The use of biomarkers in diagnosis, therapy and prognosis has gained increasing interest over the last decades. In particular, the analysis of biomarkers in cancer patients within the pre- and post-therapeutic period is required to identify several types of cells, which carry a risk for a disease progression and subsequent post-therapeutic relapse. Cancer stem cells (CSCs) are a subpopulation of tumor cells that can drive tumor initiation and can cause relapses. At the time point of tumor initiation, CSCs originate from either differentiated cells or adult tissue resident stem cells. Due to their importance, several biomarkers that characterize CSCs have been identified and correlated to diagnosis, therapy and prognosis. However, CSCs have been shown to display a high plasticity, which changes their phenotypic and functional appearance. Such changes are induced by chemo- and radiotherapeutics as well as senescent tumor cells, which cause alterations in the tumor microenvironment. Induction of senescence causes tumor shrinkage by modulating an anti-tumorigenic environment in which tumor cells undergo growth arrest and immune cells are attracted. Besides these positive effects after therapy, senescence can also have negative effects displayed post-therapeutically. These unfavorable effects can directly promote cancer stemness by increasing CSC plasticity phenotypes, by activating stemness pathways in non-CSCs, as well as by promoting senescence escape and subsequent activation of stemness pathways. At the end, all these effects can lead to tumor relapse and metastasis. This review provides an overview of the most frequently used CSC markers and their implementation as biomarkers by focussing on deadliest solid (lung, stomach, liver, breast and colorectal cancers) and hematological (acute myeloid leukemia, chronic myeloid leukemia) cancers. Furthermore, it gives examples on how the CSC markers might be influenced by therapeutics, such as chemo- and radiotherapy, and the tumor microenvironment. It points out, that it is crucial to identify and monitor residual CSCs, senescent tumor cells, and the pro-tumorigenic senescence-associated secretory phenotype in a therapy follow-up using specific biomarkers. As a future perspective, a targeted immune-mediated strategy using chimeric antigen receptor based approaches for the removal of remaining chemotherapy-resistant cells as well as CSCs in a personalized therapeutic approach are discussed.

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Cancer Stem Cells-Origins and Biomarkers: Perspectives for Targeted Personalized Therapies.

Response and Resistance to BCR-ABL1-Targeted Therapies.

Chronic myeloid leukemia (CML) is caused by BCRABL1 in a cell with the biological potential, intrinsic or acquired, to cause leukemia. This cell is commonly termed the CML leukemia stem cell (LSC). In humans a CML LSC is operationally-defined by ≥1 in vitro or in vivo assays of human leukemia cells transferred to immune-deficient mice. Results of these assays are sometimes discordant. There is also the unproved assumption that biological features of a CML LSC are stable. These considerations make accurate and precise identification of a CML LSC difficult or impossible. In this review, we consider biological features of CML LSCs defined by these assays. We also consider whether CML LSCs are susceptible to targeting by tyrosine kinase inhibitors (TKIs) and other drugs, and whether elimination of CML LSCs is needed to achieve therapy-free remission or cure CML.

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Chronic myeloid leukemia stem cells

Not all chronic myeloid leukemia (CML) patients are cured with tyrosine kinase inhibitors (TKIs), and a proportion of them develop resistance. Recently, continuous BCR-ABL gene expression has been found in resistant cells with undetectable BCR-ABL protein expression, indicating that resistance may occur through kinase independent mechanisms, mainly due to the persistence of leukemia stem cells (LSCs). LSCs reside in the bone marrow niche in a quiescent state, and are characterized by a high heterogeneity in genetic, epigenetic, and transcriptional mechanisms. New approaches based on single cell genomics have offered the opportunity to identify distinct subpopulations of LSCs at diagnosis and during treatment. In the one hand, TKIs are not able to efficiently kill CML-LSCs, but they may be responsible for the modification of some LSCs characteristics, thus contributing to heterogeneity within the tumor. In the other hand, the bone marrow niche is responsible for the interactions between surrounding stromal cells and LSCs, resulting in the generation of specific signals which could favor LSCs cell cycle arrest and allow them to persist during treatment with TKIs. Additionally, LSCs may themselves alter the niche by expressing various costimulatory molecules and secreting suppressive cytokines, able to target metabolic pathways, create an anti-apoptotic environment, and alter immune system functions. Accordingly, the production of an immunosuppressant milieu may facilitate tumor escape from immune surveillance and induce chemo-resistance. In this review we will focus on BCR-ABL-independent mechanisms, analyzing especially those with a potential clinical impact in the management of CML patients.

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BCR-ABL Independent Mechanisms of Resistance in Chronic Myeloid Leukemia.

CD26/DPP4 - a potential biomarker and target for cancer therapy.

Chronic Myeloid Leukemia (CML) patients in sustained “deep molecular response” (DMR) may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+CML CD34+/CD38-LSCs were found to specifically co-express CD26(dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+LSCs by flow-cytometry in peripheral blood. Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow-cytometry analysis on PB CD45+/CD34+/CD38- stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+LSCs (median 19.20/µL, range 0.27-698.6). PB CD26+LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/µL; range 0.0012-0.66) and in 74/112 (66%) additional patients studied on treatment free remission (TFR) (median 0.015/µL; range 0.006-0.76). Notably no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that “circulating” CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+LSCs during TKI treatment and the role of a “stem cell response” threshold to achieve and maintain TFR are ongoing.

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Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission

Despite widespread use of decitabine to treat acute myeloid leukaemia (AML), data on its effectiveness and safety in the real-world setting are scanty. Thus, to analyze the performance of decitabine in clinical practice, we pooled together patient-level data of three multicentric observational studies conducted since 2013 throughout Italy, including 306 elderly AML patients (median age 75 years), unfit for intensive chemotherapy, treated with first-line decitabine therapy at the registered schedule of 20 mg/m2 /iv daily for 5 days every 4 weeks. Overall response rate (ORR), overall survival (OS) curves, and multivariate hazard ratios (HRs) of all-cause mortality were computed. Overall, 1940 cycles of therapy were administered (median, 5 cycles/patient). A total of 148 subjects were responders and, therefore, ORR was 48.4%. Seventy-one patients (23.2%) had complete remission, 32 (10.5%) had partial remission, and 45 (14.7%) had haematologic improvement. Median OS was 11.6 months for patients with favourable-intermediate cytogenetic risk and 7.9 months for those with adverse cytogenetic risk. Median relapse-free survival after CR was 10.9 months (95% confidence interval [CI]: 8.7-16.0). In multivariate analysis, mortality was higher in patients with adverse cytogenetic risk (HR=1.58; 95% CI: 1.13-2.21) and increased continuously with white blood cell (WBC) count (HR=1.12; 95% CI: 1.06-1.18). A total of 183 infectious adverse events occurred in 136 patients mainly (>90%) within the first five cycles of therapy. This pooled analysis of clinical care studies confirmed, outside of clinical trials, the effectiveness of decitabine as first-line therapy for AML in elderly patients unfit for intensive chemotherapy. An adverse cytogenetic profile and a higher WBC count at diagnosis were, in this real life setting, unfavourable predictors of survival.

Giulia Alunni

Papers

Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission

Real‐world experience with decitabine as a first‐line treatment in 306 elderly acute myeloid leukaemia patients unfit for intensive chemotherapy