Expression of epithelial-mesenchymal transition-related markers and phenotypes during breast cancer progression.

PURPOSE: The study aimed to investigate expression of epithelial-to-mesenchymal transition (EMT)-related proteins and phenotypes during breast cancer progression and to relate this to patient outcome. METHODS: Protein expression patterns of E-cadherin, N-cadherin, twist, and vimentin were examined by immunohistochemistry on formalin-fixed paraffin-embedded samples from primary tumors (PTs) (n = 419), synchronous lymph node metastases (LNMs) (n = 131) and recurrences (n = 34) from patients included in an observational prospective primary breast cancer study. Markers were evaluated individually and combined as defined EMT phenotypes (epithelial, mesenchymal, partial EMT, and negative). EMT profiles were compared between matched tumor progression stages, and related to clinicopathological data and distant recurrence-free interval (DRFi). RESULTS: N-cadherin-positivity, vimentin-positivity, mesenchymal and partial EMT phenotypes were associated with more aggressive tumor characteristics such as triple-negative subtype. Single EMT markers and phenotype discordance rates between paired tumor samples were observed in the range of 2-35%. Non-epithelial phenotypes were more frequently identified in recurrences compared to PTs, however, no skewness of expression or phenotype was detected between PTs and matched LNMs or between PTs and matched recurrences (Exact McNemar test). Interestingly, patients with a twist positive PT had shorter DRFi, compared to patients with a twist negative PT (hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.2-5.1, P = 0.02). Essentially, the same effect was seen in multivariable analysis (HR 2.5, 95% CI 0.97-6.6, P = 0.06). CONCLUSION: The epithelial phenotype was indicated to be lost between PTs and recurrences as a reflection of tumor progression. Twist status of the PT was related to long-term prognosis warranting further investigation in larger cohorts.

Evolution of Estrogen Receptor Status from Primary Tumors to Metastasis and Serially Collected Circulating Tumor Cells.

BACKGROUND: The estrogen receptor (ER) can change expression between primary tumor (PT) and distant metastasis (DM) in breast cancer. A tissue biopsy reflects a momentary state at one location, whereas circulating tumor cells (CTCs) reflect real-time tumor progression. We evaluated ER-status during tumor progression from PT to DM and CTCs, and related the ER-status of CTCs to prognosis. METHODS: In a study of metastatic breast cancer, blood was collected at different timepoints. After CellSearch® enrichment, CTCs were captured on DropMount slides and evaluated for ER expression at baseline (BL) and after 1 and 3 months of therapy. Comparison of the ER-status of PT, DM, and CTCs at different timepoints was performed using the McNemar test. The primary endpoint was progression-free survival (PFS). RESULTS: Evidence of a shift from ER positivity to negativity between PT and DM was demonstrated (p = 0.019). We found strong evidence of similar shifts from PT to CTCs at different timepoints (p < 0.0001). ER-positive CTCs at 1 and 3 months were related to better prognosis. CONCLUSIONS: A shift in ER-status from PT to DM/CTCs was demonstrated. ER-positive CTCs during systemic therapy might reflect the retention of a favorable phenotype that still responds to therapy.

Detection of circulating tumor cells and circulating tumor DNA before and after mammographic breast compression in a cohort of breast cancer patients scheduled for neoadjuvant treatment.

PURPOSE: It is not known if mammographic breast compression of a primary tumor causes shedding of tumor cells into the circulatory system. Little is known about how the detection of circulating biomarkers such as circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) is affected by breast compression intervention. METHODS: CTCs and ctDNA were analyzed in blood samples collected before and after breast compression in 31 patients with primary breast cancer scheduled for neoadjuvant therapy. All patients had a central venous access to allow administration of intravenous neoadjuvant chemotherapy, which enabled blood collection from superior vena cava, draining the breasts, in addition to sampling from a peripheral vein. RESULTS: CTC and ctDNA positivity was seen in 26% and 65% of the patients, respectively. There was a significant increase of ctDNA after breast compression in central blood (p = 0.01), not observed in peripheral testing. No increase related with breast compression was observed for CTC. ctDNA positivity was associated with older age (p = 0.05), and ctDNA increase after breast compression was associated with high Ki67 proliferating tumors (p = 0.04). CTCs were more abundant in central compared to peripheral blood samples (p = 0.04). CONCLUSIONS: There was no significant release of CTCs after mammographic breast compression but more CTCs were present in central compared to peripheral blood. No significant difference between central and peripheral levels of ctDNA was observed. The small average increase in ctDNA after breast compression is unlikely to be clinically relevant. The results give support for mammography as a safe procedure from the point of view of CTC and ctDNA shedding to the blood circulation. The results may have implications for the standardization of sampling procedures for circulating tumor markers.

Longitudinal enumeration and cluster evaluation of circulating tumor cells improve prognostication for patients with newly diagnosed metastatic breast cancer in a prospective observational trial.

BACKGROUND: Circulating tumor cells (CTCs) carry independent prognostic information in patients with metastatic breast cancer (MBC) on different lines of therapy. Moreover, CTC clusters are suggested to add prognostic information to CTC enumeration alone but their significance is unknown in patients with newly diagnosed MBC. We aimed to evaluate whether longitudinal enumeration of circulating tumor cells (CTCs) and CTC clusters could improve prognostication and monitoring of patients with metastatic breast cancer (MBC) starting first-line therapy. METHODS: This prospective study included 156 women with newly diagnosed MBC. CTCs and CTC clusters were detected using CellSearch technology at baseline (BL) and after 1, 3, and 6 months of systemic therapy. The primary end point was progression-free survival (PFS) and the secondary end point overall survival (OS). Median follow-up time was 25 (7-69) months. RESULTS: There were 79 (52%) and 30 (20%) patients with ≥ 5 CTCs and ≥ 1 CTC cluster at baseline, respectively; both factors were significantly associated with impaired survival. Landmark analyses based on follow-up measurements revealed increasing prognostic hazard ratios for ≥ 5 CTCs and CTC clusters during treatment, predicting worse PFS and OS. Both factors added value to a prognostic model based on clinicopathological variables at all time points and ≥ 5 CTCs and presence of CTC clusters enhanced the model's C-index to > 0.80 at 1, 3, and 6 months. Importantly, changes in CTCs during treatment were significantly correlated with survival and patients with a decline from ≥ 5 CTCs at BL to < 5 CTCs at 1 month had a similar odds ratio for progression to patients with < 5 CTCs at BL and 1 month. Stratification of patients based on CTC count and CTC clusters into four groups (0 CTCs, 1-4 CTCs, ≥ 5 CTCs, and ≥ 1 CTC + CTC clusters) demonstrated that patients with CTC clusters had significantly worse survival compared to patients without clusters. CONCLUSIONS: Longitudinal evaluation of CTC and CTC clusters improves prognostication and monitoring in patients with MBC starting first-line systemic therapy. The prognostic value increases over time, suggesting that changes in CTC count are clinically relevant. The presence of CTC clusters adds significant prognostic value to CTC enumeration alone. TRIAL REGISTRATION: NCT01322893 . Registered on 25 March 2011.

The PDGF pathway in breast cancer is linked to tumour aggressiveness, triple-negative subtype and early recurrence.

PURPOSE: The platelet-derived growth factor (PDGF) signalling pathway is often dysregulated in cancer and PDGF-receptor expression has been linked to unfavourable prognostic factors in breast cancer (e.g. ER negativity, high Ki67 and high grade). This study aimed to evaluate the expression of PDGFRα, PDGFRß and ligand PDGF-CC in breast cancer in relation to molecular subtypes and prognosis. METHODS: Protein expression of tumour and/or stromal cell PDGFRα, PDGFRß and PDGF-CC was evaluated in primary tumours (N = 489), synchronous lymph node metastases (N = 135) and asynchronous recurrences (N = 39) using immunohistochemistry in a prospectively maintained cohort of primary breast cancer patients included during 1999-2003. Distant recurrence-free interval (DRFi) was the primary end-point. RESULTS: High expression of all investigated PDGF family members correlated to increasing Nottingham histopathological grade and high Ki67. Tumour cells displayed high expression of PDGFRα in 20%, and PDGF-CC in 21% of primary tumours, which correlated with the triple-negative subtype (TNBC). Patients with high PDGF-CC had inferior prognosis (P = 0.04) in terms of 5-year DRFi, whereas PDGFRα was up-regulated in lymph node metastasis and recurrences compared to primary tumours. High primary tumour PDGFRα was associated with increased risk of central nervous system (CNS) recurrence. CONCLUSIONS: High PDGFRα and PDGF-CC expression were linked to breast cancer with an aggressive biological phenotype, e.g. the TNBC subtype, and high PDGF-CC increased the risk of 5-year distant recurrence. Tumour cell PDGFRα was significantly up-regulated in lymph node metastases and asynchronous recurrences. Our findings support an active role of the PDGF signalling pathway in tumour progression.

Prognostic impact of circulating tumor cell apoptosis and clusters in serial blood samples from patients with metastatic breast cancer in a prospective observational cohort.

BACKGROUND: Presence of circulating tumor cells (CTCs) is a validated prognostic marker in metastatic breast cancer. Additional prognostic information may be obtained by morphologic characterization of CTCs. We explored whether apoptotic CTCs, CTC clusters and leukocytes attached to CTCs are associated with breast cancer subtype and prognosis at base-line (BL) and in follow-up (FU) blood samples in patients with metastatic breast cancer scheduled for first-line systemic treatment. METHODS: Patients with a first metastatic breast cancer event were enrolled in a prospective observational study prior to therapy initiation and the CellSearch system (Janssen Diagnostics) was used for CTC enumeration and characterization. We enrolled patients (N = 52) with ≥5 CTC/7.5 ml blood at BL (median 45, range 5-668) and followed them with blood sampling for 6 months during therapy. CTCs were evaluated for apoptotic changes, CTC clusters (≥3 nuclei), and leukocytes associated with CTC (WBC-CTC, ≥1 CTC + ≥1 leukocytes) at all time-points by visual examination of the galleries generated by the CellTracks Analyzer. RESULTS: At BL, patients with triple-negative and HER2-positive breast cancer had blood CTC clusters present more frequently than patients with hormone receptor-positive cancer (P = 0.010). No morphologic characteristics were associated with prognosis at BL, whereas patients with apoptotic CTCs or clusters in FU samples had worse prognosis compared to patients without these characteristics with respect to progression-free (PFS) and overall survival (OS) (log-rank test: P = 0.0012 or lower). Patients with apoptotic or clustered CTCs at any time-point had impaired prognosis in multivariable analyses adjusting for number of CTCs and other prognostic factors (apoptosis: HROS = 25, P < 0.001; cluster: HROS = 7.0, P = 0.006). The presence of WBC-CTCs was significantly associated with an inferior prognosis in terms of OS at 6 months in multivariable analysis. CONCLUSIONS: Patients with a continuous presence of apoptotic or clustered CTCs in FU samples after systemic therapy initiation had worse prognosis than patients without these CTC characteristics. In patients with ≥5 CTC/7.5 ml blood at BL, morphologic characterization of persistent CTCs could be an important prognostic marker during treatment, in addition to CTC enumeration alone. Clinical Trials (NCT01322893), registration date 21 March 2011.

A novel method for downstream characterization of breast cancer circulating tumor cells following CellSearch isolation.

BACKGROUND: Enumeration of circulating tumor cells (CTCs) obtained from minimally invasive blood samples has been well established as a valuable monitoring tool in metastatic and early breast cancer, as well as in several other cancer types. The gold standard technology for detecting CTCs in blood against a backdrop of millions of leukocytes is the FDA-approved CellSearch system (Janssen Diagnostics), which relies on EpCAM-based immunomagnetic separation. Secondary characterization of these cells could enable treatment selection based on specific targets in these cells, as well as providing a real time window into the metastatic process and offering unique insights into tumor heterogeneity. The objective of this study was to develop a method for downstream characterization of CTCs following isolation with the CellSearch system. METHODS: An in vitro CTC model system focusing on clinically useful treatment predictive biomarkers in breast cancer, specifically the estrogen receptor α (ERα) and the human epidermal growth factor receptor 2 (HER2), was established using healthy donor blood spiked with breast cancer cell lines MCF7 (ERα(+)/HER2(-)) and SKBr3 (ERα(-)/HER2(+)). Following CTC isolation by CellSearch, the captured CTCs were further enriched and fixed on a microscope slide using the in-house-developed CTC-DropMount technique. RESULTS: The recovery rate of CTCs after CellSearch Profile analysis and CTC-DropMount was 87%. A selective and consistent triple-immunostaining protocol was optimized. Cells positive for DAPI, cytokeratin (CK) 8, 18 and 19, but negative for the leukocyte-specific marker CD45, were classified as CTCs and subsequently analyzed for ERα and HER2 expression. The method was verified in breast cancer patient samples, thus demonstrating its clinical relevance. CONCLUSIONS: Our results show that it is possible to ascertain the status of important predictive biomarkers expressed in breast cancer CTCs using the newly developed CTC-DropMount technique. Downstream characterization of multiple biomarkers using a standard fluorescence microscope demonstrates that important clinical and biological information may be obtained from a single patient blood sample following either CellSearch epithelial or profile analyses. TRIAL REGISTRATION: Clinical Trials NCT01322893.

Transurethral bladder tumor resection can cause seeding of cancer cells into the bloodstream.

PURPOSE: Transurethral bladder tumor resection is the initial diagnostic procedure for bladder cancer. Hypothetically tumor resection could induce seeding of cancer cells into the circulation and subsequent metastatic disease. In this study we ascertain whether transurethral bladder tumor resection induces measurable seeding of cancer cells into the vascular system. MATERIALS AND METHODS: Patients newly diagnosed with suspected invasive bladder cancer and planned for transurethral resection of bladder tumor in 2012 to 2013 were enrolled in the study. Before transurethral bladder tumor resection a vascular surgeon placed a venous catheter in the inferior vena cava via the femoral vein. Blood samples were drawn before and during the resection from the inferior vena cava and a peripheral vein, and analyzed for circulating cancer cells using the CellSearch® system. The number of circulating tumor cells identified was compared in preoperative and intraoperative blood samples. RESULTS: The circulating tumor cell data on 16 eligible patients were analyzed. In 6 of 7 positive inferior vena cava samples (86%) the number of circulating tumor cells was increased intraoperatively (28 vs 9, 28 vs 0, 28 vs 5, 3 vs 0, 4 vs 0, 1 vs 0), and results were similar, although less conclusive, for the corresponding peripheral vein samples. CONCLUSIONS: Our study confirms that tumor cells can be released into the circulation during transurethral bladder tumor resection. It is currently unknown whether this will increase the risk of metastatic disease.

Association between insulin-like growth factor-1 receptor (IGF1R) negativity and poor prognosis in a cohort of women with primary breast cancer.

BACKGROUND: Resistance towards endocrine therapy is a great concern in breast cancer treatment and may partly be explained by the activation of compensatory signaling pathways. The aim of the present study was to investigate if the insulin-like growth factor-1 receptor (IGF1R) signaling pathway was activated or deregulated in breast cancer patients and to explore if any of the markers were prognostic, with or without adjuvant tamoxifen. This signaling pathway has been suggested to cause estrogen independent cell growth and thus contribute to resistance to endocrine treatment in estrogen receptor (ER) positive breast cancer. METHODS: The protein expression of IGF1R, phosphorylated Mammalian Target of Rapamycin (p-mTOR) and phosphorylated S6 ribosomal protein (p-S6rp) were investigated by immunohistochemistry using tissue microarrays in two patient cohorts. Cohort I (N = 264) consisted of mainly postmenopausal women with stage II breast cancer treated with tamoxifen for 2 years irrespective of ER status. Cohort II (N = 206) consisted of mainly medically untreated, premenopausal patients with node-negative breast cancer. Distant disease-free survival (DDFS) at 5 years was used as end-point for survival analyses. RESULTS: We found that lower IGF1R expression was associated with worse prognosis for tamoxifen treated, postmenopausal women (HR = 0.70, 95% CI = 0.52 - 0.94, p = 0.016). The effect was seen mainly in ER-negative patients where the prognostic effect was retained after adjustment for other prognostic markers (adjusted HR = 0.49, 95% CI = 0.29 - 0.82, p = 0.007). Expression of IGF1R was associated with ER positivity (p < 0.001) in the same patient cohort. CONCLUSIONS: Our results support previous studies indicating that IGF1R positivity reflects a well differentiated tumor with low metastatic capacity. An association between lack of IGF1R expression and worse prognosis was mainly seen in the ER-negative part of Cohort I. The lack of co-activation of downstream markers (p-mTOR and p-S6rp) in the IGF1R pathway suggested that the prognostic effect was not due to complete activation of this pathway. Thus, no evidence could be found for a compensatory function of IGF1R signaling in the investigated cohorts.

No evidence for shedding of circulating tumor cells to the peripheral venous blood as a result of mammographic breast compression.

This pilot study aimed to investigate whether mammographic compression procedures might cause shedding of tumor cells into the circulatory system as reflected by circulating tumor cell (CTC) count in peripheral venous blood samples. From March to October 2012, 24 subjects with strong suspicion of breast malignancy were included in the study. Peripheral blood samples were acquired before and after mammography. Enumeration of CTCs in the blood samples was performed using the CellSearch(®) system. The pressure distribution over the tumor-containing breast was measured using thin pressure sensors. The median age was 66.5 years (range, 51-87 years). In 22 of the 24 subjects, breast cancer was subsequently confirmed. The difference between the average mean tumor pressure 6.8 ± 5.3 kPa (range, 1.0-22.5 kPa) and the average mean breast pressure 3.4 ± 1.6 kPa (range, 1.5-7.1 kPa) was statistically significant (p < 0.001), confirming that there was increased pressure over the tumor. The median pathological tumor size was 19 mm (range, 9-30 mm). Four subjects (17 %) were CTC positive before compression and two of these (8 %) were also CTC positive after compression. A total of seven CTCs were isolated with a mean size of 8 × 6 µm(2) (range of the longest diameter, 5-12 µm). The study supports the view that mammography is a safe procedure from the point of view of tumor cell shedding to the peripheral blood.

Increased gene copy number of KIT and VEGFR2 at 4q12 in primary breast cancer is related to an aggressive phenotype and impaired prognosis.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and no targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and KIT, have shown some promising results for patients with TNBC. The aim of the study was to investigate whether gains and/or amplifications of VEGFR2 and KIT, located at 4q12, occur in TNBC. Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and KIT in 83 primary human breast cancers including 31 TNBCs. Gains were defined as ≥ 4 gene copies in >40% of the cancer cells, whereas amplification was defined as CEP >2 in more than 10% of the cancer cells. A tumor was considered FISH positive for KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and KIT in nine (18%) cases for both genes, but no significant difference between TNBCs and non-TNBCs was found. FISH positivity for VEGFR2 and KIT was significantly correlated (χ(2) test, P < 0.001), and significantly related to ER negativity and high Nottingham histological grade (NHG). A significantly worse 5-year breast cancer specific survival (BCSS) was seen for FISH positive cases. Increased copy number of VEGFR2 and KIT thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category.

Gene expression in metastatic breast cancer-patterns in primary tumors and metastatic tissue with prognostic potential.

Background: Metastatic breast cancer (MBC) is the main cause of breast cancer-related death. The outcome of MBC varies, and there is a lack of biomarkers to aid in prognostication. The primary aim of this study was to evaluate the prognostic value of gene expression (GEX) signatures in the primary tumor (PT) and distant metastasis (DM) for progression-free survival (PFS) and overall survival (OS). The secondary aim was to describe GEX changes through MBC evolution and to identify MBC subtypes. Methods: RNA was extracted from the PT, lymph node metastasis (LNM), and DM from MBC patients in a prospective observational study (n = 142; CTC-MBC NCT01322893) and was subjected to GEX analysis retrospectively using the NanoString Breast Cancer 360™ panel. 31 continuous GEX variables in DMs and PTs were analyzed for PFS and OS by Cox regression analysis and Kaplan-Meier estimates. Multivariable Cox regressions were adjusted for number of DM sites and CTCs, visceral metastasis, ECOG status, age at MBC diagnosis and, in additional analyses, PAM50 subtype. Differential GEX analyses and Euclidean distances were used to describe subgroup differences and visualize within-patient heterogeneity. Results: Compared to DM GEX, GEX of the PT was at least equally useful for predicting MBC outcome. The strongest marker for a favorable PFS, both when expressed in the PT and the DM was AR, even after adjustment for prognostic markers including PAM50. GEX signatures related to hormone responsiveness, including ESR1, FOXA1, PGR, and AR were favorable prognostic markers, and the p53 signature was unfavorable for PFS when expressed in PT or DM. The previously published PAM50MET signature was prognostic for both PFS and OS. We established five distinct DM GEX profiles where two associated with liver and bone metastases, respectively. Finally, we identified four DM GEX profiles able to identify MBCs with poor OS in this cohort. Conclusion: GEX of both DM and PT are useful in MBC prognostication. GEX of AR adds prognostic information for MBC. Our descriptive analyses illuminate the biological differences between MBCs in relation to outcome and metastatic site.

Macrophage infiltration promotes regrowth in MYCN-amplified neuroblastoma after chemotherapy.

Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment.

Patient-derived models: Advanced tools for precision medicine in neuroblastoma.

Neuroblastoma is a childhood cancer derived from the sympathetic nervous system. High-risk neuroblastoma patients have a poor overall survival and account for ~15% of childhood cancer deaths. There is thus a need for clinically relevant and authentic models of neuroblastoma that closely resemble the human disease to further interrogate underlying mechanisms and to develop novel therapeutic strategies. Here we review recent developments in patient-derived neuroblastoma xenograft models and in vitro cultures. These models can be used to decipher mechanisms of metastasis and treatment resistance, for drug screening, and preclinical drug testing. Patient-derived neuroblastoma models may also provide useful information about clonal evolution, phenotypic plasticity, and cell states in relation to neuroblastoma progression. We summarize current opportunities for, but also barriers to, future model development and application. Integration of patient-derived models with patient data holds promise for the development of precision medicine treatment strategies for children with high-risk neuroblastoma.

Clinically relevant treatment of PDX models reveals patterns of neuroblastoma chemoresistance.

Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB). Here, we developed a clinically relevant in vivo treatment protocol mimicking the first-line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN-amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal NB chemoresistance mechanisms. Intrinsic resistance was associated with high genetic diversity and an embryonic phenotype. Relapsed NB with acquired resistance showed a decreased adrenergic phenotype and an enhanced immature mesenchymal-like phenotype, resembling multipotent Schwann cell precursors. NBs with a favorable treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts. Novel integrated phenotypic gene signatures reflected treatment response and patient prognosis. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states. This work sheds light on the mechanisms of NB chemotherapy response and emphasizes the importance of transcriptional cell states in chemoresistance.

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis.

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.

PAM50 Intrinsic Subtype Profiles in Primary and Metastatic Breast Cancer Show a Significant Shift toward More Aggressive Subtypes with Prognostic Implications.

BACKGROUND: PAM50 breast cancer intrinsic subtyping adds prognostic information in early breast cancer; however, the role in metastatic disease is unclear. We aimed to identify PAM50 subtypes in primary tumors (PTs) and metastases to outline subtype changes and their prognostic role. METHODS: RNA was isolated from PTs, lymph node metastases (LNMs), and distant metastases (DMs) in metastatic breast cancer patients (n = 140) included in a prospective study (NCT01322893). Gene expression analyses were performed using the Breast Cancer 360 (BC360) assay from Nano-String. The subtype shifts were evaluated using McNemar and symmetry tests, and clinical outcomes were evaluated with log-rank tests and Cox regression. RESULTS: The PAM50 subtype changed in 25/59 of paired samples between PTs and LNMs (Psymmetry = 0.002), in 31/61 between PTs and DMs (Psymmetry < 0.001), and in 16/38 between LNMs and DMs (Psymmetry = 0.004). Shifts toward subtypes with worse outcomes were the most common. Patients with shifts from the luminal PT to non-luminal DM subtypes had worse progression-free survival compared to patients with a stable subtype (hazard ratio (HR): 2.3; 95% confidence interval (CI): 1.14-4.68, p = 0.02). CONCLUSION: Strong evidence of PAM50 subtype shifts toward unfavorable subtypes were seen between PTs and metastatic samples. For patients with a shift in subtype from luminal PT to non-luminal DM, a worse prognosis was noted.

Anti-tumor effects of rigosertib in high-risk neuroblastoma.

High-risk neuroblastoma has a poor prognosis despite intense treatment, demonstrating the need for new therapeutic strategies. Here we evaluated the effects of rigosertib (ON-01910.Na) in preclinical models of high-risk neuroblastoma. Among several hundred cancer cell lines representing 24 tumor types, neuroblastoma was the most sensitive to rigosertib. Treatment of MYCN-amplified neuroblastoma organoids resulted in organoid disintegration, decreased cell viability, and increased apoptotic cell death. Neuroblastoma response to rigosertib involved G2M cell cycle arrest and decreased phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Rigosertib delayed tumor growth and prolonged survival of mice carrying neuroblastoma MYCN-amplified PDX tumors (median survival: 31 days, treated; 22 days, vehicle) accompanied with increased apoptosis in treated tumors. We further identified vincristine and rigosertib as a potential promising drug combination treatment. Our results show that rigosertib might be a useful therapeutic agent for MYCN-amplified neuroblastomas, especially in combination with existing agents.

Inhibition of fatty acid synthesis induces differentiation and reduces tumor burden in childhood neuroblastoma.

Many metabolic pathways, including lipid metabolism, are rewired in tumors to support energy and biomass production and to allow adaptation to stressful environments. Neuroblastoma is the second deadliest solid tumor in children. Genetic aberrations, as the amplification of the MYCN-oncogene, correlate strongly with disease progression. Yet, there are only a few molecular targets successfully exploited in the clinic. Here we show that inhibition of fatty acid synthesis led to increased neural differentiation and reduced tumor burden in neuroblastoma xenograft experiments independently of MYCN-status. This was accompanied by reduced levels of the MYCN or c-MYC oncoproteins and activation of ERK signaling. Importantly, the expression levels of genes involved in de novo fatty acid synthesis showed prognostic value for neuroblastoma patients. Our findings demonstrate that inhibition of de novo fatty acid synthesis is a promising pharmacological intervention strategy for the treatment of neuroblastoma independently of MYCN-status.

Serial evaluation of serum thymidine kinase activity is prognostic in women with newly diagnosed metastatic breast cancer.

The rapid development of new therapies in metastatic breast cancer (MBC), entails a need for improved prognostic and monitoring tools. Thymidine kinase 1 (TK1) is involved in DNA synthesis and its activity correlates to outcome in cancer patients. The aim of this study was to evaluate serum TK1 activity (sTK1) levels in MBC patients as a tool for prognostication and treatment monitoring. 142 women with MBC scheduled for 1st line systemic treatment were included in a prospective observational study. sTK1 was measured at baseline (BL) and at 1, 3 and 6 months and correlations to progression-free and overall survival (PFS, OS) evaluated. High sTK1 levels (above median) correlated to worse PFS and OS at BL, also after adjusting for other prognostic factors. sTK1 levels were significantly associated with PFS and OS measured from follow-up time points during therapy. Changes from 3 to 6 months during therapy significantly correlated to PFS and OS, whereas early changes did not. We could demonstrate sTK1 level as an independent prognostic factor in patients with newly diagnosed MBC. Changes in sTK1 levels from 3 to 6 months correlated to PFS and OS. Future studies of sTK1 are warranted to further define its clinical utility.