G.J. Schuurhuis

Cancer stem cell definitions and terminology: the devil is in the details

The cancer stem cell (CSC) concept has important therapeutic implications, but its investigation has been hampered both by a lack of consistency in the terms used for these cells and by how they are defined. Evidence of their heterogeneous origins, frequencies and their genomic, as well as their phenotypic and functional, properties has added to the confusion and has fuelled new ideas and controversies. Participants in The Year 2011 Working Conference on CSCs met to review these issues and to propose a conceptual and practical framework for CSC terminology. More precise reporting of the parameters that are used to identify CSCs and to attribute responses to them is also recommended as key to accelerating an understanding of their biology and developing more effective methods for their eradication in patients.

Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells.

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects.

Aberrant marker expression patterns on the CD34+CD38− stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. In CD34-positive AML, the leukemic stem cell has been recognized as CD38 negative. This CD34+CD38− population survives chemotherapy and is most probable the cause of minimal residual disease (MRD). The outgrowth of MRD causes relapse and MRD can therefore serve as a prognostic marker. The key role of leukemogenic CD34+CD38− cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34+CD38− population in AML and high-risk MDS samples at diagnosis, including C-type lectin-like molecule-1 and several lineage markers/marker-combinations. Fluorescent in situ hybridization analysis revealed that marker-positive cells were indeed of malignant origin. The markers were neither expressed on normal CD34+CD38− cells in steady-state bone marrow (BM) nor in BM after chemotherapy. We found that these markers were indeed expressed in part of the patients on malignant CD34+CD38− cells in complete remission, indicating the presence of malignant CD34+CD38− cells. Thus, by identifying residual malignant CD34+CD38− cells after chemotherapy, MRD detection at the stem cell level turned out to be possible. This might facilitate characterization of these chemotherapy-resistant leukemogenic cells, thereby being of help to identify new targets for therapy.

Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein.

Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance.

Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen-thawed peripheral blood stem cell transplants

Apoptosis is the common cell death pathway which is initiated by a variety of different stimuli. The recognition of early apoptotic events would markedly improve reliability and convenience of apoptosis assays. In the present study the vital stain SytoR16 in combination with the permeability marker 7-amino actinomycin D, (7-AAD) has been used to identify an early stage of apoptosis, not detected with trypan blue or 7-AAD alone or with conventional apoptosis tests and not consistently and only partly detected by the early apoptosis marker annexin V. The method was established using solid tumour cell lines treated with TNF. Subsequently we applied it to determine apoptotic populations in CD34+ peripheral blood progenitor cells obtained from growth factor and/or chemotherapy mobilised patients and frozen/thawed according to standard stem cell transplantation protocols. In a cell line model as well as CD34+ progenitor cells, different subpopulations with decreased SytoR16 fluorescence (SytoR16int or SytoR16low, compared with the normal SytoR16high) appeared which are not, or only partly, apoptotic using conventional techniques including morphology or 7-AAD staining: eg percentages of SytoR16int/7-AAD− and SytoR16low/7-AAD− may amount to the majority of cells present in a particular CD34+ sample. Second, upon further incubation these subpopulations become late apoptotic/secondary necrotic much faster than the unmodified SytoR16high population, as determined with 7-AAD staining and morphology. Third, these cells have strongly or completely reduced clonogenic capacity for committed (CFU-GM) and early (LTC-IC, determined only for CD34+ cells) progenitors. This technique needs the inclusion of a blocker of P-glycoprotein, which is highly active in CD34+ progenitor cells. This prevents the interference of the detection of SytoR16low apoptotic cells by SytoR16lowcells resulting from P-glycoprotein activity. By comparison with other apoptosis markers we found that early apoptotic subpopulations were detected in the order SytoR16>annexin V>7-AAD. In conclusion, the combination of SytoR16 and 7-AAD detects apoptotic events earlier than conventional apoptosis techniques or annexin V. Compared to the presently available viability tests, it allows a much better estimation of the number of viable clonogenic CD34+ cells after freeze/thawing. Bone Marrow Transplantation (2001) 27, 487–498.

Extensive early apoptosis in frozen–thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation

Stem cell doses necessary for engraftment after myelo-ablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze–thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen–thawed grafts, threshold numbers for adequate haematological recovery of 2.8–3.0 × 106 CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2–1.3 × 106 CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34+ cells were sufficient for recovery (0.3–0.4 × 106CD34+ cells/kg). In contrast to freeze–thawing of leucapheresis material, a high viability of CD34+ cells was preserved during storage for 3 days at 4°C, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen–thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery.Bone Marrow Transplantation (2002) 29, 249–255. doi:10.1038/sj.bmt.1703357

High-frequency type I/II mutational shifts between diagnosis and relapse are associated with outcome in pediatric AML: Implications for personalized medicine

Although virtually all pediatric patients with acute myeloid leukemia (AML) achieve a complete remission after initial induction therapy, 30%-40% of patients will encounter a relapse and have a dismal prognosis. To prevent relapses, personalized treatment strategies are currently being developed, which target specific molecular aberrations. To determine relevance of established AML type I/II mutations that may serve as therapeutic targets, we assessed frequencies of these mutations and their persistence during disease progression in a large group (n = 69) of paired diagnosis and relapse pediatric AML specimens. In 26 of 42 patients (61%) harboring mutations at either stage of the disease, mutation status changed between diagnosis and relapse, particularly in FLT3, WT1, and RAS genes. Presence or gain of type I/II mutations at relapse was associated with a shorter time to relapse (TTR), whereas absence or loss correlated with longer TTR. Moreover, an adverse outcome was found for patients with activating mutations at relapse, which was statistically significant for FLT3/ITD and WT1 mutations. These findings suggest that mutational shifts affect disease progression. We hence propose that risk stratification, malignant cell detection, and selection of personalized treatment should be based on status of type I/II mutations both at initial diagnosis and during follow-up.

The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse

The majority of pediatric and younger adult (<60 years) AML patients achieve complete remission. However, 30–40% of patients relapse and display a dismal outcome. Recently we described a frequent instability of type I/II mutations between diagnosis and relapse. Here, we explored the hypothesis that these mutational shifts originate from clonal selection during treatment/disease progression. Subfractions of blasts from initial diagnosis samples were cell sorted and their mutational profiles were compared with those of the corresponding relapse samples of 7 CD34+ AML patients. At diagnosis, subfractions of the CD45dimCD34+CD38dim/− compartment were heterogeneous in the distribution of mutations, when compared to the whole CD45dimCD34+ blast compartment in 6 out of 7 patients. Moreover, within CD45dimCD34+CD38dim/− fraction of initial samples of 5 of these 6 AML patients, we found evidence for the presence of a minor, initially undetected subpopulation with a specific mutational profile that dominated the bulk of leukemic blasts at relapse. In conclusion, our findings lend support to the AML oligoclonality concept and provide molecular evidence for selection and expansion of a chemo-resistant subpopulation towards development of relapse. These results imply that early detection of pre-existing drug-resistant leukemic subpopulations is crucial for relapse prevention by proper timing of targeted treatment.

Early Apoptosis Largely Accounts for Functional Impairment of CD34+ Cells in Frozen-Thawed Stem Cell Grafts

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.

In vitro drug resistance profiles of adult versus childhood acute lymphoblastic leukaemia

The difference in the current cure rates between adult and childhood acute lymphoblastic leukaemia (ALL) may be caused by differences in drug resistance. Earlier studies showed that in vitro cellular drug resistance is a strong independent adverse risk factor in childhood ALL. Knowledge about cellular drug resistance in adult ALL is still limited. The present study compared the in vitro drug resistance profiles of 23 adult ALL patients with that of 395 childhood ALL patients. The lymphoblasts were tested by the MTT assay. The group of adult ALL samples was significantly more resistant to cytosine arabinoside, l‐asparaginase, daunorubicin, dexamethasone and prednisolone. The resistance ratio (RR) was highest for prednisolone (31·7‐fold) followed by dexamethasone (6·9‐fold), l‐asparaginase (6·1‐fold), cytosine arabinoside (2·9‐fold), daunorubicin (2·5‐fold) and vincristine (2·2‐fold). Lymphoblasts from adult patients were not more resistant to mercaptopurine, thioguanine, 4‐HOO‐ifosfamide, mitoxantrone and teniposide. There were no significant differences in drug resistance between adult T‐cell (T‐) ALL (n = 11) and adult common/pre‐B‐cell (B‐) ALL (n = 10). Additionally, adult T‐ALL did not differ from childhood T‐ALL (n = 69). There were significant differences between adult common/pre‐B‐ALL and childhood common/pre‐B‐ALL (n = 310) for prednisolone (RR = 302, P = 0·008), dexamethasone (RR = 20·9, P = 0·017) and daunorubicin (RR = 2·7, P = 0·009). Lymphoblasts from adults proved to be relatively resistant to drugs commonly used in therapy. This might contribute to the difference in outcome between children and adults with ALL.