Tumor Dormancy Research Articles

Low-dose neoadjuvant chemotherapy dominates Ki67− quiescent tumor dormancy for an effective immunotherapy of breast cancer

Abstract Breast cancer mortality is mainly due to distant recurrence of the disease. The disease recurrence is usually because of the establishment of tumor dormancy following successful treatment of early stage breast cancer. In current clinical practice, dormant tumor cells are not actively treated, because they do not respond to chemotherapy or radiation therapies. Immunotherapeutic targeting of tumor dormancy is also challenging because of the induction of tumor immunoediting, leading to tumor escape and relapse. Recently, we have characterized Ki67− quiescent and Ki67low indolent types of tumor dormancy. We demonstrated that quiescent, but not indolent, dormant tumor cells fail to undergo immunoediting and remain highly susceptible to immunotherapy. Here, we sought to develop neoadjuvant therapies for dominating a quiescent type of tumor dormancy as the best target for immunotherapy. We showed that a low dose 5-FU+Adriamycin+Cyclophosphamide (FAC) dominated Ki67− quiescent type of tumor dormancy in the neu-overexpression mouse mammary carcinoma (MMC) tumor cells. These dormant cells were effectively controlled by immunotherapy in FVBN202 transgenic mouse model of spontaneous breast cancer. Since the antibiotic azithromycin has been shown to effectively eliminate senescent cells, we also sought to determine its efficacy against dormant tumor cells. We demonstrated that azithromycin specifically induced apoptosis in FAC-induced dormant tumor cells. All together, the results suggest that use of less toxic, low dose chemotherapy combined with azithromycin in a neoadjuvant setting could lead to the elimination of dormant tumor cells by immunotherapy.

Tumor Cell Dormancy—Triggered by the Niche

Dormant cancer cells often survive treatment and increase the risk for tumor relapse, associated with dismal prognosis. Two recent papers describe mechanisms used by the bone marrow niche to regulate leukemia dormancy. The findings provide a molecular basis for niche-targeting therapies that may enable elimination of dormant tumor cells.

150 Cancer immune control requires interferon- dependent activation of the p16Ink4a senescence signaling pathways

Immunotherapy with monoclonal antibodies against exhaustion-associated surface molecules, immune checkpoint blockade (ICB), reactivates T cells and induces durable therapeutic stability in a variety of metastatic cancers. Cancer regression with ICB requires cancer cell killing but additional, unknown mechanisms are needed to establish the long-lasting tumor dormancy in the responder patients. To analyze cancer cell dormancy induced by ICB we used a model of adoptive transfer of tumor-associated antigen (TAA)-specific T-helper-1 (Th1) cells in transgenic tumor-bearing mice. Th1 immunotherapy prolongs the life of mice by induction tumor cell senescence. Combining adoptive transfer of Th1 cells with ICB significantly increased life time. The therapy restored a normal health status in the mice, destroyed the cancers partly and induced a p16Ink4a+/Ki67- senescent phenotype in the remaining cancer cells. The therapy was dependent on an intact interferon Stat1-signaling pathway and the activation of p16Ink4a in the cancer cells. While TAA-specific Th1 cells similarly migrated into cancers of either wildtype or Stat1.ko cancers, they failed to induce p16Ink4a+/Ki67- senescent tumor cells. Stat1.ko and p16Ink4a.ko cancer cells were fully susceptible to apoptosis and T cell-mediated killing. In contrast, both, Stat1.ko and p16Ink4a.ko cancer were resistant to senescence induction, in vitro and in vivo. To determine whether this is also valid for ICB therapy of human melanomas, we analyzed metastatic melanomas that progressed within < 3 months during ICB. These metastasis had losses of senescence-inducing genes or ≥ 4fold amplifications of senescence inhibitors. In contrast such mutations were infrequent in metastatic melanomas that regressed during ICB for > 1 year. Thus, cancer immune control with ICB requires, beside cancer cell killing, the interferon- and p16Ink4a- dependent senescence induction in cancer cells.

T-cell recognition of non-mutated tumor antigens in healthy individuals: connecting endogenous immunity and tumor dormancy.

The concept of a dual functional programme of the immune system to destroy malignant cells but also to edit their immunogenic profile, considerably improved our understanding of the process of tumor evolution in the context of a continuum of interactions between tumor cells and immune lymphocytes. Such an endogenous antitumor immunity throughout the period of cancer development established the concept of cancer immunomodulation which is practically based on a process of selection of more clonal tumors which are manageable by the immune system and constitute the equilibrium phase of immunoediting. The duration of this phase is very important, because the immune system keeps the tumor in a dormant state via cell interactions which establish a balanced state of tumor immunosurveillance versus tumor immune evasion. Depending on the quality and quantity of antitumor immune reactivity and the effectiveness of resistance mechanisms employed by the tumor cells to counteract this immune attack, the equilibrium phase may have shorter or longer duration. Notwithstanding its natural course, the equilibrium phase should be considered as a part of tumor evolutionary process guided by genetic as well as epigenetic changes which in turn activate endogenous cellular immunity to certain levels capable of controlling tumor growth rates and maintain tumor dormancy.

Sox2 dosage: A critical determinant in the functions of Sox2 in both normal and tumor cells.

The stem cell transcription factor Sox2 is widely recognized for its many roles during normal development and cancer. Over the last several years, it has become increasingly evident that Sox2 dosage plays critical roles in both normal and malignant cells. The work described in this review indicates that the dosage of Sox2 influences cell fate decisions made during normal mammalian development, as well as cell fate decisions in cancer, including those that influence the tumor cell of origin and progression of the cancer. Equally important, Sox2 dosage is a key determinant in the proliferation of both normal cells and tumor cells, where proliferation is restricted in Sox2high cells. Collectively, the studies reviewed here indicate that tumor cells utilize the fundamental effects of Sox2 dosage to suit their own needs. Finally, we speculate that elevated expression of Sox2 helps establish and maintain tumor dormancy in Sox2-positive cancers.

A nonlinear mathematical model of cell-mediated immune response for tumor phenotypic heterogeneity

Human cancers display intra-tumor heterogeneity in many phenotypic features, such as expression of cell surface receptors, growth, and angiogenic, proliferative, and immunogenic factors, which represent obstacles to a successful immune response. In this paper, we propose a nonlinear mathematical model of cancer immunosurveillance that takes into account some of these features based on cell-mediated immune responses. The model describes phenomena that are seen in vivo, such as tumor dormancy, robustness, immunoselection over tumor heterogeneity (also called “cancer immunoediting”) and strong sensitivity to initial conditions in the composition of tumor microenvironment. The results framework has as common element the tumor as an attractor for abnormal cells. Bifurcation analysis give us as tumor attractors fixed-points, limit cycles and chaotic attractors, the latter emerging from period-doubling cascade displaying Feigenbaum’s universality. Finally, we simulated both elimination and escape tumor scenarios by means of a stochastic version of the model according to the Doob-Gillespie algorithm.

Inhibition of TGF-β induced lipid droplets switches M2 macrophages to M1 phenotype

Lipid droplets (LD) are newly characterized dynamic cytoplasmic organelle which is the storehouse of different immunosuppressive cytokines and enzymes like cyclooxygenase and lipoxygenase. Tumors are known to modulate the immune system by immune-editing the microenvironment. Immuno-editing comprises of three steps namely cancer immune-surveillance, tumor dormancy and finally escape leading to tumor development. The latency of the tumor microenvironment is greatly contributed by the M2 polarized macrophages and TGF-β is a prime culprit. Modulating M2 macrophages to M1 can be a strategy against tumor progression. We found that tumor-conditioned medium or recombinant TGF-β was efficient to induce LD formation in Raw264.7 cells and the inhibition of LD was associated with the switch of M2 to M1 phenotype involving MEK1/2 axis. Signature molecules of M2 polarized macrophages like CD206 were also downregulated while co-stimulatory molecules like CD80, CD86 were up-regulated along with enhanced surface expression of MHCII when these macrophages were subjected to C75 treatment to reduce the LD formation. The level of pro-inflammatory cytokine, as well as ROS and NO generation, were also increased when TGF-β treated macrophages were subjected to C75 treatment. This study is probably the first report of this kind and can be used in the future in cancer treatment.

Profile of Daniel A. Haber

Research oncologist Daniel Haber may not have treated patients in years, but they are never far from his mind. “When I started my MD/PhD program at Stanford I thought I could be both a physician and a researcher,” says Haber, director of the Massachusetts General Hospital (MGH) Cancer Center, where he also runs a cancer research laboratory. “But at some point the research became dominant in terms of my interest and my ability. I tried to make up for it by directing my research toward very clinically relevant questions.” Daniel A. Haber. Image courtesy of Mark Karlsberg (Studio Eleven, Newton, MA). Haber’s research focuses on using human genetics to tackle cancer through early diagnosis and targeted treatments. He was among the first to find targetable genetic mutations that cause cancer, discovering a mutation in lung cancer that helped lay the foundation for widespread testing of cancers with treatable mutations. He is also making strides in detecting rare cancer cells circulating in blood, with the goal of early detection and treatment. Haber’s Inaugural Article (1) identifies a signaling pathway that switches on dormant metastatic tumor cells that have spread through blood. This work could lead to ways to prevent cancer from spreading through blood. Haber is the Kurt J. Isselbacher Professor of Oncology at Harvard Medical School and has been a Howard Hughes Medical Institute investigator since 2008. He was elected to the National Academy of Sciences in 2018. Haber’s father taught him the importance of helping people. The elder Haber worked for an organization founded in 1881 to assist Jews fleeing pogroms and that today resettles refugees in the United States. “He had a sense of mission,” Haber says of his father. “He wanted his life to be about doing something that was larger than himself, something meaningful. That was …

Tumor dormancy at bedside: A late awakening

Breast cancer recurrence may occur at variable times following primary tumor removal. The corresponding event dynamics displays a structured multipeak pattern, which can be explained by the occurrence of microscopic phases of metastasis quiescence (tumor dormancy) followed by wake up, growth and timed clinical appearance. This model provides a meaningful justification of the early recurrence pattern and even explains the effectiveness of adjuvant systemic therapies. Yet, late recurrences, which were less investigated, are fairly little known and a few researchers supported their steady state appearance. We report here the analysis of the late clinical course from patients who were disease-free at 5 years of follow-up, which again displays a structured pattern, supporting the view that tumor dormancy can explain the late recurrence risk as well. Tailored treatments are needed to address late clinical recurrences.

Abstract ES5-1: Tumor dormancy and recurrence

Abstract Despite early detection and adjuvant therapy, breast cancer remains the leading cause of cancer mortality in women, largely due to distant, incurable recurrences that arise years, or even decades, after treatment of the primary tumor. Recurrent, metastatic tumors arise from the pool of residual local and disseminated tumor cells (DTCs) that survive primary treatment in tissues at sites throughout the body where they persist in a presumed dormant state. Early studies demonstrated that an immunohistochemical assay can detect cytokeratin-positive DTCs in bone marrow aspirates of a substantial fraction of breast cancer patients lacking any clinical signs of metastasis. Importantly, women who harbor detectable DTCs in their bone marrow have a substantially increased risk of distant recurrence, as well as poorer breast cancer-specific and overall survival, and this measure has repeatedly been shown to be an independent predictor of recurrence-free survival and overall survival in multivariate analysis. Since DTCs constitute the reservoir from which recurrent cancers arise, understanding their biology is a critical priority in cancer research. We hypothesize that effectively disabling the survival mechanisms by which DTCs persist in breast cancer patients following treatment will deplete this critical reservoir of cells, reduce tumor recurrence, and thereby improve survival. At present, however, the mechanisms enabling these cells to survive in a dormant state and ultimately recur are poorly understood and clinical methods to detect DTCs, as well as DTC-directed therapeutic approaches, are still in their infancy. To address this critical gap and elucidate critical pathways in dormant tumor cell survival, we and others have developed a variety of mouse models for human breast cancer that recapitulate key features of human breast cancer progression, including metastasis, residual disease, dormancy and recurrence. Investigation of these models using both genetic and pharmacological approaches has identified several mediators of dormant DTC survival and recurrence, demonstrated their relevance to pathways that contribute to therapeutic resistance in human breast cancers following targeted therapy or chemotherapy, and revealed that pharmacological targets for dormancy and recurrence may be unique to these stages of tumor progression. These findings have, in turn, led to the initiation of clinical trials to test the ability of targeted therapies to deplete the burden of DTCs in the bone marrow of breast cancer patients and thereby decrease risk of relapse. If successful, the ability to therapeutically target survival mechanisms utilized by DTCs would constitute a powerful, paradigm-shifting approach to preventing cancer recurrence and the mortality associated with it. Citation Format: Chodosh L. Tumor dormancy and recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES5-1.

Abstract P1-05-01: Breast cancer cell-derived IL-1B drives metastasis and colonisation of the bone microenvironment

Abstract Background: Breast cancer bone metastases are incurable and new therapeutic targets needed. After homing and colonising bone, cancer cells remain dormant, until signals from the microenvironment stimulate their proliferation to form overt metastases. We have recently identified interleukin-1B (IL-1B) as a potential marker for predicting breast cancer patients at increased risk of skeletal relapse and established a role for IL-1 signalling in tumour dormancy in bone. Here we present novel data to support that tumour cell-derived IL-1B plays major roles in breast cancer metastasis and growth in bone. Methods: Tumour/stromal IL-B and IL-1R1 expression was assessed in samples from the AZURE study following immunohistochemical staining. A humanised mouse model of MDA-MB-231 bone metastasis was used to assess effects of the IL-1R antagonist, Anakinra, on tumour growth and spontaneous metastasis. Effects of tumour cell-derived IL-1B on parameters associated with epithelial to mesenchymal transition and metastasis were measured by ELISA, QPCR, Western blot, transwell and scratch assays in MDA-MB-231, MCF7 and T47D cells transfected with IL-1B/control. Homing of breast cancer cells to bone was monitored in BALB/c nude mice intra venously injected with IL-1B overexpressing/control cells. To assess anti-tumour effects of IL-1 inhibition combined with standard of care, BALB/c or C57BL/6 mice were injected with 4T1 or E0771 cells (intra cardiac or intra ductal) 7-days prior to administration of Anakinra, doxorubicin, zoledronic acid (ZA) or placebo, alone or in combination for 14 days. Results: In tissue samples from &amp;gt;1300 patients with stage II/III breast cancer, active IL-1B in tumour cells correlated with relapse in bone (hazard ratio 1.85; 95% CI 1.05-3.26; P=0.02) and other sites (hazard ratio 2.09; 95% CI 1.26-3.48; P=0.0016). In a model of spontaneous human breast cancer metastasis to human bone, Anakinra significantly reduced metastasis to bone (from 80% in control animals to, 20% in Anakinra treated mice) and reduced the number of tumour cells shed into the circulation. Genetic manipulation of breast cancer cells to overexpress IL-1B demonstrated that exogenous production of IL-1B promoted EMT (decreased E-Cadherin, N-Cadherin and G-Catenin), invasion, migration and organ-specific homing in ER-ve (MDA-MB-231) and ER+ve (T47D and MCF7) cells in vitro and in vivo. Contact between tumour cells and osteoblasts or bone marrow cells increased IL-1B secretion from all three cell types. Exposure of tumour cells to IL-1B in the absence of bone cells did not stimulate tumour cell proliferation. Instead, elevated concentrations of IL-1B caused expansion of the bone metastatic niche (increased osteoblasts and blood vessels) that in turn stimulated tumour proliferation. Adding Anakinra to chemotherapy and ZA to mice injected with E0771 cells completely prevented bone metastasis formation and reduced extra-skeletal metastasis by 33%. In mice injected with 4T1 cells, the triple combination therapy reduced bone metastasis by 50% and extra-skeletal metastases by 65%. Conclusion: Our novel data demonstrate that IL-1B/IL-1R1 signalling plays an important role in breast cancer metastasis to bone. Pharmacological inhibition of IL-1B has potential as a novel treatment. Citation Format: Ottewell PD, Lefley DV, Freeman K, Gregory WM, Hanby AM, Spicer-Hadlington AR, Liu X, Bradbury SM, Hambley L, Allocca G, Cookson V, Kruithof de-Julio M, Coleman RE, Brown JE, Holen I, Tulotta C. Breast cancer cell-derived IL-1B drives metastasis and colonisation of the bone microenvironment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-01.

Abstract P5-04-03: Molecular characterisation of ER+ breast cancer dormancy and acquired resistance using a clinical model: Potential involvement of epigenetic regulation

Abstract Background: The risk of recurrence for oestrogen receptor positive (ER+) breast cancer patients treated with 5 years of adjuvant endocrine therapy persists for many years or even decades following surgery and apparently successful adjuvant therapy. This period of dormancy and acquired resistance is inherently difficult to investigate. Therefore, previous efforts have been limited to in vitro or in vivo approaches. In this study sequential tumour samples from patients receiving extended neoadjuvant endocrine treatment were characterised as a novel clinical model of ER+ breast cancer dormancy and acquired resistance. Methods: Consecutive tumour samples from 62 patients undergoing extended (4-45 months) neoadjuvant letrozole therapy were subjected to transcriptomic and proteomic analysis, representing pre- (before treatment), early-on (13-120 days) and long-term (&amp;gt;120 days) neoadjuvant letrozole treatment. Patients with at least a 40% initial reduction in tumour size by 4 months of treatment were included. Of these, 42 patients with no subsequent progression were classified as “dormant”, and the remaining 20 patients as “acquired resistant”. Expression analysis was performed by using Illumina BeadChips. R and BioConductor packages were used for analysis. Differentially expressed genes were determined by using paired Rank Products (FDR, 5%). Results: Multidimensional scaling using most variant 500 genes demonstrated that long-term treated dormant samples clustered separately from their matched pre- and early-on samples whereas long-term treated resistant samples were indistinguishable from their pre-treatment counterparts. Therapy-induced changes in resistant tumours were common features of treatment, rather than being specific to resistant phenotype. Comparative analysis of long-term treated dormant and resistant tumours highlighted changes in epigenetics pathways including DNA methylation and histone acetylation. DNA methylation marks 5-methylcytosine and 5-hydroxymethylcytosine were significantly reduced in resistant tumours compared to dormant tissues after extended letrozole treatment. Decrease in 5-hydroxymethylcytosine were significant early-on. Conclusions: This is the first patient-matched gene expression study investigating long-term aromatase inhibitor-induced dormancy and acquired resistance in breast cancer. Dormant tumors exhibit distinct molecular changes under extended treatment whereas acquired resistant tumors are more similar to matched diagnostic samples supporting the molecular concordance between primary tumors and metastases. Global loss of DNA methylation was observed in resistant tumours under extended treatment which can be predicted within first 4 months of therapy. Epigenetic alterations may lead to escape from dormancy and drive acquired resistance in a subset of patients supporting a potential role for therapy targeted at these epigenetic alterations in the management of endocrine resistant breast cancer. Funding: This work was supported by Marie Skłodowska-Curie Individual Fellowship [H2020-MSCA-IF, 658170] and Welcome Trust Institutional Fund (ISSF3) to CS and AHS, Breast Cancer Now to AHS. Citation Format: Selli C, Turnbull AK, Pearce D, Fernando A, Renshaw L, Thomas JS, Dixon MJ, Sims AH. Molecular characterisation of ER+ breast cancer dormancy and acquired resistance using a clinical model: Potential involvement of epigenetic regulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-03.

Abstract P5-04-08: Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence

Abstract Precision oncology requires delivering the right drug to the right patient at the right time, but “time” is rarely studied preclinically before a drug enters a particular clinical setting. Despite showing efficacy against recurrent/metastatic solid tumors, drugs sometimes fail to prevent tumor recurrence when given (neo)adjuvantly. The biology and microenvironments of overt tumors likely differ substantially from dormant cancer cells. The development of preclinical models to investigate clinically dormant disease will increase understanding of residual tumor cell biology and enable the development of therapeutics for rational adjuvant trials. Despite treatment with adjuvant anti-estrogen therapies, ˜30% of patients with ER+ breast cancer (BC) experience recurrence. In contrast to other BC subtypes, ER+ relapses occur late, often appearing years to decades after initial diagnosis and treatment. This delay suggests that ER+ BC cells can undergo extended periods of clinical dormancy. We developed novel, clinically relevant xenograft models of dormancy in ER+ BC. Low systemic levels of estrogens in mice can be further suppressed by ovariectomy, mimicking the effects of aromatase inhibitor (AI)-induced estrogen deprivation (ED) therapy. In ovariectomized mice, luciferase-labeled MCF-7, HCC-1428, HCC-1500, and MDA-MB-415 cells, as well as the HCI-017 PDX model, formed palpable orthotopic tumors upon 17b-estradiol supplementation. ED induced rapid tumor regression and decreased bioluminescent signal. However, a small proportion of ER+ BC cells survived ED for &amp;gt;1 year in a clinically dormant, growth-suppressed state. This residual cell population stabilized after ˜90 days of ED, as evidenced by stabilization of bioluminescent signal. Transcriptional and immunohistochemical analyses revealed significant upregulation of AMP-activated protein kinase (AMPK)-alpha-2 levels and activity in clinically dormant tumor cells compared to estrogen-driven or acutely ED xenografts. Dormant tumor cells were dependent upon AMPK activity for survival, as short-term pharmacologic inhibition of AMPK reduced residual bioluminescent signal. Metformin is an AMPK-activating drug approved for the treatment of diabetes. Metformin is currently being tested as an anti-cancer agent in many clinical trials at various points in the disease progression of diverse cancer types. In our models of clinically dormant ER+ BC, AMPK activation via metformin slowed ED-induced tumor regression, promoted residual tumor cell survival, and caused earlier tumor regrowth. In vitro studies indicated that metformin promotes cell survival during ED by enhancing fatty acid β-oxidation. Conversely, metformin treatment slowed estrogen-driven tumor growth, in agreement with prior observations that metformin slows growth of various tumor subtypes. These findings suggest that AMPK activation may be efficacious against growing tumors, but deleterious when used in combination with drugs that suppress tumor growth and induce regression. More broadly, this work highlights the issue that the time in a disease course needs to be considered when testing potential anti-cancer agents such as AMPK modulators. Citation Format: Miller TW, Hampsch RA, McCleery CF, Wells JD, Fields JL, Dillon LM, Shee K. Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-08.

Abstract ES5-3: Late recurrence in ER-positive breast cancer

Abstract The risk of recurrence in early stage, ER-positive breast cancer patients persists for 15-20+ years after diagnosis, and never declines to zero. Rates of late recurrence can be reduced by extending tamoxifen therapy beyond 5 years, with additional tamoxifen or a switch to an aromatase inhibitor. The benefits of extending aromatase inhibitors beyond 5 years remains unclear. The benefit of extended adjuvant endocrine therapy for the average early stage breast cancer patient is small. Extended therapy can be associated with serious increased risks, including thromboembolic events and endometrial cancer with tamoxifen, and fractures and possibly cardiovascular events with aromatase inhibitors. Quality of life can also be impacted, including vasomotor symptoms, sexual dysfunction, and arthralgias. Determining which patients have high residual risk of recurrence beyond 5 years, and importantly whether additional therapy is likely to mitigate that risk, has been the subject of considerable recent research. Tumor size, nodal status and grade of the primary tumor at the time of diagnosis have been shown to be associated with risk of late recurrence in ER-positive breast cancer. Several multi-parameter molecular biomarker assays performed on the primary tumor are able to separate patients into groups at low and high risk for recurrence beyond 5 years when using retrospective data sets. To date, no specific assay has been felt to have high enough level evidence to recommend inclusion in treatment guidelines or use in decision-making regarding continuing, stopping or switching endocrine therapy. Recent studies have evaluated whether there are better determinants of late risk of recurrence, beyond the biology of the primary tumor, that can be performed years after the original diagnosis. Assays that may predict the presence of dormant tumor cells and late relapse include Circulating Tumor Calls (CTCs), Disseminated Tumor Cells (DTCs) in the bone marrow, and circulating tumor DNA, proteins or metabolites. While early data is promising, further research is needed before incorporating into clinical decision-making. Identifying a biomarker to determine patients most likely to benefit from longer duration endocrine therapy, or additional therapeutic approaches, could help in reducing late relapse as well as minimizing the risk of side effects and overtreatment. Citation Format: Gralow J. Late recurrence in ER-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES5-3.

AXL Is a Putative Tumor Suppressor and Dormancy Regulator in Prostate Cancer.

Prostate cancer bone metastasis remains lethal and incurable, and often arises years after elimination of the primary tumor. It is unclear what underlies the decades-long clinical latency before recurrence, but evidence points to the existence of dormant residual tumor cells that disseminated before the primary tumor was eliminated. To design therapies to prevent progression of disseminated tumor cells (DTC) into lethal metastases, it is crucial to understand the mechanism(s) underlying this dormancy. The current study functionally validated our previous observation that implicated the GAS6/AXL axis in mediating DTC dormancy in the bone marrow. AXL-null and AXL-overexpressing prostate cancer cell lines were generated to determine if AXL was necessary and/or sufficient for dormancy. Characterization of these cells in vitro and using in vivo mouse models of DTC growth demonstrated that AXL was indeed sufficient to induce dormancy, but was unable to maintain it long-term and was not absolutely required for a dormancy period. Clinically, AXL expression correlated with longer survival in prostate cancer patients, and AXL was not expressed by cancer cells in primary or metastatic tissue. These data point to a tumor-suppressive role for AXL in prostate cancer, and future work is required to determine if AXL is expressed on human bone marrow DTCs. IMPLICATIONS: The ability of AXL to initiate but not maintain dormancy, coupled with its dispensability, suggests that targeting AXL alone will not prevent lethal metastatic outgrowth, and likely a cooperative network of factors exists to mediate long-term cellular dormancy.

Abstract A144: The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine

Abstract Melanoma (MM) cells leave primary tumors early and evolve at different sites in parallel. However, timing in human MM is unknown. Lymphatic dissemination is fast shortly after the dermal invasion, but metastases may occur late. Such delay in metastases formation suggests possible host defense mechanism, or particular nature of non-actively proliferative (dormant) MM stem cell (MSC) population. T lymphocytes may also induce tumor dormancy. The proposed mechanism involves a cytostatic mode of tumor silencing resulting from the suppression of both neoplastic cell proliferation and cell cycle progression. Significant progress in the development of novel advanced MM therapies was made. However, not all patients benefit from the therapy, developing late metastases decades after treatment ends. Moreover, using the immunotherapy-based model of MM dormancy in mouse, vaccination against MM prevented tumor growth but failed to prevent tumor cell dissemination or elimination of all MM cells. We have developed therapeutic gene modified allogenic MM vaccine (AGI-101H) that has been tested since 1997 and resulted in long-term survival of a substantial fraction of patients. Genetic modification of vaccine cells to express designer cytokine cDNA encoding Hyper-IL-6 protein (comprising of IL-6 linked with a soluble IL-6 receptor) has altered AGI-101H cells phenotype towards MSC-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). We have found significant induction of anti-MSCs response in immunized patients by a generation of functionally active cytotoxic CD8+T-cells and antibodies specific for ALDH1A1 in circulation. However, the AGI-101H mode of action is complex: besides inducing a direct anticancer response to MSC population, long-term vaccination may induce MM dormancy through T-cell mediated immunity. The above hypothesis is based on clinical observations of AHI-101H treated patient who was progression-free for many years, but injury induced metastases formation in the bruise. This patient developed skin MM in 1999. In 2002 in transit metastases appeared (resected). The patient was enrolled into AGI-101H program. In 2003 metastases in liver (resected), 2004 metastasis in ovary (resected). 11 years (2004-2015) no progression. In Nov. 2015 she had bicycle accident, trauma, and MM metastasis presenting with a bruise (resected). The treatment continues with no progression. The aim was to determine the molecular mechanisms of peripheral T-cell-mediated induction of MM dormancy induced by vaccination. Therefore, T-cell mRNA expression profiling of long-term surviving patients was carried out. PBMCs were isolated from treated patients (18), untreated MM patients (13) and healthy controls (8). Untouched T-cells were separated by magnetic beads and total RNA isolated. The transcriptome profiling was performed with Affymetrix HG U219 microarray (19,285 markers). Differential gene expression (DGE) analysis between all groups was conducted and validated with RT-qPCR and FACS. The transcriptomic results were further analyzed with Gene Set Enrichment Analysis (GSEA) tool. DGE analyses comparing AGI-101H vaccinated (AV) and nonimmunized (C) patients revealed 538 differentially expressed genes (DEGs), with 373 downregulated and 165 upregulated in AV (adj p &amp;lt; 0.05). Among these 538 markers, the expression of 14 (with |log2FC| &amp;gt; 1) is now being validated with RT-qPCR and FACS. Also, GSEA analysis revealed significant enrichment of “TNFa_signaling_via_NFkB”, “TGFb_signaling” and “G2/M_checkpoint” hallmark processes (MSigDB Hallmark gene sets) in AGI-101H vaccinated patients, that indicates considerable activation of antitumor response and prevention of conversion of MM dormanT-cells into proliferative metastases. Transcriptome profiling of peripheral T-cells in long-surviving MM patients immunized with AGI-101H suggests activation of the molecular mechanism that sustains MM cells in the non-actively proliferative dormant state. Citation Format: Andrzej A. Mackiewicz, Partycja Czerwinska, Marcin Rucinski, Katarzyna Gryska, Iga Grzadzielewska, Anna Jaworska, Jacek Mackiewicz. The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A144.

Circulating miRNAs as a marker of metastatic disease and prognostic factor in metastatic breast cancer.

BackgroundCirculating miRNAs (miRs) are increasingly recognized as potential biomarkers in cancer. We aimed to evaluate the differential expression of miR-23b and miR-190 which are involved in tumor dormancy, miR-21 involved in metastasis and miR-200b and miR-200c involved in epithelial-mesenchymal transition (EMT) and metastasis, in the plasma of patients with early and metastatic breast cancer (MBC). We also aimed to identify associations of the expression levels with patient and disease characteristics and outcomes in metastatic patients treated with first-line chemotherapy.ResultsmiR-21 (p < 0.001), miR-23b (p = 0.033), miR-200b (p < 0.001) and miR-200c (p < 0.001) expression was higher in metastatic compared to early breast cancer. ROC curve analysis showed that miR-21 (AUC = 0.722; p < 0.001) and miR-200b (AUC = 0.720; p < 0.001) distinguished with high accuracy among the two disease states, whereas the combination of miR-21, miR-190, miR-200b and miR-200c, further improved accuracy (AUC = 0.797; p < 0.001). High miR-200b expression independently predicted for shorter OS (p = 0.026) in MBC. High expression of both miR23b and miR-190 emerged as a strong independent factor associated with shorter PFS (p = 0.001) in de novo metastatic patients and high miR-200b independently predicted for decreased OS in the HER2-negative subgroup (p = 0.007).Materials and MethodsBlood samples were obtained from patients with early (n = 133) and MBC (n = 110) before adjuvant or first-line chemotherapy, respectively. Plasma miRNA expression levels were assessed by RT-qPCR and were classified as high or low according to the median values.ConclusionsOur results are in support of the concept that circulating miRNAs represent a tool with significant diagnostic and prognostic implications in breast cancer.

Tumor dormancy in bone.

Bone marrow is a common site of metastasis for a number of tumor types, including breast, prostate, and lung cancer, but the mechanisms controlling tumor dormancy in bone are poorly understood. In breast cancer, while advances in drug development, screening practices, and surgical techniques have dramatically improved survival rates in recent decades, metastatic recurrence in the bone remains common and can develop years or decades after elimination of the primary tumor. It is now understood that tumor cells disseminate to distant metastatic sites at early stages of tumor progression, leaving cancer survivors at a high risk of recurrence. This review will discuss mechanisms of bone lesion development and current theories of how dormant cancer cells behave in bone, as well as a number of processes suspected to be involved in the maintenance of and exit from dormancy in the bone microenvironment. The bone is a complex microenvironment with a multitude of cell types and processes. Many of these factors, including angiogenesis, immune surveillance, and hypoxia, are thought to regulate tumor cell entry and exit from dormancy in different bone marrow niches.

Stability analysis and numerical simulations via fractional calculus for tumor dormancy models

Fractional calculus is a field of mathematics in considerable expansion and has been understood as a tool with a wide range of applications, including in biological systems. Cancer dormancy is a state in which cancer cells have an intrinsic rate of reduced proliferation over a period of time, after which they arise with an accelerated growth rate, usually triggering metastases. This work investigates via fractional calculus two proposed ordinary differential equation systems via fractional calculus, which address dynamics between tumor cells and the immune system. Analyses and comparison processes are performed through an analytical study about the equilibrium points of each model and numerical simulations by the Nonstandard Finite Difference (NSFD) method. The analytical and numerical results show two important behaviors associated with tumor dormancy. The first refers to qualitative changes in the stability of an equilibrium point, which strongly depend on the order of the derivative and represent scenarios where there is no cancer escape. The second behavior refers to changes in the damping of some solutions, which can represent longer periods of time to exit the state of dormancy. In this case, different orders used in the derivatives are investigated, as well as their influence on the behavior of the solutions against the main parameters of the model.

Bone as a Preferential Site for Metastasis.

Bone marrow provides a unique microenvironment favoring the colonization and outgrowth of metastatic tumor cells. Despite the high incidence of bone metastasis in breast and prostate cancer patients, many of the molecular mechanisms controlling metastatic progression remain unclear. Several gene signatures associated with bone metastasis have been reported, but no metastasis-specific gene alterations have been identified. Therefore, there has been considerable interest in understanding how the bone microenvironment impacts the behavior of disseminated tumor cells (DTCs) prior to and following colonization of the bone. Substantial evidence indicates that disruption of normal bone homeostasis by tumor-derived factors establishes a premetastatic niche within the bone that favors DTC colonization. Following dissemination, bone resident cells and the surrounding stroma provide critical signals that support tumor cell colonization, survival, and eventual outgrowth. Clinical data suggest that patients can harbor DTCs for years to decades prior to developing overt bone metastases, suggesting a period of tumor dormancy occurs in the bone marrow. Several dormancy-promoting factors have been recently identified; however, critical questions surrounding the molecular triggers and timing of tumor cell emergence from dormancy remain. Here, we review how metastatic tumor cells co-opt the bone marrow microenvironment for metastatic progression and discuss emerging insights into how to more effectively target DTCs and prevent metastasis. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.