Lack of TGFß signaling competency predicts immune poor cancer conversion to immune rich and response to checkpoint blockade.

Background: Transforming growth factor beta (TGFß) is well-recognized as an immunosuppressive player in the tumor microenvironment but also has a significant impact on cancer cell phenotypes. Loss of TGFß signaling impairs DNA repair competency, which is described by a transcriptomic score, ßAlt. Cancers with high ßAlt have more genomic damage and are more responsive to genotoxic therapy. The growing appreciation that cancer DNA repair deficits are important determinants of immune response prompted us to investigate the association of ßAlt with response to immune checkpoint blockade (ICB). We predicted that high ßAlt tumors would be infiltrated with lymphocytes because of DNA damage burden and hence responsive to ICB. Methods: We analyzed public transcriptomic data from clinical trials and preclinical models using transcriptomic signatures of TGFß targets, DNA repair genes, tumor educated immune cells and interferon. A high ßAlt, immune poor mammary tumor derived transplant model resistant to programmed death ligand 1 (PD-L1) antibodies was studied using multispectral flow cytometry to interrogate the immune system. Results: Metastatic bladder patients in IMvigor 210 who responded to ICB had significantly increased ßAlt scores and experienced significantly longer overall survival compared to those with low ßAlt scores (hazard ratio 0.62, P=0.011) . Unexpectedly, 75% of high ßAlt cancers were immune poor as defined by low expression of tumor educated immune cell and interferon signatures. The association of high ßAlt with immune poor cancer was also evident in TCGA and preclinical cancer models. We used a high ßAlt, immune poor cancer to test therapeutic strategies to overcome its inherent anti-PD-L1 resistance. Combination treatment with radiation and TGFß inhibition were necessary for lymphocytic infiltration and activated NK cells were required for ICB response. Bioinformatic analysis identified high ßAlt, immune poor B16 and CT26 preclinical models and paired biopsies of cancer patients that also demonstrated NK cell activation upon response to ICB. Conclusions: Our studies support ßAlt as a biomarker that predicts response to ICB albeit in immune poor cancers, which has implications for the development of therapeutic strategies to increase the number of cancer patients who will benefit from immunotherapy.

PLX038A, a long-acting SN-38, penetrates the blood-tumor-brain-barrier, accumulates and releases SN-38 in brain tumors to increase survival of tumor bearing mice.

Central nervous system tumors have resisted effective chemotherapy because most therapeutics do not penetrate the blood-tumor-brain-barrier. Nanomedicines between ~ 10 and 100 nm accumulate in many solid tumors by the enhanced permeability and retention effect, but it is controversial whether the effect can be exploited for treatment of brain tumors. PLX038A is a long-acting prodrug of the topoisomerase 1 inhibitor SN-38. It is composed of a 15 nm 4-arm 40 kDa PEG tethered to four SN-38 moieties by linkers that slowly cleave to release the SN-38. The prodrug was remarkably effective at suppressing growth of intracranial breast cancer and glioblastoma (GBM), significantly increasing the life span of mice harboring them. We addressed the important issue of whether the prodrug releases SN-38 systemically and then penetrates the brain to exert anti-tumor effects, or whether it directly penetrates the blood-tumor-brain-barrier and releases the SN-38 cargo within the tumor. We argue that the amount of SN-38 formed systemically is insufficient to inhibit the tumors, and show by PET imaging that a close surrogate of the 40 kDa PEG carrier in PLX038A accumulates and is retained in the GBM. We conclude that the prodrug penetrates the blood-tumor-brain-barrier, accumulates in the tumor microenvironment and releases its SN-38 cargo from within. Based on our results, we pose the provocative question as to whether the 40 kDa nanomolecule PEG carrier might serve as a "Trojan horse" to carry other drugs past the blood-tumor-brain-barrier and release them into brain tumors.

In situ analyses of genome instability in breast cancer.

Transition through telomere crisis is thought to be a crucial event in the development of most breast carcinomas. Our goal in this study was to determine where this occurs in the context of histologically defined breast cancer progression. To this end, we assessed genome instability (using fluorescence in situ hybridization) and other features associated with telomere crisis in normal ductal epithelium, usual ductal hyperplasia, ductal carcinoma in situ and invasive cancer. We modeled this process in vitro by measuring these same features in human mammary epithelial cell cultures during ZNF217-mediated transition through telomere crisis and immortalization. Taken together, the data suggest that transition through telomere crisis and immortalization in breast cancer occurs during progression from usual ductal hyperplasia to ductal carcinoma in situ.

Monitoring TGFß signaling in irradiated tumors.

Transforming growth factor ß (TGFß) is exquisitely regulated under physiological conditions but its activity is highly dysregulated in cancer. All cells make TGFß and have receptors for the ligand, which is sequestered in the extracellular matrix in a latent form. Ionizing radiation elicits rapid release of TGFß from these stores, so-called activation, over a wide range of doses and exposures, including low dose (<1Gy) whole-body irradiation, creating an extraordinarily potent signal in the irradiated tissue or tumor. Hence, accurate evaluation of TGFß activity is complicated because of its ubiquitous distribution as a latent complex. Here we describe conditions for assays that reveal TGFß activity in situ using either tissue preparations or functional imaging.

Inflammation Mediates the Development of Aggressive Breast Cancer Following Radiotherapy.

PURPOSE: Women treated with radiotherapy before 30 years of age have increased risk of developing breast cancer at an early age. Here, we sought to investigate mechanisms by which radiation promotes aggressive cancer. EXPERIMENTAL DESIGN: The tumor microenvironment (TME) of breast cancers arising in women treated with radiotherapy for Hodgkin lymphoma was compared with that of sporadic breast cancers. To investigate radiation effects on carcinogenesis, we analyzed tumors arising from Trp53-null mammary transplants after irradiation of the target epithelium or host using immunocompetent and incompetent mice, some of which were treated with aspirin. RESULTS: Compared with age-matched specimens of sporadic breast cancer, radiation-preceded breast cancers (RP-BC) were characterized by TME rich in TGFß, cyclooxygenase 2, and myeloid cells, indicative of greater immunosuppression, even when matched for triple-negative status. The mechanism by which radiation impacts TME construction was investigated in carcinomas arising in mice bearing Trp53-null mammary transplants. Immunosuppressive TMEs (iTME) were recapitulated in mice irradiated before transplantation, which implicated systemic immune effects. In nu/nu mice lacking adaptive immunity irradiated before Trp53-null mammary transplantation, cancers also established an iTME, which pointed to a critical role for myeloid cells. Consistent with this, irradiated mammary glands contained more macrophages and human cells cocultured with polarized macrophages underwent dysplastic morphogenesis mediated by IFNγ. Treating mice with low-dose aspirin for 6 months postirradiation prevented establishment of an iTME and resulted in less aggressive tumors. CONCLUSIONS: These data show that radiation acts via nonmutational mechanisms to promote markedly immunosuppressive features of aggressive, RP-BCs.

Positron Emission Tomography Imaging of Functional Transforming Growth Factor ß (TGFß) Activity and Benefit of TGFß Inhibition in Irradiated Intracranial Tumors.

PURPOSE: Transforming growth factor ß (TGFß) promotes cell survival by endorsing DNA damage repair and mediates an immunosuppressive tumor microenvironment. Thus, TGFß activation in response to radiation therapy is potentially targetable because it opposes therapeutic control. Strategies to assess this potential in the clinic are needed. METHODS AND MATERIALS: We evaluated positron emission tomography (PET) to image 89Zr -fresolimumab, a humanized TGFß neutralizing monoclonal antibody, as a means to detect TGFß activation in intracranial tumor models. Pathway activity of TGFß was validated by immunodetection of phosphorylated SMAD2 and the TGFß target, tenascin. The contribution of TGFß to radiation response was assessed by Kaplan-Meier survival analysis of mice bearing intracranial murine tumor models GL261 and SB28 glioblastoma and brain-adapted 4T1 breast cancer (4T1-BrA) treated with TGFß neutralizing monoclonal antibody, 1D11, and/or focal radiation (10 Gy). RESULTS: 89Zr-fresolimumab PET imaging detected engineered, physiological, and radiation-induced TGFß activation, which was confirmed by immunostaining of biological markers. GL261 glioblastoma tumors had a greater PET signal compared with similar-sized SB28 glioblastoma tumors, whereas the widespread PET signal of 4T1-BrA intracranial tumors was consistent with their highly dispersed histologic distribution. Survival of mice bearing intracranial tumors treated with 1D11 neutralizing antibody alone was similar to that of mice treated with control antibody, whereas 1D11 improved survival when given in combination with focal radiation. The extent of survival benefit of a combination of radiation and 1D11 was associated with the degree of TGFß activity detected by PET. CONCLUSIONS: This study demonstrates that 89Zr-fresolimumab PET imaging detects radiation-induced TGFß activation in tumors. Functional imaging indicated a range of TGFß activity in intracranial tumors, but TGFß blockade provided survival benefit only in the context of radiation treatment. This study provides further evidence that radiation-induced TGFß activity opposes therapeutic response to radiation.

From Mouse to Human: Cellular Morphometric Subtype Learned From Mouse Mammary Tumors Provides Prognostic Value in Human Breast Cancer.

Mouse models of cancer provide a powerful tool for investigating all aspects of cancer biology. In this study, we used our recently developed machine learning approach to identify the cellular morphometric biomarkers (CMB) from digital images of hematoxylin and eosin (H&E) micrographs of orthotopic Trp53-null mammary tumors (n = 154) and to discover the corresponding cellular morphometric subtypes (CMS). Of the two CMS identified, CMS-2 was significantly associated with shorter survival (p = 0.0084). We then evaluated the learned CMB and corresponding CMS model in MMTV-Erbb2 transgenic mouse mammary tumors (n = 53) in which CMS-2 was significantly correlated with the presence of metastasis (p = 0.004). We next evaluated the mouse CMB and CMS model on The Cancer Genome Atlas breast cancer (TCGA-BRCA) cohort (n = 1017). Kaplan-Meier analysis showed significantly shorter overall survival (OS) of CMS-2 patients compared to CMS-1 patients (p = 0.024) and added significant prognostic value in multi-variable analysis of clinical and molecular factors, namely, age, pathological stage, and PAM50 molecular subtype. Thus, application of CMS to digital images of routine workflow H&E preparations can provide unbiased biological stratification to inform patient care.

Aggressive Mammary Cancers Lacking Lymphocytic Infiltration Arise in Irradiated Mice and Can Be Prevented by Dietary Intervention.

Because the incidence of breast cancer increases decades after ionizing radiation exposure, aging has been implicated in the evolution of the tumor microenvironment and tumor progression. Here, we investigated radiation-induced carcinogenesis using a model in which the mammary glands of 10-month-old BALB/c mice were transplanted with Trp53-null mammary tissue 3 days after exposure to low doses of sparsely ionizing γ-radiation or densely ionizing particle radiation. Mammary transplants in aged, irradiated hosts gave rise to significantly more tumors that grew more rapidly than those in sham-irradiated mice, with the most pronounced effects seen in mice irradiated with densely ionizing particle radiation. Tumor transcriptomes identified a characteristic immune signature of these aggressive cancers. Consistent with this, fast-growing tumors exhibited an immunosuppressive tumor microenvironment with few infiltrating lymphocytes, abundant immunosuppressive myeloid cells, and high COX-2 and TGFß. Only irradiated hosts gave rise to tumors lacking cytotoxic CD8+ lymphocytes (defined here as immune desert), which also occurred in younger irradiated hosts. These data suggest that host irradiation may promote immunosuppression. To test this, young chimera mice were fed chow containing a honeybee-derived compound with anti-inflammatory and immunomodulatory properties, caffeic acid phenethyl ester (CAPE). CAPE prevented the detrimental effects of host irradiation on tumor growth rate, immune signature, and immunosuppression. These data indicated that low-dose radiation, particularly densely ionizing exposure of aged mice, promoted more aggressive cancers by suppressing antitumor immunity. Dietary intervention with a nontoxic immunomodulatory agent could prevent systemic effects of radiation that fuel carcinogenesis, supporting the potential of this strategy for cancer prevention.

Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition.

Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

Access to primary care for Medicare beneficiaries.

OBJECTIVES: To determine the acceptance rate of new Medicare patients by all primary care physicians. Among primary care physicians accepting new patients, to determine whether demographic and geographic factors are associated with the likelihood of accepting new Medicare patients. DESIGN: Cross-sectional. SETTING: Primary care physicians drawn from a national sample. PARTICIPANTS: Eight hundred forty-eight primary care physicians. MEASUREMENTS: Percentage of physicians accepting, not accepting, or conditionally accepting new Medicare patients. RESULTS: Of the 848 primary care physicians contacted, only 58% unconditionally accepted all new Medicare patients; 20% accepted new patients but restricted new Medicare patients using policies of nonacceptance or conditional acceptance. Of the 665 physicians accepting new patients, those in the south and west were more likely not to accept new Medicare patients than those in the northeast, with multivariable odds ratios (ORs) of 2.79 (95% confidence interval (CI)=1.34-5.78) and 3.14 (95% CI=1.35-7.33), respectively. Similarly, family physicians were more likely than internists not to accept new Medicare patients (OR=2.36, 95% CI=1.39-3.99). Primary care physicians in the Midwest were more likely to conditionally accept new Medicare patients than those in the northeast (OR=4.84, 95% CI=1.32-17.76), and primary care physicians in metropolitan areas with a population less than 100,000 were more likely to conditionally accept new Medicare patients than those in areas with a population greater than 100,000 (OR=2.39, 95% CI=1.18-4.84). CONCLUSION: Medicare beneficiaries' access to primary care is limited and varies significantly by region, population size, and type of provider.

Perceptions of physicians on the barriers and facilitators to integrating fall risk evaluation and management into practice.

BACKGROUND: Falls are common, treatable, and result in considerable morbidity in older adults. However, fall risk factor evaluation and management targeted at high-risk patients is largely neglected in clinical practice. OBJECTIVE: To identify barriers and facilitators to the implementation of fall risk management by primary care providers. DESIGN: Qualitative study using a semi-structured interview. PARTICIPANTS: Primary care providers who received an academic outreach visit. APPROACH: Self-reported facilitators and barriers to evaluating and managing fall risk in older patients. RESULTS: Physician factors, logistical factors, and patient factors intersect to either facilitate or impede fall risk evaluation and management by primary care providers. Physician factors include awareness, competing risks, appropriateness of referrals, training, and tie-in to familiar activities. Logistical factors include availability of transportation, time requirements of immobile patients, reimbursement, scheduling, family involvement, and utilization of other health care providers. Physicians' perceptions of patient factors include reporting, attitudes toward medication, and positive feedback. CONCLUSION: Strategies to improve the adoption of fall risk evaluation and management in primary care should address the specific physician, logistical, and patient barriers perceived by physicians who had received an informative, motivational intervention to assess and manage falls among their patients.

Lack of radiation dose or quality dependence of epithelial-to-mesenchymal transition (EMT) mediated by transforming growth factor ß.

PURPOSE: Epithelial-to-mesenchymal transition (EMT) is a phenotype that alters cell morphology, disrupts morphogenesis, and increases motility. Our prior studies have shown that the progeny of human mammary epithelial cells (HMECs) irradiated with 2 Gy undergoes transforming growth factor ß (TGF-ß)-mediated EMT. In this study we determined whether radiation dose or quality affected TGF-ß-mediated EMT. METHODS AND MATERIALS: HMECs were cultured on tissue culture plastic or in Matrigel (BD Biosciences, San Jose, CA) and exposed to low or high linear energy transfer (LET) and TGF-ß (400 pg/mL). Image analysis was used to measure membrane-associated E-cadherin, a marker of functional epithelia, or fibronectin, a product of mesenchymal cells, as a function of radiation dose and quality. RESULTS: E-cadherin was reduced in TGF-ß-treated cells irradiated with low-LET radiation doses between 0.03 and 2 Gy compared with untreated, unirradiated cells or TGF-ß treatment alone. The radiation quality dependence of TGF-ß-mediated EMT was determined by use of 1 GeV/amu (gigaelectron volt/atomic mass unit) (56)Fe ion particles at the National Aeronautics and Space Administration's Space Radiation Laboratory. On the basis of the relative biological effectiveness of 2 for (56)Fe ion particles' clonogenic survival, TGF-ß-treated HMECs were irradiated with equitoxic 1-Gy (56)Fe ion or 2-Gy (137)Cs radiation in monolayer. Furthermore, TGF-ß-treated HMECs irradiated with either high- or low-LET radiation exhibited similar loss of E-cadherin and gain of fibronectin and resulted in similar large, poorly organized colonies when embedded in Matrigel. Moreover, the progeny of HMECs exposed to different fluences of (56)Fe ion underwent TGF-ß-mediated EMT even when only one-third of the cells were directly traversed by the particle. CONCLUSIONS: Thus TGF-ß-mediated EMT, like other non-targeted radiation effects, is neither radiation dose nor quality dependent at the doses examined.

DNA polyplexes formed using PEGylated biodegradable hyperbranched polymers.

A novel PEGylated biodegradable hyperbranched PEG-b-PDMAEMA has been synthesized. The low toxicity, small molecular weight PDMAEMA chains were crosslinked using a biodegradable disulfide-based dimethacrylate (DSDMA) agent to yield higher molecular weight hyperbranched polymers. PEG chains were linked onto the polymer surface, masking the positive charge (as shown by Zeta potential measurements) and reducing the toxicity of the polymer. The hyperbranched structures were also cleaved under reducing conditions and analyzed, confirming the expected component structures. The hyperbranched polymer was mixed with DNA and efficient binding was shown to occur through electrostatic interactions. The hyperbranched structures could be reduced easily, generating lower toxicity oligomer chains.

Intensity-modulated radiation therapy in head and neck cancers: dosimetric advantages and update of clinical results.

Intensity-modulated radiation therapy (IMRT) is an exciting new modality in radiation therapy. The head and neck region is an ideal target for this new technology for several reasons. First, IMRT offers the potential for improved tumor control through delivery of high doses to the target volume. Second, because of sharp dose gradients, IMRT results in the relative sparing of normal structures in the head and neck region. Third, organ motion is virtually absent in the head and neck region, so, with proper immobilization, treatment can be delivered accurately. Although this is a relatively new technology, preliminary studies show excellent dosimetric profiles and clinical results. Salivary gland sparing has also resulted in reduced incidence and severity of xerostomia. Early reports of improvement in tumor control with better side effect profiles versus conventional techniques are promising, but will need to be confirmed with longer follow-up.

Intensity-modulated radiation therapy for head and neck cancer.

Radiotherapy planning studies have confirmed dosimetric advantages of intensity-modulated radiation therapy over conventional and conformal radiation therapy. Utilization of intensity-modulated radiation therapy is ideal in head and neck cancer patients. Critical structures can be spared due to sharp dose gradients and limited organ motion with correct immobilization. Initial clinical results have shown excellent locoregional control, in part due to the delivery of high doses to the target volume. Reductions in acute toxicities and xerostomia through parotid sparing have been notable benefits. However, long-term outcomes with regards to local control and late toxicities with intensity-modulated radiation therapy are still lacking. This review focuses on the implementation of intensity-modulated radiation therapy for the treatment of head and neck cancers, with a specific focus on set-up uncertainties, dose prescription and target volume determination and delineation.