Connecting blood and intratumoral Treg cell activity in predicting future relapse in breast cancer.

Regulatory T (Treg) cells play a major role in the development of an immunosuppressive tumor microenvironment. The origin of intratumoral Treg cells and their relationship with peripheral blood Treg cells remain unclear. Treg cells consist of at least three functionally distinct subpopulations. Here we show that peripheral blood CD45RA-FOXP3hi Treg cells (Treg II cells) are phenotypically closest to intratumoral Treg cells, including in their expression of CCR8. Analyses of T cell antigen receptor repertoires further support the hypothesis that intratumoral Treg cells may originate primarily from peripheral blood Treg II cells. Moreover, the signaling responsiveness of peripheral blood Treg II cells to immunosuppressive, T helper type 1 (TH1) and T helper type 2 (TH2) cytokines reflects intratumoral immunosuppressive potential, and predicts future relapse in two independent cohorts of patients with breast cancer. Together, our findings give important insights into the relationship between peripheral blood Treg cells and intratumoral Treg cells, and highlight cytokine signaling responsiveness as a key determinant of intratumoral immunosuppressive potential and clinical outcome.

Phase II Trial of Neoadjuvant Carboplatin and Nab-Paclitaxel in Patients with Triple-Negative Breast Cancer.

BACKGROUND: In this phase II clinical trial, we evaluated the efficacy of the nonanthracycline combination of carboplatin and nab-paclitaxel in early stage triple-negative breast cancer (TNBC). PATIENTS AND METHODS: Patients with newly diagnosed stage II-III TNBC (n = 69) were treated with neoadjuvant carboplatin (area under the curve 6) every 28 days for four cycles plus nab-paclitaxel (100 mg/m2 ) weekly for 16 weeks. Pathological complete response (pCR) and residual cancer burden (RCB) were analyzed with germline mutation status, tumor-infiltrating lymphocytes (TILs), TNBC molecular subtype, and GeparSixto immune signature (GSIS). RESULTS: Sixty-seven patients were evaluable for safety and response. Fifty-three (79%) patients experienced grade 3/4 adverse events, including grade 3 anemia (43%), neutropenia (39%), leukopenia (15%), thrombocytopenia (12%), fatigue (7%), peripheral neuropathy (7%), neutropenia (16%), and leukopenia (1%). Twenty-four patients (35%) had at least one dose delay, and 50 patients (72%) required dose reduction. Sixty-three (94%) patients completed scheduled treatment. The responses were as follows: 32 of 67 patients (48%) had pCR (RCB 0), 10 of 67 (15%) had RCB I, 19 of 67 (28%) had RCB II, 5 of 67 (7%) had RCB III, and 1 of 67 (2%) progressed and had no surgery. Univariate analysis showed that immune-hot GSIS and DNA repair defect (DRD) were associated with higher pCR with odds ratios of 4.62 (p = .005) and 4.76 (p = .03), respectively, and with RCB 0/I versus RCB II/III with odds ratio 4.80 (p = .01). Immune-hot GSIS was highly correlated with DRD status (p = .03), TIL level (p < .001), and TNBC molecular subtype (p < .001). After adjusting for age, race, stage, and grade, GSIS remained associated with higher pCR and RCB class 0/I versus II/III with odds ratios 7.19 (95% confidence interval [CI], 2.01-25.68; p = .002) and 8.95 (95% CI, 2.09-38.23; p = .003), respectively. CONCLUSION: The combination of carboplatin and nab-paclitaxel for early stage high-risk TNBC showed manageable toxicity and encouraging antitumor activity. Immune-hot GSIS is associated with higher pCR rate and RCB class 0/1. This study provides an additional rationale for using nonanthracycline platinum-based therapy for future neoadjuvant trials in early stage TNBCs. Clinical trial identification number: NCT01525966 IMPLICATIONS FOR PRACTICE: Platinum is an important neoadjuvant chemotherapy agent for treatment of early stage triple-negative breast cancer (TNBC). In this study, carboplatin and nab-paclitaxel were well tolerated and highly effective in TNBC, resulting in pathological complete response of 48%. In univariate and multivariate analyses adjusting for age, race, tumor stage and grade, "immune-hot" GeparSixto immune signature (GSIS) and DNA repair defect (DRD) were associated with higher pathological complete response (pCR) and residual cancer burden class 0/1. The association of immune-hot GSIS with higher pCR holds promise for de-escalating neoadjuvant chemotherapy for patients with early stage TNBC. Although GSIS is not routinely used in clinic, further development of this immune signature into a clinically applicable assay is indicated.

Insurance Status Predicts Survival in Women with Breast Cancer: Results of Breast and Cervical Cancer Treatment Program in California.

BACKGROUND AND PURPOSE: The Breast and Cervical Cancer Treatment Program (BCCTP) Act, passed by Congress in 2000, provides time-limited coverage to uninsured breast or cervical cancer patients. We examine survival differences between BCCTP cases and insured controls. METHODS: Stage I-III breast cancer patients, covered under California's BCCTP from 2005 to 2009 (N = 6343), were 1:1 matched with California Cancer Registry controls on age, race/ethnicity, and cancer stage. Overall and disease-specific (OS and DSS) survival were compared using multivariate regression. RESULTS: BCCTP cases were more often unmarried [odds ratio (OR) 2.47, 95% confidence interval (CI) 2.30-2.66], with poorly/undifferentiated tumors (OR 1.26, CI 1.13-1.40), classified as ER negative (OR 1.10, CI 1.02-1.20) and/or PR negative (OR 1.09, CI 1.01-1.17). Cases were more likely to undergo mastectomy (OR 1.13, CI 1.05-1.21) or no surgery (OR 1.64, CI 1.31-2.05) versus lumpectomy. Cases were also more likely to undergo radiation (OR 1.11, CI 1.03-1.19). Endocrine therapy rates were marginally lower in cases (OR 0.93, CI 0.86-1.00). OS and DSS were shorter in BCCTP cases on multivariate analysis (HR 1.29, CI 1.17-1.42 and HR 1.27, CI 1.14-1.42, respectively). When stratified by socioeconomic status (SES), cases had significantly shorter OS and DSS except in the lowest quintile. When stratified by stage, cases had significantly shorter OS and DSS, except for stage I. CONCLUSIONS: The BCCTP provides uninsured breast cancer patients with comprehensive and timely care. Although our results suggest that BCCTP delivers quality care, BCCTP patients have shorter survival rates, even after accounting for SES and stage differences. Further assistance to vulnerable populations is warranted, including longer duration of treatment coverage, and surveillance adhering to NCCN compliant surveillance programs.

Phase I clinical trial of the combination of eribulin and everolimus in patients with metastatic triple-negative breast cancer.

BACKGROUND: Alteration of the PI3K/AKT/mTOR pathway is a common genomic abnormality detected in triple-negative breast cancer (TNBC). Everolimus acts synergistically with eribulin in TNBC cell lines and xenograft models. This phase I trial was designed to test the safety and tolerability of combining eribulin and everolimus in patients with metastatic TNBC. METHODS: The primary objective of this study was to evaluate the safety and toxicities of the combination. Patients with metastatic TNBC who had up to four lines of prior chemotherapies were enrolled. The combination of eribulin and everolimus was tested using three dosing levels: A1 (everolimus 5 mg daily; eribulin 1.4 mg/m2 days 1 and 8 every 3 weeks), A2 (everolimus 7.5 mg daily; eribulin 1.4 mg/m2, days 1 and 8 every 3 weeks), and B1 (everolimus 5 mg daily; eribulin 1.1 mg/m2 days 1 and 8 every 3 weeks). RESULTS: Twenty-seven patients with median age 55 years were enrolled. Among 8 evaluable patients who received dose level A1, 4 had dose-limiting toxicities (DLTs). Among 3 evaluable patients treated with dose level A2, 2 had DLTs. Among 12 evaluable patients who received dose level B1, 4 had DLTs. The DLTs were neutropenia, stomatitis, and hyperglycemia. Over the study period, 59% had a ≥ grade 3 toxicity, 44% had ≥ grade 3 hematologic toxicities, and 22% had grade 4 hematologic toxicities. The most common hematological toxicities were neutropenia, leukopenia, and lymphopenia. Thirty-three percent had grade 3 non-hematologic toxicities. The most common non-hematological toxicities were stomatitis, hyperglycemia, and fatigue. The median number of cycles completed was 4 (range 0-8). Among 25 eligible patients, 9 patients (36%) achieved the best response as partial response, 9 (36%) had stable disease, and 7 (28%) had progression. The median time to progression was 2.6 months (95% CI [2.1, 4.0]), and median overall survival (OS) was 8.3 months (95% CI [5.5, undefined]). CONCLUSION: Eribulin 1.1 mg/m2 days 1 and 8 every 3 weeks with everolimus 5 mg daily was defined as the highest dose with acceptable toxicity (RP2D). The combination is safe, and efficacy is modest. A post hoc analysis showed that participants that used dexamethasone mouthwash stayed on treatment for one additional cycle. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02120469. Registered 18 April 2014.

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR.

Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.

Age greater than 60 years portends a worse prognosis in patients with papillary thyroid cancer: should there be three age categories for staging?

BACKGROUND: Age is an important prognostic factor in papillary thyroid cancer (PTC), with better survival observed in patients < 45 years of age, regardless of stage. Although the impact of increasing age on PTC-related survival is well-known, previous studies have focused on survival relative to age 45 years only. As the number of patients entering their 7th decade of life increases, PTC-related survival in this demographic becomes increasingly important. Survival in patients ≥ 60 years specifically compared to other groups has not previously been examined. We sought to determine whether age ≥ 60 years is an adverse prognostic factor for disease-specific survival and recurrence in patients with PTC. METHODS: The California Cancer Registry database was linked to inpatient and ambulatory patient records from the Office of Statewide Health Planning and Development for the years 2000-2011. This linked database was queried for patients diagnosed with papillary thyroid cancer and treated with surgery. We then identified prognostic factors related to both 5-year and 10-year disease-specific survival and disease-free survival in patients ≤ 45, 45-59, and ≥ 60 years. Multivariable Cox proportional hazard models were created to test the effect of age ≥ 60 on disease-specific and disease-free survival, controlling for clinical, treatment, and demographic factors. RESULTS: The final cohort included 15,675 patients. Of the group, 46.3% were between 18 and 44 years of age, 33.6% were 45-59 years, and 20.1% were ≥ 60. Univariate analysis showed that compared to other groups, patients ≥ 60 were more likely to be male (p < 0.001), present with tumors > 5 cm (p < 0.001), more likely to have metastatic disease (p < 0.001), less likely to receive radioactive iodine (p < 0.001), and more likely to receive external beam radiation therapy (p < 0.001). In multivariable Cox proportional hazards models for 5 and 10-year disease-free survival, age ≥ 60 was associated with higher risk of disease at 5 and 10-years (HR 2.3 and 1.9 respectively, p < 0.001). Similar results were observed for 5 and 10-year disease-specific survival (HR 38.0 and 30.0 respectively, p < 0.001) after controlling for gender, race, co-morbidity, stage, surgical procedure, radioactive iodine, insurance, and hospital volume. CONCLUSIONS: Patients ≥ 60 years of age have worse DSS and DFS after a diagnosis of PTC, across all stages of disease. Given that patients over the age of 45 years have progressively worse survival as they age, these data support having three age groups, 18-44 years of age, 45-59 years, and ≥ 60 as an independent predictor of survival and recurrence to current staging guidelines.

Cinnamon extract reduces VEGF expression via suppressing HIF-1α gene expression and inhibits tumor growth in mice.

Although many anti-VEGF agents are available for cancer treatment, side effects of these agents limit their application for cancer treatment and prevention. Here we studied the potential use of a diet-based agent as an inhibitor for VEGF production. Using a VEGF reporter assay, our data showed that an extract from cinnamon (CE) was a potent inhibitor of VEGF production in human cancer cells and suggested inhibition might be mediated through the suppression of HIF-1α gene expression and protein synthesis. Furthermore, CE treatment was found to inhibit expression and phosphorylation of STAT3 and AKT, which are key factors in the regulation of HIF-1α expression, and significantly reduce angiogenesis potential of cancer cells by migration assay. Consistent with these results, we observed significant suppression of VEGF expression, blood vessel formation, and tumor growth in a human ovarian tumor model in mice treated with CE. Cinnamaldehyde, a major component in cinnamon, was identified as one active component in CE that inhibits VEGF expression. Taken together, our findings provide a novel mechanism underlying anti-angiogenic and anti-tumor actions of CE and support the potential use of CE in cancer prevention and treatment. © 2016 Wiley Periodicals, Inc.

Time to Treatment: Measuring Quality Breast Cancer Care.

BACKGROUND: To optimize breast cancer care, several organizations have crafted guidelines to define best practices for treating breast cancer. In addition to recommended therapies, 'timeliness of treatment' has been proposed as a quality metric. Our study evaluates time to surgical treatment and its effect on overall survival (OS). METHODS: The National Cancer Data Base (NCDB) was used to identify women diagnosed with invasive breast cancer between 2004 and 2012. Time from diagnosis to surgical treatment was calculated and grouped according to predetermined time intervals. Univariate and multivariate Cox proportional hazard models were used to assess patient and treatment factors related to OS. RESULTS: Overall, 420,792 patients initially treated with surgery were identified. Increased time to surgical treatment >12 weeks was associated with decreased OS [hazard ratio (HR) 1.14, 95 % confidence interval (CI) 1.09-1.20]. When stratified by pathologic stage, stage I patients treated at 8 to <12 weeks (HR 1.07, 95 % CI 1.02-1.13) and >12 weeks (HR 1.19, 95 % CI 1.11-1.28), as well as stage II patients treated at >12 weeks (HR 1.16, 95 % CI 1.08-1.25), had decreased OS compared with patients treated at <4 weeks. Other variables associated with decreased survival were treatment at a community cancer program, Medicaid or Medicare insurance, Black race, increasing age, mastectomy, moderately and poorly differentiated tumor grade, increasing T and N stage, and higher Charlson Index Group. CONCLUSION: The survival benefit of expedited time to initial surgical treatment varies by stage and appears to have the greatest impact in early-stage disease. Prior to establishing standard metrics, further quantification of the impact on patient outcomes is needed.

Synergistic anti-tumor effect of combined inhibition of EGFR and JAK/STAT3 pathways in human ovarian cancer.

BACKGROUND: The EGFR signaling pathway is frequently activated in human ovarian cancer and associated with poor prognosis. However, inhibition of EGFR signaling in patients with recurrent ovarian cancer has been disappointing. It remains to be addressed whether ovarian cancer patients could benefit from targeting EGFR signaling. Here we investigated the mechanisms underlying the resistance to EGFR inhibition in ovarian cancer and developed a strategy to overcome it. RESULTS: We found that treatment of human ovarian cancer cells with an EGFR inhibitor, gefitinib, resulted in increased STAT3 phosphorylation in a dose- and time-dependent manner. Inhibiting STAT3 activation with a small molecule inhibitor of JAK, an upstream kinase that phosphorylates and activates STAT3, synergistically increased the anti-tumor activity of gefitinib in vitro. Similar results were obtained when STAT3 or JAK1 expression was knocked down. In contrast, inhibiting other signaling pathways, such as AKT/mTOR, MEK or SRC, was relatively less effective. The combined treatment resulted in simultaneous attenuation of multiple survival pathways and increased inhibition of ERK pathway. In addition, the dual inhibition showed a stronger suppression of xenograft tumor growth than either single inhibition. CONCLUSIONS: Our findings demonstrate that feedback activation of STAT3 pathway might contribute to the resistance to EGFR inhibition. Combined blockade of both pathways appears to be more effective against human ovarian cancer than inhibition of each pathway alone both in vitro and in vivo. This study may provide a strategy to improve clinical benefit of targeting EGFR pathway in ovarian cancer patients.

IRF-1 regulates alternative mRNA splicing of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in breast epithelial cells generating an immunoreceptor tyrosine-based inhibition motif (ITIM) containing isoform.

BACKGROUND: Interferon regulatory factor-1 (IRF-1) is a master regulator of IFN-γ induced gene transcription. Previously we have shown that IRF-1 transcriptionally induces CEACAM1 via an ISRE (Interferon-Stimulated Response Element) in its promoter. CEACAM1 pre-mRNA undergoes extensive alternative splicing (AS) generating isoforms to produce either a short (S) cytoplasmic domain expressed primarily in epithelial cells or as an ITIM-containing long (L) isoform in immune cells. METHODS: The transcriptional and molecular mechanism of CEACAM1 minigenes AS containing promoter ISREs mutations in the breast epithelial, MDA-MB-468, cell line was detected using flow cytometry. In addition, transcriptome sequencing was utilized to determine whether IRF-1 could direct the AS of other genes as well. Tumor xenografts were used to evaluate CEACAM1 isoform expression on the leading edge of breast tumor cells. RESULTS: In the present study, we provide evidence that CEACAM1's promoter and variable exon 7 cross-talk allowing IRF-1 to direct AS events. Transcriptome sequencing shows that IRF-1 can also induce the global AS of genes involved in regulation of growth and differentiation as well as genes of the cytokine family. Furthermore, MDA-MB-468 cells grown as tumor xenografts exhibit an AS switch to the L-isoform of CEACAM1, demonstrating that an in vivo inflammatory milieu is also capable of generating the AS switch, similar to that found in human breast cancers Mol Cancer 7:46, 2008. CONCLUSIONS: The novel AS regulatory activities attributed to IRF-1 indicate that the IFN-γ response involves a global change in both gene transcription and AS in breast epithelial cells.

PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer.

Tumor-associated macrophages (TAMs) are the predominant cells that express programmed cell death ligand 1 (PD-L1) within human tumors in addition to cancer cells, and PD-L1+ TAMs are generally thought to be immunosuppressive within the tumor immune microenvironment (TIME). Using single-cell transcriptomic and spatial multiplex immunofluorescence analyses, we show that PD-L1+ TAMs are mature and immunostimulatory with spatial preference to T cells. In contrast, PD-L1- TAMs are immunosuppressive and spatially co-localize with cancer cells. Either higher density of PD-L1+ TAMs alone or ratio of PD-L1+/PD-L1- TAMs correlate with favorable clinical outcome in two independent cohorts of patients with breast cancer. Mechanistically, we show that PD-L1 is upregulated during the monocyte-to-macrophage maturation and differentiation process and does not require external IFN-γ stimulus. Functionally, PD-L1+ TAMs are more mature/activated and promote CD8+ T cells proliferation and cytotoxic capacity. Together, our findings reveal insights into the immunological significance of PD-L1 within the TIME.

Baicalein activates 5' adenosine monophosphate-activated protein kinase, inhibits the mammalian target of rapamycin, and exhibits antiproliferative effects in pancreatic neuroendocrine tumors in vitro and in vivo.

BACKGROUND: The mammalian target of rapamycin inhibition has been shown to prolong progression-free survival in patients with pancreatic neuroendocrine tumors. The natural compound baicalein indirectly inhibits the mammalian target of rapamycin, but it is unknown if baicalein exhibits such effects at physiologically achievable concentrations or exhibits synergy. METHODS: Pancreatic neuroendocrine tumor cell lines were cultured with baicalein, everolimus, and/or a synthetic 5' adenosine monophosphate-activated protein kinase activating agent alone and in combination. Cell viability assays and immunoblotting were performed. Female severe combined immunodeficient-beige mice were injected with BON-1 cells and treated with baicalein and COH-SR4 solutions via oral gavage. Tumor volumes were compared at 30 days. RESULTS: Immunoblotting revealed that treatment of baicalein induced 5' adenosine monophosphate-activated protein kinase activation and the mammalian target of rapamycin inhibition. Treatment with baicalein alone led to a significant decrease in the ratio of viable cells compared with controls at 72 hours at concentrations ≥5 µM (P = .021). The addition of COH-SR4 led to significantly greater effect on cell viability than with baicalein alone (P < .001, P < .001). The combination of baicalein with everolimus resulted in significantly lower cell viability than with everolimus alone (P = .005, P < .001). Tumor volume in vivo was significantly decreased with the combination of baicalein and COH-SR4 compared with controls (P = .003). CONCLUSION: Baicalein exhibits antiproliferative effects against pancreatic neuroendocrine tumor cell lines at doses ≥5 µM and demonstrates synergy.

Tumor-infiltrating exhausted CD8+ T cells dictate reduced survival in premenopausal estrogen receptor-positive breast cancer.

CD8+ tumor-infiltrating lymphocytes (TILs) are associated with improved survival in triple-negative breast cancer (TNBC) yet have no association with survival in estrogen receptor-positive (ER+) BC. The basis for these contrasting findings remains elusive. We identified subsets of BC tumors infiltrated by CD8+ T cells with characteristic features of exhausted T cells (TEX). Tumors with abundant CD8+ TEX exhibited a distinct tumor microenvironment marked by amplified interferon-γ signaling-related pathways and higher programmed death ligand 1 expression. Paradoxically, higher levels of tumor-infiltrating CD8+ TEX associated with decreased overall survival of patients with ER+ BC but not patients with TNBC. Moreover, high tumor expression of a CD8+ TEX signature identified dramatically reduced survival in premenopausal, but not postmenopausal, patients with ER+ BC. Finally, we demonstrated the value of a tumor TEX signature score in identifying high-risk premenopausal ER+ BC patients among those with intermediate Oncotype DX Breast Recurrence Scores. Our data highlight the complex relationship between CD8+ TILs, interferon-γ signaling, and ER status in BC patient survival. This work identifies tumor-infiltrating CD8+ TEX as a key feature of reduced survival outcomes in premenopausal patients with early-stage ER+ BC.

The adrenal mass: correlation of histopathology with imaging.

BACKGROUND: Computed tomography (CT) and magnetic resonance (MR) imaging can help diagnose benign adrenal adenomas, but prior studies rely on nonoperative follow-up as proof of a lesion's benign nature. We examined adrenalectomy tissues to determine if imaging characteristics correlate with histopathologic findings. METHODS: We retrieved data for 196 consecutive adrenalectomies in 192 patients from 2000 to 2008. Imaging results were considered to signify benign adrenal adenoma if one or more of the following was present: Hounsfield units <10 on unenhanced CT, contrast-enhanced CT quantifying absolute contrast washout of >60% or relative contrast washout of >40%, or MR with chemical-shift imaging demonstrating loss of signal intensity on out-of-phase images. RESULTS: The sensitivity and specificity of preoperative imaging in predicting benign adrenal adenoma were 57 and 94%, respectively. Histopathology confirmed that all 66 adrenal masses with imaging characteristics suggesting benign adenoma were indeed benign lesions and included 61 benign adrenal adenomas and 5 benign nonadenomatous lesions (3 myelolipomas, 1 composite myelolipoma/adenoma, and 1 ganglioliponeuroma). The specificity of imaging in predicting benignity was 100%. Malignant adrenal lesions were diagnosed in 17/130 (13%) masses: 8 metastases, 7 adrenal cortical carcinomas, 1 epithelioid angiosarcoma, and 1 ganglioneuroblastoma. The sensitivity of imaging in identifying malignancy was 100%. No malignancies were diagnosed during postoperative follow-up (mean 6 months, range 0.2-67 months). CONCLUSION: CT or MR characteristics predicted the presence of benign lesions with 100% specificity. Every adrenal malignancy had CT or MR results that were inconsistent with benign adenoma (100% sensitivity). To exclude malignancy, adrenal masses with non-benign imaging characteristics should be resected.

Synergistic Anti-Tumor Activity by Targeting Multiple Signaling Pathways in Ovarian Cancer.

More effective therapy is needed to improve the survival of patients with advanced and recurrent ovarian cancer. Preclinical and early clinical studies with single molecular targeted agents have shown limited antitumor activity in ovarian cancer, likely due to compensation by alternative growth/survival pathways. An emerging strategy in overcoming resistance is to combine inhibitors targeting multiple pathways. In this study, we used a novel strategy of combining several FDA-approved targeted drugs, including sunitinib, dasatinib, and everolimus, in human ovarian cancers. Combination of the tyrosine kinase inhibitor sunitinib with the SRC inhibitor dasatinib showed synergistic anti-tumor activity in human ovarian cancer cells. The increased activity was associated with inhibition of the STAT3, SRC, and MAPK signaling pathways, but not AKT signaling. To inhibit the PI3K/AKT/mTOR pathway, we added the mTOR inhibitor everolimus, which further increased anti-tumor activity in cells. Combined treatment with sunitinib, dasatinib, and everolimus also resulted in greater inhibition of human ovarian tumor growth in mice. Furthermore, the triple combination also synergistically increased the anti-tumor activity of paclitaxel, both in vitro and in vivo. Taken together, our results demonstrate that simultaneous inhibition of several signaling pathways results in better anti-tumor activity compared to inhibiting any of these signaling pathways alone.

Inhibition of Autophagy Amplifies Baicalein-Induced Apoptosis in Human Colorectal Cancer.

Baicalein is a Chinese herbal compound extracted from Scutellaria baicalensis that has anti-tumor properties. The aim of this study was to elucidate the mechanisms of action of baicalein against human colorectal cancer cell lines and to assess whether the anti-proliferative effects of baicalein may be amplified with autophagy inhibition. Human colon cancer cell lines (HT-29, HCT-116, SW480, and SW620) were treated with baicalein alone and in combination with the autophagy inhibitor chloroquine (CQ). Baicalein reduced cell viability in all four colon cancer lines in a dose-dependent fashion. Combination treatment of baicalein and the autophagy inhibitor CQ significantly decreased cell viability compared with baicalein alone in HT-29 and HCT-116 cell lines. Western blot analysis of the HCT-116 cell line treated with both baicalein and CQ demonstrated increased expression of LC3-II, a component of autophagy. The combination of baicalein with CQ culminated in activation of caspase-3-mediated apoptosis. These findings demonstrate that inhibition of autophagy enhanced apoptotic cell death induced by baicalein treatment in colon cancer cell lines. Future work will assess other targetable apoptotic pathways activated by baicalein and autophagy inhibition.

Breast cancer induces systemic immune changes on cytokine signaling in peripheral blood monocytes and lymphocytes.

BACKGROUND: It is increasingly recognized that cancer progression induces systemic immune changes in the host. Alterations in number and function of immune cells have been identified in cancer patients' peripheral blood and lymphoid organs. Recently, we found dysregulated cytokine signaling in peripheral blood T cells from breast cancer (BC) patients, even those with localized disease. METHODS: We used phosphoflow cytometry to determine the clinical significance of cytokine signaling responsiveness in peripheral blood monocytes from non-metastatic BC patients at diagnosis. We also examined the correlation between cytokine signaling in peripheral monocytes and the number of tumor-infiltrating macrophages in paired breast tumors. FINDINGS: Our results show that cytokine (IFNγ) signaling may also be dysregulated in peripheral blood monocytes at diagnosis, specifically in BC patients who later relapsed. Some patients exhibited concurrent cytokine signaling defects in monocytes and lymphocytes at diagnosis, which predict the risk of future relapse in two independent cohorts of BC patients. Moreover, IFNγ signaling negatively correlates with expression of CSF1R on monocytes, thus modulating their ability to infiltrate into tumors. INTERPRETATION: Our results demonstrate that tumor-induced systemic immune changes are evident in peripheral blood immune cells for both myeloid and lymphoid lineages, and point to cytokine signaling responsiveness as important biomarkers to evaluate the overall immune status of BC patients. FUNDING: This study was supported by the Department of Defense Breast Cancer Research Program (BCRP), The V Foundation, Stand Up to Cancer (SU2C), and Breast Cancer Research Foundation (BCRF).

Localization of parathyroid adenomas by sonography and technetium tc 99m sestamibi single-photon emission computed tomography before minimally invasive parathyroidectomy: are both studies really needed?

OBJECTIVE: The purpose of this study was to determine the utility of radiologist-performed sonography as the principal modality for parathyroid localization before minimally invasive parathyroidectomy. METHODS: Both sonography and technetium Tc 99m sestamibi single-photon emission computed tomography (SPECT) are commonly performed during imaging evaluation of patients with primary hyperparathyroidism (HPTH). Sonographic examinations ordered during the study period were performed by 1 author (M.E.T.), and results were immediately reported. Findings of a subsequent Tc 99m sestamibi study were recorded blinded to the sonographic results. The sensitivity and specificity of sonography and Tc 99m sestamibi SPECT were assessed with the use of surgery and pathology reports as a reference standard. The 2007 global Medicare reimbursement rates were used to assess the costs of preoperative localization. RESULTS: Parathyroidectomy was performed in 144 of 172 patients evaluated by both modalities. The sensitivity, specificity, and positive predictive value of sonography for identifying abnormal parathyroid glands were 74%, 96%, and 90%, respectively. Sonography correctly localized a single adenoma or suggested multiglandular disease in 112 of 144 patients (78%). The sensitivity, specificity, and positive predictive value of SPECT were 58%, 96%, and 89%. Technetium 99m sestamibi SPECT correctly predicted an adenoma or multiglandular disease in 88 of 144 patients (61%). Five patients with negative sonographic findings were shown to have uniglandular disease on Tc 99m sestamibi SPECT. Selective use of Tc 99m sestamibi SPECT (ie, when sonographic findings were negative or equivocal) would have decreased the cost of imaging by 53%. CONCLUSIONS: Radiologist-performed sonography may potentially be used as a principal imaging modality for patients with HPTH. Selective use of Tc 99m sestamibi in cases with negative or equivocal sonographic findings can decrease the cost of imaging before parathyroid resection considerably.

Combination therapy with BYL719 and LEE011 is synergistic and causes a greater suppression of p-S6 in triple negative breast cancer.

A third of patients with triple negative breast cancer (TNBC) have relapsed disease within 2-5 years from initial diagnosis, leaving an unmet need for therapeutic targets. TNBC frequently harbors alterations of the PI3K/AKT/mTOR pathway, but single agent PI3K/AKT/mTOR inhibitors have not shown marked efficacy. In this study, we investigated a strategy to improve efficacy of PI3K-α inhibitor BYL719 (alpelisib) in TNBC. While BYL719 is effective at inhibiting cell proliferation in T47D, a triple positive cell line, it had limited activity in TNBC. This may be partially due to persistent phosphorylation of RB, and incomplete inhibition of p-S6 in TNBC, since the inhibitory effect of BYL719 on p-RB and p-S6 was significantly reduced in TNBC compared to T47D cells. Addition of the CDK4/6 inhibitor LEE011 to BYL719 caused a simultaneous reduction of p-RB and p-S6, and a more complete inhibition of p-S6, leading to decreased expression of the pro-survival protein MCL-1, an induction of apoptosis, and an enhanced reduction of tumor growth in a PDX model of TNBC. These findings suggest that inhibition of p-RB and p-S6 is important for an effective response to the treatment of TNBC, and provides a strong rationale for clinical development of combination therapy with BYL719 and LEE011 for treatment of metastatic TNBC with intact RB.Presentation: This study was presented in part as an abstract at the 2016 San Antonio Breast Cancer Symposium (P3-03-15) and the 2018 Cancer Research and Targeted Therapy in London.

Eribulin Synergistically Increases Anti-Tumor Activity of an mTOR Inhibitor by Inhibiting pAKT/pS6K/pS6 in Triple Negative Breast Cancer.

Unlike other breast cancer subtypes, patients with triple negative breast cancer (TNBC) have poor outcomes and no effective targeted therapies, leaving an unmet need for therapeutic targets. Efforts to profile these tumors have revealed the PI3K/AKT/mTOR pathway as a potential target. Activation of this pathway also contributes to resistance to anti-cancer agents, including microtubule-targeting agents. Eribulin is one such microtubule-targeting agent that is beneficial in treating taxane and anthracycline refractory breast cancer. In this study, we compared the effect of eribulin on the PI3K/AKT/mTOR pathway with other microtubule-targeting agents in TNBC. We found that the phosphorylation of AKT was suppressed by eribulin, a microtubule depolymerizing agent, but activated by paclitaxel, a microtubule stabilizing agent. The combination of eribulin and everolimus, an mTOR inhibitor, resulted in an increased reduction of p-S6K1 and p-S6, a synergistic inhibition of cell survival in vitro, and an enhanced suppression of tumor growth in two orthotopic mouse models. These findings provide a preclinical foundation for targeting both the microtubule cytoskeleton and the PI3K/AKT/mTOR pathway in the treatment of refractory TNBC.