Synthetic Epigenetic Reprogramming of Mesenchymal to Epithelial States Using the CRISPR/dCas9 Platform in Triple Negative Breast Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor-outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9-mediated epigenetic editing, resulting in highly-specific and nearly complete suppression of ZEB1 in vivo, accompanied by long-lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) enabled the discovery of a ZEB1-dependent-signature of 26 genes differentially-expressed and -methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally-spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1-silencing are enriched in a subset of human breast tumors, illuminating a clinically-relevant hybrid-like state. Thus, the synthetic epi-silencing of ZEB1 induces stable "lock-in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.

Second generation anti-androgens and androgen deprivation therapy with radiation therapy in the definitive management of high-risk prostate cancer.

BACKGROUND: Evolving data suggest that men with high-risk localized prostate cancer may benefit from more potent androgen receptor inhibition in the context of curative intent radiotherapy. Recently updated American Society for Clinical Oncology (ASCO) evidence-based guidelines and the National Comprehensive Cancer Network (NCCN) Guidelines have updated recommendations for the consideration of adding second generation anti-androgens to androgen deprivation therapy (ADT) in men receiving radiation therapy (RT) for noncastrate locally advanced high and very high risk nonmetastatic or node positive prostate cancer. METHODS AND RESULTS: We conducted a comprehensive review of existing published and abstract presented evidence behind RT with ADT for the definitive management of high-risk prostate cancer, particularly focused on the current phase II and III trial evidence for the addition of second generation anti-androgens to ADT in definitive RT treatment of high-risk prostate cancer and specifically focused on the recent STAMPEDE trial results with abiraterone acetate. We review the biological mechanisms in which second generation anti-androgens may help mitigate ADT resistance and provide radiosensitization through inhibition of DNA repair. Finally, we discuss ongoing clinical trials of potent androgen receptor (AR) inhibitors with ADT in this non-metastatic high-risk radiotherapy setting that may inform on future treatment guidelines. CONCLUSIONS: Recent data suggest an overall survival benefit as well as increased probabilities of disease free and metastasis free survival in men with high and very high-risk localized, node positive, and oligometastatic hormone sensitive prostate cancer with abiraterone acetate and prednisone and support the use of potent AR inhibitors in this setting after informed decision making.

Prostate MRI in Stereotactic Body Radiation Treatment Planning and Delivery for Localized Prostate Cancer.

Prostate MRI is increasingly being used to make diagnoses and guide management for patients receiving definitive radiation treatment for prostate cancer. Radiologists should be familiar with the potential uses of prostate MRI in radiation therapy planning and delivery. Radiation therapy is an established option for the definitive treatment of localized prostate cancer. Stereotactic body radiation therapy (SBRT) is an external-beam radiation therapy method used to deliver a high dose of radiation to an extracranial target in the body, often in five or fewer fractions. SBRT is increasingly being used for prostate cancer treatment and has been recognized by the National Comprehensive Cancer Network as an acceptable definitive treatment regimen for low-, intermediate-, and high-risk prostate cancer. MRI is commonly used to aid in prostate radiation therapy. The authors review the uses of prostate MRI in SBRT treatment planning and delivery. Specific topics discussed include the use of prostate MRI for identification of and dose reduction to the membranous and prostatic urethra, which can decrease the risk of acute and late toxicities. MRI is also useful for identification and appropriate dose coverage of the prostate apex and areas of extraprostatic extension or seminal vesicle invasion. In prospective studies, prostate MRI is being validated for identification of and dose intensification to dominant intraprostatic lesions, which potentially can improve oncologic outcomes. It also can be used to evaluate the placement of fiducial markers and hydrogel spacers for radiation therapy planning and delivery. ©RSNA, 2022.

Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor.

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer.

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.

Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer.

Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers.

Peptides, proteins and nanotechnology: a promising synergy for breast cancer targeting and treatment.

INTRODUCTION: The use of nanoparticles for breast cancer targeting and treatment has become a reality. They are safe and possess interesting peculiarities such as the unspecific accumulation into the tumor site and the possibility to activate controlled drug release as compared to free drugs. However, there are still many areas of improvement which can certainly be addressed with the use of peptide-based elements. AREAS COVERED: The article reviews different preclinical strategies employing peptides and proteins in combination with nanoparticles for breast cancer targeting and treatment as well as peptide and protein-targeted encapsulated drugs, and it lists the current clinical status of therapies using peptides and proteins for breast cancer. EXPERT OPINION: The conjugation of protein and peptides can improve tumor homing of nanoparticles, increase cellular penetration and attack specific drivers and vulnerabilities of the breast cancer cell to promote tumor cytotoxicity while reducing secondary effects in healthy tissues. Examples are the use of antibodies, arginylglycylaspartic acid (RGD) peptides, membrane disruptive peptides, interference peptides, and peptide vaccines. Although their implementation in the clinic has been relatively slow up to now, we anticipate great progress in the field which will translate into more efficacious and selective nanotherapies for breast cancer.

Novel Coronavirus International Public Health Emergency: Guidance on Radiation Oncology Facility Operation.

PURPOSE: The current situation of coronavirus disease 2019 (COVID-19) is rapidly evolving. Radiation therapy facilities are places of concentrated patient interactions. Oncology patients with immunosuppression are at a higher risk for contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and easily developing severe postinfection events during the SARS-CoV-2 outbreaks. This review aimed to provide some guidance and be a reference to medical professionals in radiation oncology so that they may provide oncology patients with safe and high-quality care. METHODS AND MATERIALS: This paper discussed how radiation therapy departments or centers can most effectively respond to this public health emergency through summarizing the procedures and protocols implemented at hospitals in ShenZhen, China. RESULTS: The impact of the virus in radiation therapy facilities can be mitigated and managed with appropriate and timely implementation of infection control procedures and protocols. CONCLUSIONS: In the face of acute infectious disease, it is critical to maintain strict infectious disease control procedures and to create a clear clinical workflow protocol to best protect medical staff and patients from the effect of acute infectious diseases.

From Seabed to Bedside: A Review on Promising Marine Anticancer Compounds.

The marine environment represents an outstanding source of antitumoral compounds and, at the same time, remains highly unexplored. Organisms living in the sea synthesize a wide variety of chemicals used as defense mechanisms. Interestingly, a large number of these compounds exert excellent antitumoral properties and have been developed as promising anticancer drugs that have later been approved or are currently under validation in clinical trials. However, due to the high need for these compounds, new methodologies ensuring its sustainable supply are required. Also, optimization of marine bioactives is an important step for their success in the clinical setting. Such optimization involves chemical modifications to improve their half-life in circulation, potency and tumor selectivity. In this review, we outline the most promising marine bioactives that have been investigated in cancer models and/or tested in patients as anticancer agents. Moreover, we describe the current state of development of anticancer marine compounds and discuss their therapeutic limitations as well as different strategies used to overcome these limitations. The search for new marine antitumoral agents together with novel identification and chemical engineering approaches open the door for novel, more specific and efficient therapeutic agents for cancer treatment.

Sensitizing endometrial cancer to ionizing radiation by multi-tyrosine kinase inhibition.

OBJECTIVE: Endometrial carcinoma is the most frequent gynecological cancer. About 15% of these cancers are of high risk and radiotherapy still remains the most suitable treatment. In this context, agents able to promote radiosensitization are of great interest. Here, we describe for the first time the radiosensitization ability of sunitinib in endometrial carcinoma. METHODS: Four endometrial carcinoma cell lines were used for the study. The activation of apoptosis signalling pathways and tyrosine kinase receptors were analysed by Western blot, luciferase assays and Immunoprecipitation. Radiosensitization effects were assessed using clonogenic assays. p65 and phosphatase and tensin homolog (PTEN) were upregulated by lentiviral transduction. RESULTS: We discovered that ionizing radiation activates the pro-oncogenic proteins and signalling pathways KIT, protein kinase B (AKT), and nuclear factor kappa B (NF-κB) and these activations were abrogated by sunitinib, resulting in a radiosensitization effect. We found out that AKT pathway is greatly involved in this process as PTEN restoration in the PTEN-deficient cell line RL95-2 is sufficient to inhibit AKT, rendering these cells more susceptible to ionizing radiation and sunitinib-induced radiosensitization. In Ishikawa 3-H-12 cells, radiosensitization effects and inhibition of AKT were achieved by PTEN restoration plus treatment with the phosphoinositide-3-kinase inhibitor LY294002. This suggests that endometrial tumors could have different sensitivity degree to radiotherapy and susceptibility to sunitinib-induced radiosensitization depending on their AKT activation levels. CONCLUSIONS: Our results provide the rationale of using sunitinib as neoadjuvant treatment prior radiotherapy which could be a starting point for the implementation of sunitinib and radiotherapy in the clinic for the treatment of recalcitrant endometrial cancers.

Ten-Year Single Institutional Analysis of Geographic and Demographic Characteristics of Patients Treated With Stereotactic Body Radiation Therapy for Localized Prostate Cancer.

OBJECTIVES: Stereotactic Body Radiation Therapy (SBRT) offers definitive treatment for localized prostate cancer with comparable efficacy and toxicity to conventionally fractionated radiotherapy. Decreasing the number of treatment visits from over 40 to five may ease treatment burden and increase accessibility for logistically challenged patients. Travel distance is one factor that affects a patient's access to treatment and is often related to geographic location and socioeconomic status. In this study, we review the demographic and geographic factors of patients treated with SBRT for prostate cancer for a single institution with over a decade of experience. METHODS: Patient zip codes from one thousand and thirty-five patients were derived from a large, prospectively maintained quality of life database for patients treated for prostate cancer with SBRT from 2008 to 2017. The geospatial distance between the centroid of each zip code to our institution was calculated using the R package Geosphere. Characteristics for seven hundred and twenty-one patients were evaluated at the time of analysis including: race, age, and insurance status. To assess the geographic reach of our institution, we evaluated the demographic features of each zip code using US Census data. Statistical comparisons for these features and their relation to distance traveled for treatment was performed using the Mann-Whitney U test. Finally, an unsupervised learning algorithm was performed to identify distinct clusters of patients with respect to median income, racial makeup, educational level, and rural residency. RESULTS: Patients traveled from 246 distinct zip codes at a median distance of 11.35 miles. Forty percent of patients were African American, 6.9% resided in a rural region, and 22% were over the age of 75. Using K-means cluster analysis, four distinct patient zip-code groups were identified based on the aforementioned demographic features: Suburban/high-income (45%), Urban (30%), Suburban/low-income (17%), and Rural (8%). For each of the clusters, the average travel distance for SBRT was significantly different at 11.17, 9.26, 11.75, and 40.2 miles, respectively (p-value: <0.001). CONCLUSIONS: Distinct demographic features are related to travel distance for prostate SBRT. In our large cohort, travel distance did not prevent uptake of prostate SBRT in African American, elderly or rural patient populations. Prostate SBRT offers a diverse population modern treatment for their localized prostate cancer and particularly for those who live significant distances from a treatment center.

Rationale for Involved Field Stereotactic Body Radiation Therapy-Enhanced Intermittent Androgen Deprivation Therapy in Hormone-Sensitive Nodal Oligo-Recurrent Prostate Cancer Following Prostate Stereotactic Body Radiation Therapy.

Lymph node recurrent prostate cancer is a common clinical scenario that is likely to increase significantly with the widespread adoption of novel positron emission tomography (PET) agents. Despite increasing evidence that localized therapy is disease modifying, most men with lymph node recurrent prostate cancer receive only systemic therapy with androgen deprivation therapy (ADT). For men who receive localized therapy the intent is often to delay receipt of systemic therapy. Little evidence exists on the optimal combination of local and systemic therapy in this patient population. In this hypothesis generating review, we will outline the rationale and propose a framework for combining involved field SBRT with risk adapted intermittent ADT for hormone sensitive nodal recurrent prostate cancer. In patients with a limited number of nodal metastases, involved field stereotactic body radiation therapy (SBRT) may have a role in eliminating castrate-resistant clones and possibly prolonging the response to intermittent ADT. We hypothesize that in a small percentage of patients, such a treatment approach may lead to long term remission or cure.

Precision medicine by designer interference peptides: applications in oncology and molecular therapeutics.

In molecular cancer therapeutics only 10% of known cancer gene products are targetable with current pharmacological agents. Major oncogenic drivers, such as MYC and KRAS proteins are frequently highly overexpressed or mutated in multiple human malignancies. However, despite their key role in oncogenesis, these proteins are hard to target with traditional small molecule drugs due to their large, featureless protein interfaces and lack of deep pockets. In addition, they are inaccessible to large biologicals, which are unable to cross cell membranes. Designer interference peptides (iPeps) represent emerging pharmacological agents created to block selective interactions between protein partners that are difficult to target with conventional small molecule chemicals or with large biologicals. iPeps have demonstrated successful inhibition of multiple oncogenic drivers with some now entering clinical settings. However, the clinical translation of iPeps has been hampered by certain intrinsic limitations including intracellular localization, targeting tissue specificity and pharmacological potency. Herein, we outline recent advances for the selective inhibition of major cancer oncoproteins via iPep approaches and discuss the development of multimodal peptides to overcome limitations of the first generations of iPeps. Since many protein-protein interfaces are cell-type specific, this approach opens the door to novel programmable, precision medicine tools in cancer research and treatment for selective manipulation and reprogramming of the cancer cell oncoproteome.

Tumour suppression by targeted intravenous non-viral CRISPRa using dendritic polymers.

Aberrant gene expression is a hallmark of cancer. Although transcription is traditionally considered 'undruggable', the development of CRISPR-associated protein 9 (Cas9) systems offers enormous potential to rectify cancer-associated transcriptional abnormalities in malignant cells. However delivery of this technology presents a critical challenge to overcome in order to realize clinical translation for cancer therapy. In this article we demonstrate for the first time, a fully synthetic strategy to enable CRISPR-mediated activation (CRISPRa) of tumour suppressor genes in vivo using a targeted intravenous approach. We show this via highly efficient transcriptional activation of two model tumour suppressor genes, Mammary Serine Protease Inhibitor (MASPIN, SERPINB5) and cysteine-rich 61/connective tissue growth factor/nephroblastoma-overexpressed 6 (CCN6, WISP3), in a mouse model of breast cancer. In particular, we demonstrate that targeted intravenous delivery of can be achieved using a novel nanoscale dendritic macromolecular delivery agent, with negligible toxicity and long lasting therapeutic effects, outlining a targeted effective formulation with potential to treat aggressive malignancies.

Triple-hit therapeutic approach for triple negative breast cancers using docetaxel nanoparticles, EN1-iPeps and RGD peptides.

Triple negative breast cancers (TNBC) are aggressive malignancies for which chemotherapy is the only treatment option. Many TNBC acquire chemotherapy resistance, notably docetaxel, which has been associated with the overexpression of transcription factors (TFs), such as ENGRAILED1 (EN1). Here, we have developed a tumor delivery system for docetaxel-PGMA-PAA-nanoparticles and interference peptides designed to specifically inhibit EN1 (EN1-iPeps). To promote tumor specific targeting, we functionalized these nanoparticles with EN1-iPeps engineered with RGD sequences. We found that these peptides reduce cell viability and induce apoptosis in TNBC cells with negligible effects on normal cells (EN1-). Moreover, EN1-RGD-iPeps-mediated nanoparticle internalization into breast cancer cells was via integrins and intravenous injection of this nanoformulation increased tumor accumulation. Furthermore, docetaxel nanoparticles functionalized with EN1-RGD-iPeps significantly reduced TNBC growth both in vitro and in vivo without showing toxicity. Our results suggest that this targeted nanoformulation represents a new and safe therapeutic approach for chemoresistant TNBCs.

Tumor penetrating peptides inhibiting MYC as a potent targeted therapeutic strategy for triple-negative breast cancers.

Overexpression of MYC oncogene is highly prevalent in many malignancies such as aggressive triple-negative breast cancers (TNBCs) and it is associated with very poor outcome. Despite decades of research, attempts to effectively inhibit MYC, particularly with small molecules, still remain challenging due to the featureless nature of its protein structure. Herein, we describe the engineering of the dominant-negative MYC peptide (OmoMYC) linked to a functional penetrating 'Phylomer' peptide (FPPa) as a therapeutic strategy to inhibit MYC in TNBC. We found FPPa-OmoMYC to be a potent inducer of apoptosis (with IC50 from 1-2 µM) in TNBC cells with negligible effects in non-tumorigenic cells. Transcriptome analysis of FPPa-OmoMYC-treated cells indicated that the fusion protein inhibited MYC-dependent networks, inducing dynamic changes in transcriptional, metabolic, and apoptotic processes. We demonstrated the efficacy of FPPa-OmoMYC in inhibiting breast cancer growth when injected orthotopically in TNBC allografts. Lastly, we identified strong pharmacological synergisms between FPPa-OmoMYC and chemotherapeutic agents. This study highlights a novel therapeutic approach to target highly aggressive and chemoresistant MYC-activated cancers.

Mesenchymal stem cells support growth and organization of host-liver colorectal-tumor organoids and possibly resistance to chemotherapy.

Despite having yielded extensive breakthroughs in cancer research, traditional 2D cell cultures have limitations in studying cancer progression and metastasis and screening therapeutic candidates. 3D systems can allow cells to grow, migrate, and interact with each other and the surrounding matrix, resulting in more realistic constructs. Furthermore, interactions between host tissue and developing tumors influence the susceptibility of tumors to drug treatments. Host-liver colorectal-tumor spheroids composed of primary human hepatocytes, mesenchymal stem cells (MSC) and colon carcinoma HCT116 cells were created in simulated microgravity rotating wall vessel (RWV) bioreactors. The cells were seeded on hyaluronic acid-based microcarriers, loaded with liver-specific growth factors and ECM components. Only in the presence of MSC, large tumor foci rapidly formed inside the spheroids and increased in size steadily over time, while not greatly impacting albumin secretion from hepatocytes. The presence of MSC appeared to drive self-organization and formation of a stroma-like tissue surrounding the tumor foci and hepatocytes. Exposure to a commonly used chemotherapeutic 5-FU showed a dose-dependent cytotoxicity. However, if tumor organoids were allowed to mature in the RWV, they were less sensitive to the drug treatment. These data demonstrate the potential utility of liver tumor organoids for cancer progression and drug response modeling.

Leptomeningeal failure in patients with breast cancer receiving stereotactic radiosurgery for brain metastases.

PURPOSE: Prior studies suggest a high incidence of leptomeningeal failure (LMF) in breast cancer metastatic to brain. This study examines breast cancer-specific variables affecting development of LMF and survival after Gamma-Knife Radiosurgery (GKS). METHODS: Between 2000-2010, 149 (breast) and 658 other-histology patients were treated with GKS. Hormone/HER2, age, local/distant brain failure, prior craniotomy, and prior whole-brain radiotherapy (WBRT) were assessed. Median follow-up was 54months (range, 0-106). Serial MRI determined local and distant-brain failure and LMF. Statistical analysis with categorical/continuous data comparisons were done with Fisher's-exact, Wilcoxon rank-sum, log-rank tests, and Cox-Proportional Hazard models. RESULTS: Of 149 patients, 21 (14%) developed LMF (median time of 11.9months). None of the following predicted for LMF: Her2-status (HR=0.49, p=0.16), hormone-receptor status (HR=1.15, p=0.79), prior craniotomy (HR=1.58, p=0.42), prior WBRT (HR=1.36, p=0.55). Non-significant factors between patients that did (n=21) and did not (n=106) develop LMF included neurologic death (p=0.34) and median survival (8.6 vs 14.2months, respectively). Breast patients who had distant-failure after GKS (65/149; 43.6%) were more likely to later develop LMF (HR 4.2, p=0.005); including 15/65 (23%) patients who had distant-failure and developed LMF. Median time-to-death for patients experiencing LMF was 6.1months (IQR 3.4-7.8) from onset of LMF. Median survival from LMF to death was much longer in breast (6.1months) than in other (1.7months) histologies CONCLUSION: Breast cancer patients had a longer survival after diagnosis of LMF versus other histologies. Neither ER/PR/HER2 status, nor prior surgery or prior WBRT predicted for development of LMF in breast patients.