic factors that may play a role. Growing evidence now suggests that, in addition to these endogenous factors, the exogenous environment within solid tumors may by itself be mutagenic and constitute a significant source of genetic instability. The tumor microenvironment is characterized by regions of fluctuating hypoxia, low pH, and nutrient deprivation. Each of these microenvironmental factors has been shown to cause severe disturbance in cell metabolism and physiology. Both in vivo and in vitro data demonstrate that exposure of tumor cells to adverse conditions can directly cause mutations, contributing to genetic instability. In this review, we will reexamine the current body of evidence on the role of the tumor microenvironment in inducing mutagenesis and consequent tumor progression.

Radiobiology of stereotactic body radiation therapy/stereotactic radiosurgery and the linear-quadratic model

Received Feb 26, 2013, and in revised form Mar 11, 2013. Accepted for publication Mar 12, 2013The validity of the linear-quadratic (LQ) model for calculatingisoeffect doses in radiation therapy has been intensivelydebated in recent issues of the International Journal of Radi-ation Oncology, Biology, Physics (1-3).TheLQmodelissimple and convenient, and by far it has been the most usefulmeans for isodose calculation in treating tumors with conven-tional fractionated radiation therapy (2-4). The LQ modelsolely depends on the expected incidence of direct interactionsof radiation with specific cellular targets (ie, DNA strands).Because the LQ survival curve continuously bends downwardwith increasing radiation dose, many assume that the LQcalculation will inherently overestimate cell death caused byhigh-dose-per-fraction radiation therapy. Interestingly, however,clinical results have shown that the LQ model actually under-estimates tumor control by stereotactic body radiation therapy(SBRT) or stereotactic radiosurgery (SRS) (5), indicating thatmechanism(s) in addition to DNA strand breaks and/or chro-mosome aberrations may be involved in response of tumors toSBRT or SRS. Therefore, it has been hypothesized that SBRTor SRS may cause significant vascular damage in tumors,leading to indirect cell death (5, 6). We have recently reviewedprevious studies on the radiation-induced vascular damage intumors and pointed out the potentially important role of indi-rect/necrotic cell death due to the vascular damage in tumorcontrol with SBRT and SRS (7). We further discussed theradiobiological principles of SBRT and SRS in relation toradiation-induced vascular damage and resultant indirect celldeath (8). Interestingly, some 35 years ago we (C.W.S. andS.H.L.) realized that irradiation of rodent tumors with 10-20Gy in a single dose caused severe vascular injury, leading tonecrotic cell death in significant fractions of tumor cells 2 to 3days after the treatment (9, 10). Figure 1 summarizes theobservations we made on the effects of 10 Gy (1000 rads) ofx-rays in a single dose on the clonogenic surviving cells inWalker 256 rat tumors (10). The surviving cell fraction, asmeasured with an in vivoein vitro excision method, wasapproximately 2.6 10

Evaluation of surface and superficial dose for head and neck treatments using conventional or intensity-modulated techniques

With increased use of intensity-modulated radiation therapy (IMRT) for head and neck treatment questions have arisen as to selection of an optimum treatment approach when either superficial sparing or treatment is desired. Other work has pointed out the increased superficial dose resulting from obliquity effects when multiple tangential beams are applied to head and neck treatment, as is the general case in IMRT planning. Helical tomotherapy might be expected to result in even further enhanced superficial dose compared with conventional bilateral field treatment. We have designed a typical right oropharynx target volume in an anthropomorphic head and neck phantom. Three different treatment techniques have been used to optimally treat this target, including bilateral static fields, eight-field IMRT and helical tomotherapy. The phantom was immobilized in a standard treatment position and treated on a Varian 2300cd linear accelerator and on a Hi-Art Helical Tomotherapy unit. 1 mm3 lithium-fluoride thermoluminescent dosimeters (TLDs) were placed on the surface of the phantom at a number of axial test positions. Film strips (Kodak EDR2) were either wrapped around the surface or sandwiched within the phantom. Measured doses at the surface and as a function of depth are compared with the planning system predictions for each treatment technique. The maximum surface doses on the proximal treatment side, averaged from TLDs and films, were measured to be 69-82% of the target dose with the bilateral fields yielding the lowest surface doses (69%), tomotherapy about 2% more than that (71%) and IMRT 13% more (82%). Anterior to the target volume, doses are always low for bilateral treatment. In this case the minimum anterior surface dose (chin area) was 6% of the prescription dose from that technique as compared with 26% and 35% from the IMRT and tomotherapy methods, respectively. The Eclipse and Tomotherapy planning systems both modelled deep and superficial doses well. Surface doses were better modelled by Eclipse at the test points, while the tomotherapy plans consistently overestimated the measured doses by 10% or more. Depth dose measurements, extracted from embedded films, indicated the depth of dose build-up to >99% to be the shallowest for IMRT (2-5 mm) followed by tomotherapy (5-8 mm) and bilateral fields (10-15 mm). The amount of surface dose is clearly technique dependent and should be taken into account in the planning stage.

Omission of radiation therapy after breast-conserving surgery in the United States: A population-based analysis of clinicopathologic factors

Radiation therapy (RT) after breast‐conserving surgery (BCS) is associated with a significant reduction in ipsilateral breast tumor recurrence and breast cancer mortality rates in patients with early stage breast cancer. The authors of this report sought to determine which patients with breast cancer do not receive RT after BCS in the United States.

Management of Unplanned Excision for Soft-Tissue Sarcoma With Preoperative Radiotherapy Followed by Definitive Resection.

Background and Objectives:The purpose of this study was to review the outcomes after preoperative radiotherapy and definitive surgery for patients who initially had inadvertent excision for sarcoma. Materials and Methods:Treatment records of 44 consecutive patients, who initially underwent unplanned excision of soft-tissue sarcoma between January 2004 and January 2012, were reviewed. All patients had clinically localized disease before treatment and received preoperative external-beam radiotherapy followed by definitive oncologic surgery at our institution. Results:The median follow-up was 36 months. Residual tumor after preoperative radiotherapy and wound bed excision was identified in 39% (17/44) of the cases. Kaplan-Meier estimates for 5-year local control, recurrence-free survival, and overall survival are 95% (95% confidence interval [CI], 80-99), 86% (95% CI, 69-94), and 94% (95% CI, 79-99) respectively. Perioperative morbidity occurred in 25% of patients (11/44.) All patients with perioperative wound complications had lower extremity sarcomas. Conclusions:Optimal management for unplanned excision of soft-tissue sarcoma is unknown. Our institution has adopted the approach of preoperative radiotherapy, followed by definitive surgery. In our series of 44 patients, local control was excellent at 95%, with perioperative complications seen only in patients with lower extremity sarcomas, suggesting that this is a reasonable approach to manage inadvertently resected sarcoma.

BRAF Mutation is associated with improved local control of Melanoma Brain Metastases Treated with gamma Knife radiosurgery

Objectives Evidence has implicated a possible role of tumor mutation status on local control (LC) with radiotherapy. BRAF is a proto-oncogene that is mutated in approximately 50% of patients with melanoma. We sought to analyze the influence of BRAF status on LC of melanoma brain metastases (MBM) following Gamma Knife radiosurgery (GK). Methods Among 125 patients treated with GK for MBM at our institution between 2006 and 2015, we identified 19 patients with 69 evaluable metastases whose BRAF mutation status was known and follow-up imaging was available. LC of individual metastases was compared based on BRAF mutation status using statistical techniques to control for measurements of multiple metastases within each patient. CNS progression was defined as either local failure or development of new lesions. Results Of the 69 metastases, BRAF was mutated in 30 and wild-type in 39. With a median follow-up of 30 months for all patients and a median follow-up of 5.5 months for treated lesions, 1-year LC was significantly better among metastases with mutated vs. wild-type BRAF (69 vs. 34%, RR = 0.3, 95% CI = 0.1–0.7, p = 0.01). BRAF mutation was found to be a significant predictor of LC after stereotactic radiosurgery (SRS) in both univariate [RR = 0.3 (95% CI 0.1–0.7, p = 0.01)] and multivariate [RR = 0.2 (95% CI 0.1–0.7, p = 0.01)] analyses. There was also a trend toward improved CNS progression free survival (PFS) at 1 year (26 vs. 0%, p = 0.06), favoring BRAF-mutated patients. Conclusion In this retrospective study, MBM treated with GK had significantly improved LC for patients with BRAF mutation vs. wild-type. Our data suggest that BRAF mutation may sensitize tumors to radiosurgery, and that BRAF wild-type tumors may be more radioresistant.

Role of Consolidative Radiation Therapy After Autologous Hematopoietic Cell Transplantation for the Treatment of Relapsed or Refractory Hodgkin Lymphoma

Purpose To evaluate the role of the addition of consolidative radiation therapy after high-dose chemotherapy and autologous hematopoietic cell transplantation (AHCT) for relapsed or refractory Hodgkin lymphoma (HL). Methods and Materials Medical records were reviewed from a total of 80 consecutive patients who underwent high-dose chemotherapy with AHCT treated under a single protocol at University of Minnesota between November 2005 and January 2014. Of these, 32 patients received radiation therapy after AHCT as planned consolidation. Results At a median follow-up of 25 months, the 2-year overall survival (OS) and progression-free survival (PFS) for the entire cohort was 96% and 52%, respectively. Consolidative radiation therapy was found to significantly improve the 2-year PFS (67% vs 42%, P<.01) without a significant change in OS (100% vs 93%, P=.15). On subgroup analysis, consolidative radiation therapy was shown to improve PFS in patients with bulky disease (62% vs 39%, P=.02), B-symptoms (48% vs 28%, P=.05), primary refractory disease (47% vs 32%, P=.02), and those with a partial response on pretransplant imaging (47% vs 32%, P=.02). The improvement seen on 2-year PFS with consolidative radiation therapy remained significant on multivariate analysis (hazard ratio 4.64, 95% confidence interval 1.98–10.88). Minimal toxicity was observed among the patients receiving radiation therapy. Conclusions The addition of consolidative radiation therapy after high-dose chemo-therapy and AHCT demonstrated a significant improvement in 2-year PFS and no impact on OS. Radiation therapy was well tolerated, with minimal toxicity. Our study supports a role of consolidative radiation therapy in patients with HL treated with AHCT.

SU‐FF‐T‐228: Evaluation of Surface and Superficial Dose for Head and Neck Treatments Using Conventional Or Intensity‐Modulated Techniques

With increased use of intensity-modulated radiation therapy (IMRT) for head and neck treatment questions have arisen as to selection of an optimum treatment approach when either superficial sparing or treatment is desired. Other work has pointed out the increased superficial dose resulting from obliquity effects when multiple tangential beams are applied to head and neck treatment, as is the general case in IMRT planning. Helical tomotherapy might be expected to result in even further enhanced superficial dose compared with conventional bilateral field treatment. We have designed a typical right oropharynx target volume in an anthropomorphic head and neck phantom. Three different treatment techniques have been used to optimally treat this target, including bilateral static fields, eight-field IMRT and helical tomotherapy. The phantom was immobilized in a standard treatment position and treated on a Varian 2300cd linear accelerator and on a Hi-Art Helical Tomotherapy unit. 1 mm3 lithium-fluoride thermoluminescent dosimeters (TLDs) were placed on the surface of the phantom at a number of axial test positions. Film strips (Kodak EDR2) were either wrapped around the surface or sandwiched within the phantom. Measured doses at the surface and as a function of depth are compared with the planning system predictions for each treatment technique. The maximum surface doses on the proximal treatment side, averaged from TLDs and films, were measured to be 69-82% of the target dose with the bilateral fields yielding the lowest surface doses (69%), tomotherapy about 2% more than that (71%) and IMRT 13% more (82%). Anterior to the target volume, doses are always low for bilateral treatment. In this case the minimum anterior surface dose (chin area) was 6% of the prescription dose from that technique as compared with 26% and 35% from the IMRT and tomotherapy methods, respectively. The Eclipse and Tomotherapy planning systems both modelled deep and superficial doses well. Surface doses were better modelled by Eclipse at the test points, while the tomotherapy plans consistently overestimated the measured doses by 10% or more. Depth dose measurements, extracted from embedded films, indicated the depth of dose build-up to >99% to be the shallowest for IMRT (2-5 mm) followed by tomotherapy (5-8 mm) and bilateral fields (10-15 mm). The amount of surface dose is clearly technique dependent and should be taken into account in the planning stage.

Diminished DNA repair and elevated mutagenesis in mammalian cells exposed to hypoxia and low pH

The tumor microenvironment is characterized by regions of fluctuating and chronic hypoxia, low pH, and nutrient deprivation. It has been proposed that this unique tissue environment itself may constitute a major cause of the genetic instability seen in cancer. To investigate possible mechanisms by which the tumor microenvironment might contribute to genetic instability, we asked whether the conditions found in solid tumors could influence cellular repair of DNA damage. Using an assay for repair based on host cell reactivation of UV-damaged plasmid DNA, cells exposed to hypoxia and low pH were found to have a diminished capacity for DNA repair compared with control cells grown under standard culture conditions. In addition, cells cultured under hypoxia at pH 6.5 immediately after UV irradiation had elevated levels of induced mutagenesis compared with those maintained in standard growth conditions. Taken together, the results suggest that cellular repair functions may be impaired under the conditions of the tumor microenvironment, causing hypermutability to DNA damage. This alteration in repair capacity may constitute an important mechanism underlying the genetic instability of cancer cells in vivo.