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Metastasis is the cause of over 90% of all deaths associated with breast cancer, yet the strategies to predict cancer spreading based on primary tumor profiles and therefore prevent metastasis are egregiously limited. As rare precursor cells to metastasis, circulating tumor cells (CTCs) in multicellular clusters in the blood are 20-50 times more likely to produce viable metastasis than single CTCs. However, the molecular mechanisms underlying various CTC clusters, such as homotypic tumor cell clusters and heterotypic tumor-immune cell clusters, are yet to be fully elucidated. Combining machine learning-assisted computational ranking with experimental demonstration to assess cell adhesion candidates, we identified a transmembrane protein Plexin- B2 (PB2) as a new therapeutic target that drives the formation of both homotypic and heterotypic CTC clusters. High PB2 expression in human primary tumors predicts an unfavorable distant metastasis-free survival and is enriched in CTC clusters compared to single CTCs in advanced breast cancers. Loss of PB2 reduces formation of homotypic tumor cell clusters as well as heterotypic tumor-myeloid cell clusters in triple-negative breast cancer. Interactions between PB2 and its ligand Sema4C on tumor cells promote homotypic cluster formation, and PB2 binding with Sema4A on myeloid cells (monocytes) drives heterotypic CTC cluster formation, suggesting that metastasizing tumor cells hijack the PB2/Sema family axis to promote lung metastasis in breast cancer. Additionally, using a global proteomic analysis, we identified novel downstream effectors of the PB2 pathway associated with cancer stemness, cell cycling, and tumor cell clustering in breast cancer. Thus, PB2 is a novel therapeutic target for preventing new metastasis.

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Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44–CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44–PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis.Significance:CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44–PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes.See related commentary by Rodrigues and Vanharanta, p. 22.This article is highlighted in the In This Issue feature, p. 1

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conference program

The second International Cancer Stem Cell Conference in Cleveland, Ohio, on September 20–23, 2016, convened 330 attendees from academic, industrial, and clinical organizations. It featured a debate on the concepts and challenges of the cancer stem cells (CSC) as well as CSC-centered scientific sessions on clinical trials, genetics and epigenetics, tumor microenvironment, immune suppression, metastasis, therapeutic resistance, and emerging novel concepts. The conference hosted 35 renowned speakers, 100 posters, 20 short talks, and a preconference workshop. The reported advances of CSC research and therapies fostered new collaborations across national and international borders, and inspired the next generation's young scientists. Cancer Res; 77(19); 5222–7. ©2017 AACR.

We report a case of a 58-year-old woman with a history of hypertension diagnosed at aged 35 years, on 5 antihypertensive agents and a history of intermittent spontaneous hypokalemia, was found to have a 6-cm left adrenal mass on computed tomography scan of the abdomen. The unenhanced computed tomography attenuation of the adrenal mass was −16 Hounsfield units (HU). The biochemical evaluation showed potassium of 2.8 mEq/L (SI unit, mmol/L) (reference range, 3.5-5.0), plasma aldosterone concentration of 61.3 ng/dL (SI unit, 1701 pmol/L) with plasma renin activity of 0.4 ng/mL/h (SI unit, μg/L/h). An overnight 1-mg dexamethasone suppression test showed nonsuppressible serum cortisol of 10.8 μg/dL (SI unit, 298 nmol/L). Dehydroepiandrosterone sulfate and ACTH were measured at 24.5 μg/dL (age-adjusted, 26-200) (SI unit, 0.66 μmol/L; 0.70-5.43) and

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC., Circulating tumor cell (CTC) clusters are more efficient at mediating metastasis as compared to single cells and are associated with poor prognosis in breast cancer. Here, the authors show that ICAM1 is enriched in CTC clusters and its loss suppresses cell-cell interaction and CTC cluster formation, and propose ICAM1 as a therapeutic target for treating breast cancer metastasis.

Cancer metastasis is largely incurable and accounts for 90% of breast cancer deaths, especially for the aggressive basal-like or triple negative breast cancer (TNBC). Combining patient database analyses and functional studies, we examined the association of integrin family members with clinical outcomes as well as their connection with previously identified microRNA regulators of metastasis, such as miR-206 that inhibits stemness and metastasis of TNBC. Here we report that the integrin receptor CD49b-encoding ITGA2, a direct target of miR-206, promotes breast cancer stemness and metastasis. ITGA2 knockdown suppressed self-renewal related mammosphere formation and pluripotency marker expression, inhibited cell cycling, compromised migration and invasion, and therefore decreased lung metastasis of breast cancer. ITGA2 overexpression reversed miR-206-caused cell cycle arrest in G1. RNA sequencing analyses revealed that ITGA2 knockdown inhibits genes related to cell cycle regulation and lipid metabolism, including CCND1 and ACLY as representative targets, respectively. Knockdown of CCND1 or ACLY inhibits mammosphere formation of breast cancer cells. Overexpression of CCND1 rescues the phenotype of ITGA2 knockdown-induced cell cycle arrest. ACLY-encoded ATP citrate lyase is essential to maintain cellular acetyl-CoA levels. CCND1 knockdown further mimics ITGA2 knockdown in abolishing lung colonization of breast cancer cells. We identified that the low levels of miR-206 as well as high expression levels of ITGA2, ACLY and CCND1 are associated with an unfavorable relapse-free survival of the patients with estrogen receptor-negative or high grade breast cancer, especially basal-like or TNBC, possibly serving as potential biomarkers of cancer stemness and therapeutic targets of breast cancer metastasis.

Accumulating evidence have demonstrated that circulating tumor cell (CTC) clusters have higher metastatic ability than single CTCs, and correlates with worse cancer patient’s outcomes. The heterotypic CTC clusters such as neutrophil-CTC clusters recently were identified in both cancer mouse models and cancer patients, leading to more efficient metastasis formation compared with homotypic CTC clusters. However, the mechanism by which neutrophils are associated with CTCs remains elusive. In this study, we found that the intercellular adhesion molecule (ICAM-1) on triple-negative breast cancer (TNBC) cells mediates their binding with CD11b+ neutrophils, and CD11b deficiency inhibited TNBC metastasis In vivo. Additionally, CD11b mediated H2O2 production of neutrophils. Further studies indicated that ICAM-1 promotes uPAR secretion, which functions as a chemoattractant for neutrophils. Knockdown of uPAR in ICAM-1+ TNBC cells reduced lung-infiltrating neutrophils, and lung metastasis. The bioinformatics analysis showed uPAR is highly expressed in TNBCs, which positively correlates with higher neutrophil infiltration and negatively with breast cancer patient’s survival. Together, our findings discover a novel chemoattractant role of suPAR in TNBC metastasis, and provides a rationale for targeting ICAM-1-uPAR-CD11b axis to block CTC-neutrophil cluster formation, and its-mediated metastasis.

Accumulating evidence demonstrates that circulating tumor cell (CTC) clusters have higher metastatic ability than single CTCs and negatively correlate with cancer patient outcomes. Along with homotypic CTC clusters, heterotypic CTC clusters (such as neutrophil–CTC clusters), which have been identified in both cancer mouse models and cancer patients, lead to more efficient metastasis formation and worse patient outcomes. However, the mechanism by which neutrophils bind to CTCs remains elusive. In this study, we found that intercellular adhesion molecule-1 (ICAM-1) on triple-negative breast cancer (TNBC) cells and CD11b on neutrophils mediate tumor cell–neutrophil binding. Consequently, CD11b deficiency inhibited tumor cell–neutrophil binding and TNBC metastasis. Furthermore, CD11b mediated hydrogen peroxide (H2O2) production from neutrophils. Moreover, we found that ICAM-1 in TNBC cells promotes tumor cells to secrete suPAR, which functions as a chemoattractant for neutrophils. Knockdown of uPAR in ICAM-1+ TNBC cells reduced lung-infiltrating neutrophils and lung metastasis. Bioinformatics analysis confirmed that uPAR is highly expressed in TNBCs, which positively correlates with higher neutrophil infiltration and negatively correlates with breast cancer patient survival. Collectively, our findings provide new insight into how neutrophils bind to CTC to facilitate metastasis and discover a novel potential therapeutic strategy by blocking the ICAM-1-suPAR-CD11b axis to inhibit TNBC metastasis.

Triple-negative breast cancer (TNBC) is one of the most aggressive and metastatic breast cancer subtypes lacking targeted therapy. Our recent work demonstrated that circulating tumor cell (CTC) clusters and polyclonal metastasis of TNBC are driven by aggregation of CD44+ cancer stem cells (CSC) and associated with an unfavorable prognosis, such as low overall survival. However, there is no existing therapeutic that can specifically block CTC or CSC cluster formation. Methods: Using patient-derived xenograft (PDX) models, we established an ex vivo tumor cell clustering assay for a pilot screening of blockade antibodies. After identifying EGFR as a target candidate, we modulated the gene expression and inhibited its kinase activity to determine its functional importance in tumor cell clustering and therapeutic inhibition of lung metastasis. We also examined the molecular regulation network of EGFR and a potential connection to CSC marker CD44 and microRNAs, which regulate CTC clustering. Results: We report here that EGFR inhibition successfully blocks circulating CSC (cCSC) clustering and lung metastasis of TNBC. EGFR enhances CD44-mediated tumor cell aggregation and CD44 stabilizes EGFR. Importantly, blocking EGFR by a novel anti-EGFR monoclonal antibody (clone LA1) effectively blocked cell aggregation in vitro and reduced lung metastasis in vivo. Furthermore, our data demonstrated that the tumor suppressor microRNA-30c serves as another negative regulator of cCSC clustering and lung metastasis by targeting CD44 as well as its downstream effector EGFR. Conclusion: Our studies identify a novel anti-EGFR therapeutic strategy to inhibit cCSC aggregation and therefore abolish cCSC cluster-mediated metastasis of TNBC.

Background Adrenal lipomatous tumors are uncommon fatty tumors of theadrenal glands with myelolipoma being the most common histologic feature.Adrenal myelolipoma is a benign adrenal cortical neoplasm composed of fat andmyeloid tissue, commonly diagnosed if fat composition of tumor is more than 50% on cross-sectional imaging studies. The myelolipomatous metaplasia is asecondary degenerative change in primary adrenal tumor, usually seen in benignadrenocortical adenomas and rarely in adrenocortical carcinomas and corticalneoplasms of uncertain malignant potential. Adrenocortical neoplasms areclassified as benign or malignant based on modified Weiss score, when the criteriaare not clear, the tumor is classified as a neoplasm of uncertain malignant lesion.As adrenal lipomatous tumors are usually benign and non-functional, patients withthese tumors often do not undergo hormonal evaluation. Clinical Case A 58-year-old female with a history of hypertension diagnosed atage 35 years, on five antihypertensive agents and a history of intermittentspontaneous hypokalemia, was found to have a 6 cm left adrenal myelolipoma onnon-contrast computed tomography of the abdomen performed when she presentedfor left flank pain due to nephrolithiasis. The biochemical evaluation showedpotassium of 2.8 meq/l, plasma aldosterone of 65.9 ng/dl with plasma renin activityof 0.1 ng/ml/hr. An overnight 1 mg dexamethasone suppression test showed nonsuppressible serum cortisol of 10.8 μg/dl. DHEAS, and ACTH were measured at24.5 μg/dl and Conclusion We report a rare case of aldosterone and cortisol co-secreting adrenalcortical neoplasm of uncertain malignant potential with lipomatous andmyelolipomatous metaplasia. While the majority of cases of myelolipoma arebenign and non-functioning, this case emphasizes the importance of looking forsymptoms and signs pointing to adrenal hyperfunction, thorough hormonal andmorphologic evaluation and, definitive treatment is curative unilateraladrenalectomy. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Tumor-initiating cells with reprogramming plasticity are thought to be essential for cancer development and metastatic regeneration in many cancers; however, the molecular mechanisms are not fully understood. This study reports that CD81, a tetraspanin protein marker of small extracellular vesicles (exosomes), functions as a binding partner of CD44 and facilitates self-renewal of tumor initiating cells. Using machine learning-assisted protein structure modeling, co-immunoprecipitation, and mutagenesis approaches, we further demonstrate that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. In-depth global and phosphoproteomic analyses of clustering tumor cells unveils endocytosis-related signature pathways of proteins and phosphorylation patterns regulated by CD81 and CD44 specifically or shared between two. Notably, CRISPR Cas9-mediated depletion of either CD44 or CD81 results in loss of both proteins in cancer cell-secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells for mammosphere formation. CD81 is expressed in >80% of human circulating tumor cells (CTCs) and specifically enriched in clustered CTCs along with CD44 isolated from breast cancer patients. Mimicking the phenotypes of CD44 deficiency, loss of CD81 also inhibits tumor cluster aggregation, tumorigenesis, and lung metastasis of triple negative breast cancer (TNBC), supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights the novel role of CD81 and its partnership with CD44 in cancer exosomes, self-renewal, CTC clustering, and metastasis initiation of TNBC.

This protocol for a Cochrane Review is out of date. Auhors have abandoned it.

Circulating tumor cells (CTCs) are vital components of liquid biopsies for diagnosis of residual cancer, monitoring of therapy response, and prognosis of recurrence. Scientific dogma focuses on metastasis mediated by single CTCs, but advancement of CTC detection technologies has elucidated multicellular CTC clusters, which are associated with unfavorable clinical outcomes and a 20- to 100-fold greater metastatic potential than single CTCs. While the mechanistic understanding of CTC cluster formation is still in its infancy, multiple cell adhesion molecules and tight junction proteins have been identified that underlie the outperforming attributes of homotypic and heterotypic CTC clusters, such as cell survival, cancer stemness, and immune evasion. Future directions include high-resolution characterization of CTCs at multiomic levels for diagnostic/prognostic evaluations and targeted therapies.

Tumor-initiating cells with reprogramming plasticity and/or de-differentiation attributes have been thought to initiate primary tumor development as well as to regenerate secondary tumors in metastatic organs; however, the molecular mechanisms are not fully understood. We previously found that breast tumor-initiating cell marker, CD44, directs multicellular aggregation and cluster formation of circulating tumor cells (CTCs), which further enhance stemness and survival of such cells, enabling metastatic colonization to the lungs. To further elucidate the molecular network underlying CTC cluster formation, we performed global proteomic profiling and discovered that the tetraspanin protein CD81, which is normally enriched in exosomes (small extracellular vesicles), is a new driver of cancer initiation and metastasis as a facilitator and target of CD44. Loss of CD81 compromises tumorigenicity and mammosphere formation of triple negative breast cancer (TNBC) cells. Assisted by machine learning-based algorithms and mutagenesis approach, we found that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. Notably, genetic knockout of CD44 or CD81 results in loss of both CD81 and CD44 in secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells, such as mammosphere formation. In addition, RNA sequencing analysis showed that CD81 knockdown up-regulates expression of a cell differentiation marker, SEMA7a, whose down-regulation partially rescues mammosphere formation inhibition by CD81 depletion. Clinically, CD81 expression was observed in >80% of CTCs and specifically enriched and co-expressed along with CD44 in clustered CTCs of breast cancer patients. Mimicing the phenotypes of CD44 deficiency, loss of CD81 also inhibited tumor cell aggregation and lung metastasis of TNBC in both human and mouse tumor models, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights a new driving role of CD81 in cancer exosome-induced stemness, clustered CTCs, and metastasis initiation of TNBC, reported for the first time to our knowledge.

Introduction: Circulating tumor cells (CTCs) mediate metastases, which account for 90% of solid tumor-related mortality. Compared to single cells, clustered CTCs mediate metastasis at a 20-100 times higher efficiency and are associated with lower overall survival in breast cancer (BC). We have recently identified a new mechanism of CTC cluster formation through cellular aggregation instead of cohesive shedding, and demonstrated that CTC clusters have enhanced stemness (Cancer Discovery, 2019). However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and polyclonal metastasis have yet to be fully elucidated. We hypothesize that molecular drivers of metastasis initiation enhance cancer stemness and CTC cluster formation, and serve as a novel therapeutic target for BC metastasis. Methods: Using single cell RNA sequencing, we compared tumor cells from the primary breast tumor site and lung metastases of BC patient-derived xenografts. We identified genes specifically expressed in the lung metastases and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. We performed proteomic and transcriptomic analyses as well as machine learning to elucidate the downstream signaling pathways and protein structural basis involved in CTC cluster formation and lung metastasis. Finally, we explored therapeutic intervention options in blocking CTC cluster formation and lung metastasis. New results: Compared to the primary breast tumor cells, we identified a stemness gene signature enriched in a subpopulation of the CD44+ lung metastases, with 30-60 fold higher expression of CD34, CD36, ICAM1, VCAM1, ZEB1, ALDH1A1, TGFBR2 and TSPAN8. Analysis of patient blood samples (N=40) revealed that CD44 and many of these new candidate proteins were enriched in CTC clusters in comparison to single CTCs. We then examined the CSC-related properties of these tumor cells, such as tumorigenesis, sphere formation, and lung metastasis. Knockdown of selected surface molecules (e.g. ICAM1) significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of ICAM1+/CD44+ tumor cells had increased stemness and tumor growth upon orthotopic implantation in vivo. Knockdown of ICAM1 dramatically reduced the self-renewal ability in mammosphere formation of breast tumor cells in vitro. In addition, our studies also revealed that these tumor cells cluster through CD44 and other surface protein-mediated homophilic binding between two neighboring tumor cells. Neutralizing antibodies significantly blocked tumor cluster formation and lung colonization. Conclusions: We identified new molecular mediators of CTC aggregation and lung metastasis in BC. We anticipate that specific blockade of tumor clustering could decrease cancer progression and improve survival of BC patients. Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Youbin Zhang, Andrew Davis, Carolina Reduzzi, Yue Cao, Yang Shen, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Single cell RNA sequencing-based identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2603.

Introduction: Breast cancer (BC) is the leading type of cancer and the second leading cause of cancer-related deaths among women in the US. Metastasis accounts for 90% of solid tumor-related mortality and is mainly mediated by hematogenous spread of circulating tumor cells (CTCs). Compared to single cells, clustered CTCs mediate metastasis at a 20-100 times higher efficiency and are associated with lower overall survival. We have recently identified a new mechanism of CTC cluster formation through cellular aggregation instead of cohesive shedding and demonstrated that CTC clusters have enhanced stemness (Cancer Discovery 2019). However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and polyclonal metastasis have yet to be fully elucidated. We hypothesize that molecular drivers of metastasis initiation enhance cancer stemness and CTC cluster formation and serve as a novel therapeutic target for BC metastasis. Methods: Using single-cell RNA sequencing, we compared tumor cells from the primary breast tumor site and lung metastases of breast cancer patient-derived xenograft. We identified genes with differential expression levels in the lung mets and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. Upon the candidate gene modulation, we performed proteomic and transcriptome analyses to elucidate the downstream signaling pathways involved in CTC cluster formation and lung metastasis. Finally, we explored therapeutic intervention options in blocking CTC cluster formation and lung metastasis. New Results: Compared to the primary breast tumor cells, we identified a stemness gene signature enriched in a subpopulation of the CD44+ lung metastases, with 30- to 60-fold higher expression of CD34, CD36, VCAM1, ZEB1, ALDH1A1, TGFBR2, TSPAN8, and ICAM1. Patient blood analyses (N=40) revealed that CD44 and many of these new candidate proteins are enriched in CTC clusters in comparison to single CTCs. We then examined the CSC-related properties of these tumor cells, such as tumorigenesis, sphere formation, and lung metastasis. Knockdown of selected surface molecules from the signature genes significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of these tumor cells had increased stemness and highest tumor growth upon orthotopic implantation in vivo. Knockdown of the signature genes dramatically reduced the self-renewal ability in mammosphere formation of breast tumor cells in vitro. In addition, our studies also revealed that these tumor cells cluster through CD44 and other surface protein-mediated homophilic binding between two neighboring tumor cells. Neutralizing antibodies significantly blocked tumor cluster formation and lung colonization. Conclusion: We identified new molecular mediators of CTC aggregation and lung metastasis in breast cancer. We anticipate that a specific blocking of tumor clustering could decrease cancer progression and improve survival of breast cancer patients. Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B42.

Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44–CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44–PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis. Significance: CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44–PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes. See related commentary by Rodrigues and Vanharanta, p. 22. This article is highlighted in the In This Issue feature, p. 1

Background: Circulating tumor cells (CTCs) are cells shed from primary tumor and circulate in the peripheral blood. They are considered the seeds of metastasis. Compared to single CTCs, clusters of multiple CTCs possess 50 times higher metastatic capacity in mouse breast cancer models. However, the mechanisms underlying the metastatic promotion effect of CTC clusters are unclear. In addition, whether clustered CTCs have cancer stem cell (CSC) properties and what stem cell markers they express have not been determined. Methods: Immunohistochemistry (IHC) was used to detect CTCs in vascular structures, and the stem cell markers in single and clustered CTCs. Lung metastases were either monitored by bioluminescence imaging (BLI) or visualized by fluorescence microscopy. Stem cell properties were examined by mammosphere assays in vitro and tumorigenic assays in vivo. Clustering assay was performed by culturing cells in Poly-HEMA coated plates (for cell lines) or collagen-coated plates (for primary cells derived from patient-derived breast tumor xenografts, PDXs), and then monitored by IncuCyte live cell dynamic imaging. Results: CD44+ enriched circulating tumor cell (CTC) clusters were found in the lung/liver vascular structures in vivo in both metastatic breast cancer patients and PDXs that develop spontaneous lung metastases. Comparing to single CD44+ cells, clustered CD44+ cells formed more mammospheres, increased tumorigenic potential, and promoted lung colonization. Combining siRNA-mediated knockdown and CRISPR/Cas-based knockdout, we found that CD44 is required for breast tumor cell cluster formation and lung colonization upon tail vein seeding. During cluster formation, EGFR was activated, which improved both stemness and survival of clustered CTCs. Anti-EGFR antibody mimicked CD44 knockdown to inhibit cluster formation of PDX-derived tumor cells. Administration of EGFR inhibitor Erlotinib efficiently inhibited CD44+ cells-mediated spontaneous metastases to the lungs without affecting primary tumor growth. Conclusions: Our data provide new insights into the cellular and molecular mechanisms of stem-like clustered CD44+ cells-seeded metastasis, and implicate that targeting CD44+ CTC clusters by inhibition of EGFR activity could be a new therapeutic strategy to treat metastatic breast cancer. Citation Format: Xia Liu, Wenjing Chen, Rokana Taftaf, Huiping Liu. Stem-like clusters of CD44+ circulating tumor cells seed metastases in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1921. doi:10.1158/1538-7445.AM2017-1921

Benign metastasizing leiomyoma (BML) is a rare disease that usually occurs in women of reproductive age. They typically have history of uterine leiomyoma treated with hysterectomy. BML can metastasize to distant organs, with the lung being the most common organ. We report two patients who presented with benign metastasizing leiomyoma to the lung. Our first case was a fifty-two-year-old female who presented with multiple lung masses, with a past medical history of uterine leiomyoma who underwent hysterectomy 17 years ago. A CT-guided biopsy showed benign appearing spindle cells and pathology confirmed her diagnosis with additional positive estrogen/progesterone receptor stains. Our second case was a fifty-six-year-old female who presented with multiple cavitary pulmonary nodules. She subsequently underwent a video-assisted thoracoscopic surgery (VATS) with wedge resection of one of the nodules. Pathology confirmed the diagnosis based on morphology and immunohistochemical staining strongly positive for estrogen/progesterone receptors. Benign metastasizing leiomyoma is a rare condition which may affect women of reproductive age. This should be considered in the differential in patients who present with multiple pulmonary nodules, especially with a history of uterine leiomyoma. Additional stains, such as estrogen/progesterone receptors, may need to be done to confirm the diagnosis.

VIPomas are rare neuroendocrine tumors that secrete vasoactive intestinal peptide. They are detected in 1 in ten million people per year. The majority of these tumors arises within the pancreas, but may also arise as other VIP-secreting tumors as in bronchogenic carcinoma, colon carcinoma, ganglioneuroblastoma, pheochromocytoma, hepatoma and adrenal tumors. They are characterized by profound diarrhea, hypokalemia, and achlorhydria and were first described by Verner and Morrison in 1958. It was also named pancreatic cholera as the cholera toxin acts; the Vasoactive Intestinal Peptide (VIP) causes elevation of the cyclic adenosine monophosphate, resulting in intestinal smooth muscle relaxation, inhibition of electrolyte absorption and profound secretory diarrhea. We are reporting a case involving a 42 year old male patient who presented with intermittent attacks of watery diarrhea up to 10 times per day for 3 weeks mostly worsening in the last 3 days prior to seeking medical advice. Stool studies were negative, but patient had hyperglycemia and mild hyponatremia. Further evaluation revealed a pancreatic mass with multiple hepatic focal lesions encountered in abdominal ultrasound and CT scan. Ultrasound guided biopsy revealed metastatic pancreatic endocrine carcinoma with VIP expression. Serum VIP level was elevated to 496 (Normal

Ruxolitinib is the only JAK 1/2 inhibitor approved by the FDA for the treatment of myelofibrosis (MF). It has been established that ruxolitinib helps improve disease-related constitutional symptoms and splenomegaly. However, studies have shown that ruxolitinib affects several cytokines (IL1, IL6, and TNF-α) and other immune processes (dendritic cells function and T-cell response) and has been linked to increased incidence of opportunistic and non-opportunistic infections. Here we report our experience at the Cleveland Clinic. A total of 50 patients (pts) with MF treated with ruxolitinib were included. The median age of the cohort was 68 years (range: 41-89), 28 of them were males and 22 were females. According to the Dynamic International Prognostic Scoring System (DIPSS), 5 pts had high risk, 23 had intermediate-2 risk, 21 had intermediate-1 risk and 1 had low risk disease. Of these pts, 18 developed infections during their treatment course. Of these infections, 12 were CTCAEv4.0 grade 1-2 and 6 were grade 3-4. Median age of the group was 66 years-old ranging between 50 and 83. Males=9 and females =9. The group was risk-stratified with DIPSS into high risk (N=3), intermediate-2 risk (N=7) and intermediate-1 risk (N=8).The infection started several months after initiation of JAK inhibitor treatment ranging between 2 weeks to 22 months (median of 8 months). Of note, 10 pts had episodes of infections prior to starting ruxolitinib (pneumonia N=5, cellulitis N=3, oral herpes N=2, UTI N=2, genital herpes N=1, Clostiridium difficile diarrhea N=1, viral diarrhea N=1, neutropenic fever N=1, spontaneous bacterial peritonitis N=1 and psoas muscle abscess N=1) . None of the patients had an active infection at the time of starting ruxolitinib. The median total dose given to the pts who developed grade1-2 infection was 20 mg daily, and for those who had grade 3-4 infection it was 15 mg daily. A variety of infections have been reported including pneumonia (N=4), cellulitis\folliculitis (N=4), Clostridium Difficile diarrhea (N=2), URI (N=1), neutropenic fever (N=1), MSSA bacteremia (N=1), herpes zoster activation (N=2), spontaneous bacterial peritonitis (N=1), perineal abscess (N=1) and one pt had a gluteal abscess after a bone marrow biopsy. Most of these infections resolved using oral or topical antibiotics (N=12). However, 6 pts required hospital admissions, and 5 of them required intravenous antibiotics. One of them required admission to the intensive care unit and two others required surgical interventions. Duration of treatment ranged between 1 to 8 weeks (median 2 weeks). Ruxolitinib had to be discontinued due to the severity of the infection in 2 pts, while the others either had a dose reduction or no dose alteration. Two pts required prophylactic antibiotics; one had recurrent SBP despite antimicrobial prophylaxis, while another pt required acyclovir prophylaxis while receiving glucocorticoids with ruxolitinib with no reactivation of Zoster infection. Predisposing factors for infections included a procedure (BMBx N=1), the concurrent use of other immunosuppressive drugs (steroids N=3, other agents N=3) and the prior use of systemic antibiotics in the pts who had C Diff infections (N=2). Only 2 pts had subsequent infections, one had oral herpes and the other had candida esophagitis which required hospitalization and treatment with IV antifungal drugs while on high dose steroids (dexamethasone 4 mg PO twice daily) for immune thrombocytopenia at the time of infection. Though 5 pts had already died at the time of the chart review, none of them died of infectious complications. In summary, infections can occur in patients treated with ruxolitinib but are generally mild. Most infections resolve after an adequate course of oral antimicrobial therapy suggesting that prophylactic antimicrobial agents may not be necessary or cost-effective in the vast majority of MF pts treated with ruxolitinib. Disclosures No relevant conflicts of interest to declare.

Myelofibrosis (MF) is a hematologic neoplasm characterized by variable degrees of cytopenias/ myeloproliferation, extramedullary hematopoiesis, disease-related constitutional symptoms and an increased risk for acute myeloid leukemia (AML) transformation. Ruxolitinib, a JAK1/2 inhibitor is FDA approved for the management of intermediate to high-risk MF. However, intermediate-2 and high-risk MF patients accounted for 99% of patients (pts) in the 2 pivotal trials that led to the approval of ruxolitinib in North America, Australia and Europe (COMFORT 1 and COMFORT 2 studies). However, even low and intermediate-1 (int-1) risk MF pts could have a high burden of disease. Here we report our experience of using ruxolitinib in MF pts stratified as low and int-1 risk by the Dynamic International Prognostic Scoring System (DIPSS). A total of 25 pts with low and Int-1 risk disease were treated with ruxolitinib at the Cleveland Clinic and Northwestern University. The median age of the cohort was 61 yrs (range=33-87; males=9, females=16). The median total symptom score (TSS) by Myeloproliferative Neoplasm Symptom Assessment Form (MPN SAF) was 20. The median pre-treatment spleen size by palpation was 13 cm below the left costal margin. Baseline median bone marrow (BM) fibrosis grading was 2 (on a scale 0-3; grade 1 N=3, grade 2 N=11, and grade 3 N=11). The indications for starting ruxolitinib were constitutional symptoms (fatigue N=20, night sweats N=4, pruritus N=1) and splenomegaly (N=13). The median starting dose was 5 mg twice daily, 10mg twice daily at 3 months and 6 months, and 5mg twice daily at 12 months. The median dose at last follow up was 10 mg twice daily. The median duration of treatment was 12 months. There was a median 73% reduction in the MPN SAF TSS compared to baseline (P< 0.001). There was improvement in each of the parameters of the TSS. The percentage of reduction in spleen size by palpation at 3, 6, 12 months was 49, 57, and 64%, respectively. In 5 patients who had a repeat BM biopsy, there was an improvement in their BM fibrosis by at least 1 grade (N=3), stable fibrosis (N=1) or increased fibrosis (N=1). Hematologic adverse events were reported at 3, 6, 12 months of treatment. Using Common Terminology Criteria for Adverse Events (CTCAEv4.0), there were seven with grade 3/4 anemia and six with grade 3/4 thrombocytopenia. Non-hematologic adverse events reported at 3, 6, 12 months of treatment included dizziness (N=1), headaches (N=1) and infections (pneumonia N=2, cellulitis N=2, perineal abscess N=1, Herpes Zoster N=2, and gluteal abscess after BM biopsy N=1; only one required hospitalization). Ruxolitinib was discontinued in 5 patients due to lack of clinical response (N=3) and grade 3/4 anemia/thrombocytopenia (N=2). The remaining 20 patients are still on treatment. None of the pts progressed to AML or died. The median follow up of our cohort was 27 months. Further, using a multi-Analyte ELISArray Kit, plasma concentrations of interleukins (IL):IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12, IL17A, INFg, TNFa and GM-CSF were measured in low/int-1 risk pts (N=3) and intermediate-2/high risk patients (N=4) and no statistically significant difference was found between the two risk groups (P>0.05) further supporting the high burden of disease observed even in low and int-1 risk MF patients. In conclusion, pts with low/int-1 risk MF have a high burden of disease and can achieve meaningful clinical responses to ruxolitinib similar to int-2/ high risk MF pts without severe toxicity. Larger studies are needed to further evaluate the safety and efficacy of ruxoltinib in this patient population. Disclosures No relevant conflicts of interest to declare.