The Hedgehog pathway plays an important role in stem-cell biology and malignant transformation. Therefore, we investigated the expression and prognostic impact of Hedgehog pathway members in acute ...myeloid leukemia (AML).
Pretreatment samples from 104 newly diagnosed AML patients (AMLSG 07-04 trial) were analyzed by qPCR, and expression of Hedgehog family members was correlated with clinical outcome. Inhibition of GLI by GANT61 or shRNA was investigated in AML cells in vitro and in vivo.
Expression of receptors Smoothened and Patched-1 and their downstream mediators, GLI1, GLI2, and GLI3, was found in AML patients in contrast to Hedgehog ligands. GLI2 expression had a significant negative influence on event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS; P = 0.037, 0.026, and 0.013, respectively) and was correlated with FLT3 mutational status (P < 0.001). Analysis of a second, independent patient cohort confirmed the negative impact of GLI2 on EFS and OS (P = 0.007 and 0.003, respectively; n = 290). Within this cohort, GLI1 had a negative prognostic impact (P < 0.001 for both EFS and OS). Although AML cells did not express Hedgehog ligands by qPCR, AML patients had significantly increased Desert Hedgehog (DHH) plasma levels compared with healthy subjects (P = 0.002), in whom DHH was presumably provided by bone marrow niche cells. Moreover, the GLI inhibitor GANT61 or knockdown of GLI1/2 by shRNA caused antileukemic effects, including induction of apoptosis, reduced proliferation, and colony formation in AML cells, and a survival benefit in mice.
GLI expression is a negative prognostic factor and might represent a novel druggable target in AML.
Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral ...blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal ...influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring.
The transforming and tumor growth–promoting properties of Axl, a member of the Tyro3, Axl, and Mer (TAM) family of receptor tyrosine kinases (TAMRs), are well recognized. In contrast, little is known ...about the role of the TAMR ligand growth arrest–specific gene 6 (Gas6) in tumor biology. By using Gas6-deficient (Gas6−/−) mice, we show that bone marrow–derived Gas6 promotes growth and metastasis in different experimental cancer models, including one resistant to vascular endothelial growth factor inhibitors. Mechanistic studies reveal that circulating leukocytes produce minimal Gas6. However, once infiltrated in the tumor, leukocytes up-regulate Gas6, which is mitogenic for tumor cells. Consistent herewith, impaired tumor growth in Gas6−/− mice is rescued by transplantation of wild-type bone marrow and, conversely, mimicked by transplantation of Gas6−/− bone marrow into wild-type hosts. These findings highlight a novel role for Gas6 in a positive amplification loop, whereby tumors promote their growth by educating infiltrating leukocytes to up-regulate the production of the mitogen Gas6. Hence, inhibition of Gas6 might offer novel opportunities for the treatment of cancer.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Inhibition of tumor angiogenesis emerged as valuable strategy to treat cancer and has revolutionized the face of clinical
oncology by prolonging the life of numerous cancer patients. However, the ...duration of this response is rather short and tumors
rapidly evade treatment, leaving antiangiogenic treatment thus far unable to cure cancer. Hence, novel targets are needed
to diversify antiangiogenic treatments and to overcome resistance. Recent data support the concept that tumor infiltration
by bone marrow-derived myeloid cells confers resistance to current antiangiogenic drugs targeting primarily vascular endothelial
growth factor (VEGF). In this review, we will summarize (pre)clinical data on the role of PlGF and its receptor VEGFR-1 in
promoting angiogenesis and inflammation, and the “antimyeloangiogenic” activity of an antibody against PlGF (αPlGF), which
may help to overcome resistance against VEGF(R)Is. Because of these promising results, a humanized αPlGF antibody (TB403)
is currently evaluated in different phase I clinical trials in cancer patients.
Circulating tumor cells (CTCs) hold immense promise for unraveling tumor heterogeneity and understanding treatment resistance. However, conventional methods, especially in cancers like non-small cell ...lung cancer (NSCLC), often yield low CTC numbers, hindering comprehensive analyses. This study addresses this limitation by employing diagnostic leukapheresis (DLA) to cancer patients, enabling the screening of larger blood volumes. To leverage DLA's full potential, this study introduces a novel approach for CTC enrichment from DLAs.
DLA was applied to six advanced stage NSCLC patients. For an unbiased CTC enrichment, a two-step approach based on negative depletion of hematopoietic cells was used. Single-cell (sc) whole-transcriptome sequencing was performed, and CTCs were identified based on gene signatures and inferred copy number variations.
Remarkably, this innovative approach led to the identification of unprecedented 3,363 CTC transcriptomes. The extensive heterogeneity among CTCs was unveiled, highlighting distinct phenotypes related to the epithelial-mesenchymal transition (EMT) axis, stemness, immune responsiveness, and metabolism. Comparison with sc transcriptomes from primary NSCLC cells revealed that CTCs encapsulate the heterogeneity of their primary counterparts while maintaining unique CTC-specific phenotypes.
In conclusion, this study pioneers a transformative method for enriching CTCs from DLA, resulting in a substantial increase in CTC numbers. This allowed the creation of the first-ever single-cell whole transcriptome in-depth characterization of the heterogeneity of over 3,300 NSCLC-CTCs. The findings not only confirm the diagnostic value of CTCs in monitoring tumor heterogeneity but also propose a CTC-specific signature that can be exploited for targeted CTC-directed therapies in the future. This comprehensive approach signifies a major leap forward, positioning CTCs as a key player in advancing our understanding of cancer dynamics and paving the way for tailored therapeutic interventions.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Forty percent of non-small cell lung cancer (NSCLC) patients develop brain metastases, resulting in a dismal prognosis. However, patients in an oligo-metastatic brain disease setting seem to have ...better outcomes. Here, we investigate the possibility of using circulating tumor cells (CTCs) as biomarkers to differentiate oligo-metastatic patients for better risk assessment. Using the CellSearch
system, few CTCs were detected among NSCLC patients with brain metastases (
= 52, 12.5% ≥ two and 8.9% ≥ five CTC/7.5 mL blood) and especially oligo-metastatic brain patients (
= 34, 5.9%, and 2.9%). Still, thresholds of both ≥ two and ≥ five CTCs were independent prognostic indicators for shorter overall survival time among all of the NSCLC patients (
= 90, two CTC ≥ HR: 1.629,
= 0.024, 95% CI: 1.137⁻6.465 and five CTC ≥ HR: 2.846,
= 0.0304, CI: 1.104⁻7.339), as well as among patients with brain metastases (two CTC ≥ HR: 4.694,
= 0.004, CI: 1.650⁻13.354, and five CTC ≥ HR: 4.963,
= 0.003, CI: 1.752⁻14.061). Also, oligo-brain NSCLC metastatic patients with CTCs had a very poor prognosis (
= 0.019). Similarly, in other tumor entities, only 9.6% of patients with brain metastases (
= 52) had detectable CTCs. Our data indicate that although patients with brain metastases more seldom harbor CTCs, they are still predictive for overall survival, and CTCs might be a useful biomarker to identify oligo-metastatic NSCLC patients who might benefit from a more intense therapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision-making and disease monitoring. A suitable diagnostic-grade platform is required ...for the detection of tumor-specific mutations with high sensitivity in the circulating cell-free DNA (ccfDNA) of cancer patients. In this multicenter study, the ccfDNA of 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC) was evaluated using the UltraSEEK® Lung Panel on the MassARRAY® System, covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2, and PIK3CA against mutation-specific droplet digital PCR (ddPCR) and routine tumor tissue NGS. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy, were analyzed. A multicenter evaluation using reference material demonstrated an overall detection rate of over 90% for variant allele frequencies (VAFs) > 0.5%, irrespective of ccfDNA input. A comparison of UltraSEEK® and ddPCR analyses revealed a 90% concordance. An 80% concordance between therapeutically targetable mutations detected in tumor tissue NGS and ccfDNA UltraSEEK® analysis at baseline was observed. Nine of 84 (11%) tumor tissue mutations were not covered by UltraSEEK®. A decrease in ctDNA levels at 4–6 weeks after treatment initiation detected with UltraSEEK® correlated with prolonged median PFS (46 vs. 6 weeks; p < 0.05) and OS (145 vs. 30 weeks; p < 0.01). Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect mutations both at diagnosis and during disease progression and to monitor treatment response.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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