Itai Pashtan

Isolation of Hyperactive Mutants of the MAPK p38/Hog1 That Are Independent of MAPK Kinase Activation*

Mitogen-activated protein kinases (MAPKs) play pivotal roles in growth, development, differentiation, and apoptosis. The exact role of a given MAPK in these processes is not fully understood. This question could be addressed using active forms of these enzymes that are independent of external stimulation and upstream regulation. Yet, such molecules are not available. MAPK activation requires dual phosphorylation, on neighboring Tyr and Thr residues, catalyzed by MAPK kinases (MAPKKs). It is not known how to force MAPK activation independent of MAPKK phosphorylation. Here we describe a series of nine hyperactive (catalytically and biologically), MAPKK-independent variants of the MAPK Hog1. Each of the active molecules contains just a single point mutation. Six mutations are in the conserved L16 domain of the protein. The active Hog1 mutants were obtained through a novel genetic screen that could be applied for isolation of active MAPKs of other families. Equivalent mutations, introduced to the human p38α, rendered the enzyme active even when produced in Escherichia coli, showing that the mutations increased the intrinsic catalytic activity of p38. It implies that the activating mutations could be directly used for production of active forms of MAPKs from yeasts to humans and could open the way to revealing their biological functions.

Long-term cosmetic outcomes and toxicities of proton beam therapy compared with photon-based 3-dimensional conformal accelerated partial-breast irradiation: a phase 1 trial.

PURPOSE To present long-term outcomes of a prospective feasibility trial using either protons or 3-dimensional conformal photon-based (accelerated partial-breast irradiation [APBI]) techniques. METHODS AND MATERIALS From October 2003 to April 2006, 98 evaluable patients with stage I breast cancer were treated with APBI (32 Gy in 8 fractions given twice daily) on a prospective clinical trial: 19 with proton beam therapy (PBT) and 79 with photons or mixed photons/electrons. Median follow-up was 82.5 months (range, 2-104 months). Toxicity and patient satisfaction evaluations were performed at each visit. RESULTS At 7 years, the physician rating of overall cosmesis was good or excellent for 62% of PBT patients, compared with 94% for photon patients (P=.03). Skin toxicities were more common for the PBT group: telangiectasia, 69% and 16% (P=.0013); pigmentation changes, 54% and 22% (P=.02); and other late skin toxicities, 62% and 18% (P=.029) for PBT and photons, respectively. There were no significant differences between the groups in the incidences of breast pain, edema, fibrosis, fat necrosis, skin desquamation, and rib pain or fracture. Patient-reported cosmetic outcomes at 7 years were good or excellent for 92% and 96% of PBT and photon patients, respectively (P=.95). Overall patient satisfaction was 93% for the entire cohort. The 7-year local failure rate for all patients was 6%, with 3 local recurrences in the PBT group (7-year rate, 11%) and 2 in photon-treated patients (4%) (P=.22). CONCLUSIONS Local failure rates of 3-dimensional APBI and PBT were similar in this study. However, PBT, as delivered in this study, led to higher rates of long-term telangiectasia, skin color changes, and skin toxicities. We recommend the use of multiple fields and treatment of all fields per treatment session or the use of scanning techniques to minimize skin toxicity.

DNA Repair Pathway Gene Expression Score Correlates with Repair Proficiency and Tumor Sensitivity to Chemotherapy

Measuring DNA repair pathway choice determines the sensitivity of cancers to individual classes of treatment. Navigating Tumor Therapy with an RPS For cancer patients, DNA damage is a double-edged sword. Mutations can contribute to carcinogenesis, but the cancer cell can’t survive with too much DNA damage. Indeed, many cancer therapies aim at increasing DNA damage, but selecting the correct therapies for individual patients can be hit-or-miss. Pitroda et al. proposed that mechanisms of double-strand DNA break repair could correlate with cancer prognosis and could guide choices in cancer therapy. The authors devised a recombination proficiency score (RPS) based on the expression levels for four genes involved in DNA repair pathway preference. They then validated the RPS in patients with either breast or non–small cell lung cancer. Tumors with low RPS—suppressed homologous recombination (HR)—were associated with greater mutagenesis and decreased likelihood of patient survival. This prognosis could be counteracted with platinum-based adjuvant chemotherapy—patients with suppressed HR were more sensitive to this treatment. These data suggest that RPS can help determine treatment course for cancer patients. Mutagenesis is a hallmark of malignancy, and many oncologic treatments function by generating additional DNA damage. Therefore, DNA damage repair is centrally important in both carcinogenesis and cancer treatment. Homologous recombination (HR) and nonhomologous end joining are alternative pathways of double-strand DNA break repair. We developed a method to quantify the efficiency of DNA repair pathways in the context of cancer therapy. The recombination proficiency score (RPS) is based on the expression levels for four genes involved in DNA repair pathway preference (Rif1, PARI, RAD51, and Ku80), such that high expression of these genes yields a low RPS. Carcinoma cells with low RPS exhibit HR suppression and frequent DNA copy number alterations, which are characteristic of error-prone repair processes that arise in HR-deficient backgrounds. The RPS system was clinically validated in patients with breast or non–small cell lung carcinomas (NSCLCs). Tumors with low RPS were associated with greater mutagenesis, adverse clinical features, and inferior patient survival rates, suggesting that HR suppression contributes to the genomic instability that fuels malignant progression. This adverse prognosis associated with low RPS was diminished if NSCLC patients received adjuvant chemotherapy, suggesting that HR suppression and associated sensitivity to platinum-based drugs counteract the adverse prognosis associated with low RPS. Therefore, RPS may help oncologists select which therapies will be effective for individual patients, thereby enabling more personalized care.

External Beam Accelerated Partial-Breast Irradiation Using 32 Gy in 8 Twice-Daily Fractions: 5-Year Results of a Prospective Study

PURPOSE External beam accelerated partial breast irradiation (APBI) is an increasingly popular technique for treatment of patients with early stage breast cancer following breast-conserving surgery. Here we present 5-year results of a prospective trial. METHODS AND MATERIALS From October 2003 through November 2005, 98 evaluable patients with stage I breast cancer were enrolled in the first dose step (32 Gy delivered in 8 twice-daily fractions) of a prospective, multi-institutional, dose escalation clinical trial of 3-dimensional conformal external beam APBI (3D-APBI). Median age was 61 years; median tumor size was 0.8 cm; 89% of tumors were estrogen receptor positive; 10% had a triple-negative phenotype; and 1% had a HER-2-positive subtype. Median follow-up was 71 months (range, 2-88 months; interquartile range, 64-75 months). RESULTS Five patients developed ipsilateral breast tumor recurrence (IBTR), for a 5-year actuarial IBTR rate of 5% (95% confidence interval [CI], 1%-10%). Three of these cases occurred in patients with triple-negative disease and 2 in non-triple-negative patients, for 5-year actuarial IBTR rates of 33% (95% CI, 0%-57%) and 2% (95% CI, 0%-6%; P<.0001), respectively. On multivariable analysis, triple-negative phenotype was the only predictor of IBTR, with borderline statistical significance after adjusting for tumor grade (P=.0537). CONCLUSIONS Overall outcomes were excellent, particularly for patients with estrogen receptor-positive disease. Patients in this study with triple-negative breast cancer had a significantly higher IBTR rate than patients with other receptor phenotypes when treated with 3D-APBI. Larger, prospective 3D-APBI clinical trials should continue to evaluate the effect of hormone receptor phenotype on IBTR rates.

Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors

Hepatocyte growth factor/scatter factor (HGF/SF) receptor c-Met is implicated in growth, invasion and metastasis of many tumors. Tumor cells harboring MET gene amplification are initially sensitive to c-Met tyrosine kinase inhibitors (TKI), but escape from long-term treatment has not been investigated. C-Met is a client of heat shock protein 90 (Hsp90) and is destabilized by Hsp90 inhibitors, suggesting that these drugs may inhibit tumors driven by MET amplification, although tumor escape under these conditions also has not been explored. Here, we evaluated the initial inhibitory effects of, and the likelihood of escape from, the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and the c-Met TKI SU11274, using two cell lines harboring MET gene amplification. 17-AAG inhibited cell growth in both cell lines and induced substantial apoptosis, whereas SU11274 was only growth inhibitory in one cell line. In both cell lines, c-Met-dependent Akt, Erk and/or STAT3 signaling, as well as activation of the EGFR family, resumed shortly after treatment with c-Met TKI despite sustained c-Met inhibition. PKC δ upregulation may participate in reactivation of c-Met downstream signaling in both cell lines. In contrast to c-Met TKI, 17-AAG destabilized c-Met protein and durably blocked reactivation of downstream signaling pathways and EGFR family members. Our data demonstrate that downstream signaling in tumor cells over-expressing c-Met is not stably suppressed by c-Met TKI, even though c-Met remains fully inhibited. In contrast, Hsp90 inhibitors provide long-lasting suppression of c-Met-dependent signaling, and these drugs should be further evaluated in tumors driven by MET gene amplification.

Endometrial Carcinoma Recurrence Score (ECARS) validates to identify aggressive disease and associates with markers of epithelial–mesenchymal transition and PI3K alterations

PURPOSE Our previously reported 29-gene expression signature identified an aggressive subgroup of endometrial cancer patients with PI3K activation. We here wanted to validate these findings by independent patient series. PATIENTS AND METHODS The 29-gene expression signature was assessed in fresh frozen tumor tissue from 280 primary endometrial carcinomas (three independent cohorts), 19 metastatic lesions and in 333 primary endometrial carcinomas using TCGA data, and expression was related to clinico-pathologic features and survival. The 29-gene signature was assessed by real-time quantitative PCR, DNA oligonucleotide microarrays, or RNA sequencing. PI3K alterations were assessed by immunohistochemistry, DNA microarrays, DNA sequencing, SNP arrays or fluorescence in situ hybridization. A panel of markers of epithelial-mesenchymal transition (EMT) was also correlated to the 29-gene signature score. RESULTS High 29-gene Endometrial Carcinoma Recurrence Score (ECARS) values consistently validated to identify patients with aggressive clinico-pathologic phenotype and reduced survival. Within the presumed favorable subgroups of low grade, endometrioid tumors confined to the uterus, high ECARS still predicted a poor prognosis. The score was higher in metastatic compared to primary lesions (P<0.001) and was significantly associated with potential measures of PI3K activation, markers of EMT and vascular invasion as an indicator of metastatic spread (all P<0.001). CONCLUSIONS ECARS validates to identify aggressive endometrial carcinomas in multiple, independent patients cohorts. The higher signature score in metastatic compared to primary lesions, and the potential link to PI3K activation and EMT, support further studies of ECARS in relation to response to PI3K and EMT inhibitors in clinical trials of metastatic endometrial carcinoma.

Radiotherapy in the management of localized primary cutaneous B-cell lymphoma

Abstract The optimal therapy and radiation dose for patients with localized primary cutaneous B-cell lymphoma (PCBCL) are unknown. We retrospectively identified 23 patients with localized (T1–T2) PCBCL treated with definitive radiation to doses ranging from 30 to 44 Gy (median, 36 Gy). With a median follow-up of 4.8 years, the 5-year overall survival rate was 100%, the relapse-free survival rate was 71% (95% confidence interval, 46–86%) and there were no local recurrences, suggesting that radiotherapy to a dose of 30 Gy may be sufficient for cure.

Chapter 21 – Radiation therapy in the management of pituitary adenomas

Radiation therapy in the form of fractionated treatment or radiosurgery has an important role in the management of pituitary adenomas. Radiation is a reliable way of gaining local control for radiographically progressing pituitary adenomas. For functioning adenomas that are biochemically recurrent or persistent, radiation therapy is less consistent in offering biochemical normalization and often requires a latency period of years or decades. The decision of when to use radiation therapy is a delicate balance between its benefits and late sequelae, especially in the context of benign disease. Recent technological advances in radiation oncology hold the potential to minimize dose to uninvolved normal tissue and therefore reduce the risk of toxicity.

The impact of PSA and digital rectal examination on the risk of prostate cancer specific mortality in men with a PSA level <2.5 ng/ml

INTRODUCTION It is unknown whether a normal range, diagnostic serum prostate specific antigen (PSA) levels influence on prostate cancer specific mortality (PCSM) is dependent upon digital rectal examination (DRE) findings. METHODS Between 2004 and 2007, 9081 men diagnosed with non-palpable (T1c, N=1710) or palpable (T2-T4, N=7371) and non-metastatic prostate cancer (PC) were identified from surveillance, epidemiology, and end results data, selected based on pre-treatment PSA<2.5 ng/ml. A multivariable competing risks regression model evaluated whether DRE findings interacted with PSA level in predicting risk of PCSM. RESULTS After median follow-up of 2.83 years, 118 of 548 deaths (21.5%) were due to PC. Increasing diagnostic PSA was associated with increased risk of PCSM (AHR=3.52; 95% CI: 1.25-9.89; P=.017) in men with T1c, Gleason score 7-10 PC, but decreased PCSM risk (AHR=0.66; 95% CI: 0.52-0.83; P<.001) for men with T2-T4 PC and any Gleason score. DISCUSSION For men with diagnostic PSA level <2.5 ng/ml and palpable PC, risk of early PCSM increases by 34% for a 1 point decrease in PSA from 2. This suggests the existence of clinically detectable, low PSA secreting disease with an elevated risk of early PCSM, highlighting the importance of the DRE in men with PC and normal range, diagnostic PSA.

Very low-dose versus standard dose radiation therapy for indolent primary cutaneous B-cell lymphomas: A retrospective study

Limitations include the fact that roughly 90% of US allopathic dermatologists are members of the American Academy of Dermatology; our results may underestimate provider counts. Our ideal dermatologist:population ratio of 3.5 per 100,000 people is somewhat arbitrary, given that an appropriate ratio has never been validated. Patients in rural, poor, and high-minority areas lack an acceptable level of access to dermatologists. Minority physicians are more likely to care for patients of their own race and practice in underserved areas. Sadly, only 3% and 4.1% of US dermatologists are African American and Latin American, respectively. Physicians with rural backgrounds and clinical experience are more likely to practice in those areas, as evidenced by Michigan State University’s Rural Physician Program. Initiatives to train more dermatologists from rural, poor, or high-minority areas, and methods to incentivize practice in these areas, such as loan forgiveness, should be considered. The AccessDerm teledermatology initiative has expanded access to dermatologic care by helping patients unable to receive in-person consultation. However, teledermatology alone without access to definitive treatments, including obtaining biopsy specimens, surgical services, and medication monitoring, will not adequately serve the needs of these populations. In addition, dermatologists often serve as advocates in their communities for skin health education. Residency-trained dermatologists are leaders of important public health initiativeswhen they are active participants in the communities they serve.