Clinicopathological and molecular characteristics of extramedullary acute myeloid leukaemia.

AIMS: Myeloid sarcoma (MS) is a rare extramedullary neoplasm composed of immature myeloid precursor cells thought to be a unique clinical presentation of acute myeloid leukaemia (AML). Like AML, MS has a poor prognosis, but due to the rare nature of MS there are limited studies examining potential prognostic factors. We report our institutional experience, with the aim of investigating and establishing salient clinicopathological and molecular features of MS. METHODS AND RESULTS: We retrospectively examined all clinicopathological and molecular data on MS patients between 2001 and 2017 from the University of Alabama at Birmingham (UAB) electronic medical records. The UAB electronic medical records were also reviewed and compared with the literature for other potential prognostic factors. Sixty-three patients were included in the study. The median overall survival was 24 months in the group with normal karyotype and 12 months in patients with an abnormal karyotype. CONCLUSIONS: We found that an abnormal karyotype was associated with a statistically significant worse prognosis.

Clinical Utility and Biologic Implications of Phosphatase and Tensin Homolog (PTEN) and ETS-related Gene (ERG) in Prostate Cancer.

Phosphatase and tensin homolog (PTEN) and ETS-related gene (ERG) mutations are commonly found in prostate cancer. Although mouse studies have demonstrated that PTEN and ERG cooperatively interact during tumorigenesis, human studies examining these genes have been inconclusive. A systematic PubMed search including original articles assessing the pathogenesis of PTEN and ERG in prostate cancer was performed. Studies examining ERG's prognostic significance have conflicting results. Studies examining PTEN and ERG simultaneously found these genes are likely to occur together, but cooperative tumorigenesis functions have not been conclusively established. PTEN mutations are associated with a range of prognostic features. However, the practical clinical utility of this information remains to be determined.

GATA3 expression in benign prostate glands with radiation atypia: a diagnostic pitfall.

AIMS: Patients who have undergone prostate radiation are at increased risk for developing urothelial carcinoma. Radiation changes can cause significant cytological atypia in benign prostate glands that may mimic pagetoid spread of urothelial carcinoma. GATA3 is a common marker for differentiating prostatic adenocarcinoma from urothelial carcinoma. The aim of this study was to investigate the expression of GATA3 in the epithelium of irradiated benign and malignant prostate tissue. METHODS AND RESULTS: We performed a retrospective review of prostate cases with radiation atypia. GATA3 staining was performed on benign prostate tissue with and without radiation atypia, as well as on residual/recurrent prostatic adenocarcinoma after radiation therapy. PIN4 immunohistochemical (IHC) staining was performed on prostate tissue with radiation atypia to confirm the presence of both basal and luminal cells. We identified 31 cases with benign prostate glands containing radiation atypia. The average time between treatment with radiation therapy and the surgical procedure was 10 years (range 5-20 years). PIN4 IHC staining confirmed the presence of basal and luminal cells in benign prostate glands with radiation atypia. Thirty-one of 31 (100%) cases of benign prostate tissue with radiation atypia showed staining for GATA3 in both basal and luminal cells. Eight of eight (100%) cases of benign prostate glands without radiation atypia and no inflammatory changes showed GATA3 positivity limited to basal cells. GATA3 was negative in 12/12 (100%) cases of prostatic adenocarcinoma after radiation therapy. CONCLUSION: Benign prostate glands with radiation atypia show diffuse positivity for GATA3. This staining pattern, along with the cytological atypia resulting from radiation, can mimic urothelial carcinoma.

Stem cell autotomy and niche interaction in different systems.

The best known cases of cell autotomy are the formation of erythrocytes and thrombocytes (platelets) from progenitor cells that reside in special niches. Recently, autotomy of stem cells and its enigmatic interaction with the niche has been reported from male germline stem cells (GSCs) in several insect species. First described in lepidopterans, the silkmoth, followed by the gipsy moth and consecutively in hemipterans, foremost the milkweed bug. In both, moths and the milkweed bug, GSCs form finger-like projections toward the niche, the apical cells (homologs of the hub cells in Drosophila). Whereas in the milkweed bug the projection terminals remain at the surface of the niche cells, in the gipsy moth they protrude deeply into the singular niche cell. In both cases, the projections undergo serial retrograde fragmentation with progressing signs of autophagy. In the gipsy moth, the autotomized vesicles are phagocytized and digested by the niche cell. In the milkweed bug the autotomized vesicles accumulate at the niche surface and disintegrate. Autotomy and sprouting of new projections appears to occur continuously. The significance of the GSC-niche interactions, however, remains enigmatic. Our concept on the signaling relationship between stem cell-niche in general and GSC and niche (hub cells and cyst stem cells) in particular has been greatly shaped by Drosophila melanogaster. In comparing the interactions of GSCs with their niche in Drosophila with those in species exhibiting GSC autotomy it is obvious that additional or alternative modes of stem cell-niche communication exist. Thus, essential signaling pathways, including niche-stem cell adhesion (E-cadherin) and the direction of asymmetrical GSC division - as they were found in Drosophila - can hardly be translated into the systems where GSC autotomy was reported. It is shown here that the serial autotomy of GSC projections shows remarkable similarities with Wallerian axonal destruction, developmental axon pruning and dying-back degeneration in neurodegenerative diseases. Especially the hypothesis of an existing evolutionary conserved "autodestruction program" in axons that might also be active in GSC projections appears attractive. Investigations on the underlying signaling pathways have to be carried out. There are two other well known cases of programmed cell autotomy: the enucleation of erythroblasts in the process of erythrocyte maturation and the segregation of thousands of thrombocytes (platelets) from one megakaryocyte. Both progenitor cell types - erythroblasts and megakaryocytes - are associated with a niche in the bone marrow, erythroblasts with a macrophage, which they surround, and the megakaryocytes with the endothelial cells of sinusoids and their extracellular matrix. Although the regulatory mechanisms may be specific in each case, there is one aspect that connects all described processes of programmed cell autotomy and neuronal autodestruction: apoptotic pathways play always a prominent role. Studies on the role of male GSC autotomy in stem cell-niche interaction have just started but are expected to reveal hitherto unknown ways of signal exchange. Spermatogenesis in mammals advance our understanding of insect spermatogenesis. Mammal and insect spermatogenesis share some broad principles, but a comparison of the signaling pathways is difficult. We have intimate knowledge from Drosophila, but of almost no other insect, and we have only limited knowledge from mammals. The discovery of stem cell autotomy as part of the interaction with the niche promises new general insights into the complicated stem cell-niche interdependence.

CDK4/6 Inhibitor PD 0332991 Sensitizes Acute Myeloid Leukemia to Cytarabine-Mediated Cytotoxicity.

Cyclin-dependent kinase (CDK)4 and CDK6 are frequently overexpressed or hyperactivated in human cancers. Targeting CDK4/CDK6 in combination with cytotoxic killing therefore represents a rational approach to cancer therapy. By selective inhibition of CDK4/CDK6 with PD 0332991, which leads to early G1 arrest and synchronous S-phase entry upon release of the G1 block, we have developed a novel strategy to prime acute myeloid leukemia (AML) cells for cytotoxic killing by cytarabine (Ara-C). This sensitization is achieved in part through enrichment of S-phase cells, which maximizes the AML populations for Ara-C incorporation into replicating DNA to elicit DNA damage. Moreover, PD 0332991 triggered apoptosis of AML cells through inhibition of the homeobox (HOX)A9 oncogene expression, reducing the transcription of its target PIM1. Reduced PIM1 synthesis attenuates PIM1-mediated phosphorylation of the proapoptotic BAD and activates BAD-dependent apoptosis. In vivo, timely inhibition of CDK4/CDK6 by PD 0332991 and release profoundly suppresses tumor growth in response to reduced doses of Ara-C in a xenograft AML model. Collectively, these data suggest selective and reversible inhibition of CDK4/CDK6 as an effective means to enhance Ara-C killing of AML cells at reduced doses, which has implications for the treatment of elderly AML patients who are unable to tolerate high-dose Ara-C therapy.

Predictors of repeat transarterial chemoembolization in the treatment of hepatocellular carcinoma.

OBJECTIVES: Repeat transarterial chemoembolization (TACE) is a common intervention performed for hepatocellular carcinoma (HCC). The aim of this study was to identify predictors of the need for repeat TACE. METHODS: Between 2008 and 2012, data on patient and tumour variables were collected for 262 patients treated with a first TACE procedure for HCC. The decision to perform repeat TACE procedures was made at the completion of the first TACE or after follow-up imaging demonstrated the subtotal treatment of HCC tumours. RESULTS: Repeat TACE was performed in 67 of 262 (25.6%) patients. Necrosis of HCC, measured after the first TACE, was lower in patients who subsequently received repeat TACE (P = 0.042). On multivariable analysis, total tumour diameter of >5 cm [odds ratio (OR) 2.76, 95% confidence interval (CI) 1.45-5.25; P = 0.002] and increasing age (OR 1.04/year, 95% CI 1.00-1.07; P = 0.030) were predictive of the need for repeat TACE. Measures of liver function and TACE approach (selective versus non-selective) were not predictive of repeat TACE. Median survival did not differ significantly between patients who did (median survival: 21.1 months) and did not (median survival: 26.1 months) receive a repeat TACE procedure (P = 0.574). CONCLUSIONS: The requirement for repeat TACE is associated with older age, increased HCC tumour burden and subtotal TACE-induced HCC necrosis. Importantly, repeat TACE was not associated with reduced survival.

Chemoembolization outcomes for hepatocellular carcinoma in cirrhotic patients with compromised liver function.

BACKGROUND: Transarterial chemoembolization (TACE) is recommended as a treatment for unresectable hepatocellular carcinoma (HCC) in patients with normal underlying liver function. The efficacy of TACE in cirrhotic patients with compromised liver function is unknown. METHODS: All 'first' TACE interventions for HCC performed at a single institution from 2008 to 2012 were retrospectively reviewed (n = 190). Liver function was quantified via the Child's score. Tumour necrosis after TACE was quantified via the mRECIST criteria. RESULTS: The 'first' TACE procedures of 100 Child's A and 90 Child's B/C cirrhotic patients were evaluated. As expected, the lab-model for end-stage liver disease (MELD) score was significantly higher in the Child's B/C group. Although the number of tumours were similar between the groups, both the size of the largest tumour and the total tumour diameter were greater in the Child's A group. There were no significant differences in post-TACE tumour necrosis between groups. The median survival after TACE was significantly longer in the Child's A compared with Child's B/C patients (21.9 versus 13.7 months, P = 0.03). CONCLUSIONS: TACE appears to be equally efficacious in cirrhotic patients regardless of their Child's classification based upon equivalent mRECIST measures of tumour necrosis. However, inferior survival after TACE was observed in the Child's B/C group.

Resource utilization associated with procurement of transplantable organs from donors that do not meet OPTN eligible death criteria.

BACKGROUND: The strategy of evaluating every donation opportunity warrants an investigation into the financial feasibility of this practice. The purpose of this investigation is to measure resource utilization required for procurement of transplantable organs in an organ procurement organization (OPO). METHODS: Donors were stratified into those that met OPTN-defined eligible death criteria (ED donors, n=589) and those that did not (NED donors, n=703). Variable direct costs and time utilization by OPO staff for organ procurement were measured and amortized per organ transplanted using permutation methods and statistical bootstrapping/resampling approaches. RESULTS: More organs per donor were procured (3.66±1.2 vs. 2.34±0.8, P<0.0001) and transplanted (3.51±1.2 vs. 2.08±0.8, P<0.0001) in ED donors compared with NED donors. The variable direct costs were significantly lower in the NED donors ($29,879.4±11590.1 vs. $19,019.6±7599.60, P<0.0001). In contrast, the amortized variable direct costs per organ transplanted were significantly higher in the NED donors ($8,414.5±138.29 vs. $9,272.04±344.56, P<0.0001). The ED donors where thoracic organ procurement occurred were 67% more expensive than in abdominal-only organ procurement. The total time allocated per donor was significantly shorter in the NED donors (91.2±44.9 hr vs. 86.8±78.6 hr, P=0.01). In contrast, the amortized time per organ transplanted was significantly longer in the NED donors (23.1±0.8 hr vs. 36.9±3.2 hr, P<0.001). DISCUSSION: The variable direct costs and time allocated per organ transplanted is significantly higher in donors that do not meet the eligible death criteria.

Computed tomography predictors of hepatocellular carcinoma tumour necrosis after chemoembolization.

BACKGROUND: Radiographical features associated with a favourable response to trans-arterial chemoembolization (TACE) are poorly defined for patients with hepatocellular carcinoma (HCC). METHODS: From 2008 to 2012, all first TACE interventions for HCC performed at the University of Alabama at Birmingham (UAB) were retrospectively reviewed. Only patients with a pre-TACE and a post-TACE computed tomography (CT) scan were included in the analyses (n = 115). HCC tumour response to TACE was quantified via the the modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria. Univariate and multivariable analyses were constructed. RESULTS: The index HCC tumours experienced a > 90% or complete tumour necrosis in 59/115 (51%) of patients after the first TACE intervention. On univariate analysis, smaller tumour size, peripheral tumour location and arterial enhancement were associated with a > 90% or complete tumour necrosis, whereas, only smaller tumour size [odds ratio (OR) 0.62; 95% confidence interval (CI) 0.48, 0.81] and peripheral location (OR 6.91; 95% CI 1.75, 27.29) were significant on multivariable analysis. There was a trend towards improved survival in the patients that experienced a > 90% or complete tumour necrosis (P = 0.08). CONCLUSIONS: Peripherally located smaller HCC tumours are most likely to experience a > 90% or complete tumour necrosis after TACE. Surprisingly, arterial-phase enhancement and portal venous-phase washout were not significantly predictive of TACE-induced tumour necrosis. The TACE response was not statistically associated with improved survival.

Structural characterization and primary in vitro cell culture of locust male germline stem cells and their niche.

The establishment of in vitro culture systems to expand stem cells and to elucidate the niche/stem cell interaction is among the most sought-after culture systems of our time. To further investigate niche/stem cell interactions, we evaluated in vitro cultures of isolated intact male germline-niche complexes (i.e., apical complexes), complexes with empty niche spaces, and completely empty niches (i.e., isolated apical cells) from the testes of Locusta migratoria and the interaction of these complexes with isolated germline stem cells, spermatogonia (of transit-amplifying stages), cyst progenitor cells, cyst progenitor cell-like cells, cyst cells, and follicle envelope cells. The structural characteristics of these cell types allow the identification of the different cell types in primary cultures, which we studied in detail by light and electron microscopy. In intact testes germline stem cells strongly adhere to their niche (the apical cell), but emigrate from their niche and form filopodia if the apical complex is put into culture with "standard media." The lively movements of the long filopodia of isolated germline stem cells and spermatogonia may be indicative of their search for specific signals to home to their niche. All other incubated cell types (except for follicle envelope cells) expressed rhizopodia and lobopodia. Nevertheless isolated germline stem cells in culture do not migrate to empty niche spaces of nearby apical cells. This could indicate that apical cells lose their germline stem cell attracting ability in vitro, although apical cells devoid of germline stem cells either by emigration of germline stem cells or by mechanical removal of germline stem cells are capable of surviving in vitro up to 56 days, forming many small lobopodia and performing amoeboid movements. We hypothesize that the breakdown of the apical complex in vitro with standard media interrupts the signaling between the germline stem cells and the niche (and conceivably the cyst progenitor cells) which directs the typical behavior of the male regenerative center. Previously we demonstrated the necessity of the apical cell for the survival of the germline stem cell. From these studies we are now able to culture viable isolated germline stem cells and all cells of its niche complex, although DNA synthesis stops after Day 1 in culture. This enables us to examine the effects of supplements to our standard medium on the interaction of the germline stem cell with its niche, the apical cell. The supplements we evaluated included conditioned medium, tissues, organs, and hemolymph of male locusts, insect hormones, mammalian growth factors, Ca(2+) ion, and a Ca(2+) ionophore. Although biological effects on the germline stem cell and apical cell could be detected with the additives, none of these supplements restored the in vivo behavior of the incubated cell types. We conclude that the strong adhesion between germline stem cells and apical cells in vivo is actively maintained by peripheral factors that reach the apical complex via hemolymph, since a hemolymph-testis barrier does not exist. The in vitro culture model introduced in this study provides a platform to scan for possible regulatory factors that play a key role in a feedback loop that keeps germline stem cell division and sperm disposal in equilibrium.

The effect of cantharidins on leukemic stem cells.

To identify an agent with specific activity against leukemic stem cells (LSCs), we evaluated compounds that targeted hepatic leukemia factor (HLF), a gene implicated in hematopoietic stem cell (HSCs) regulation, that we found overexpressed in LSCs. Cantharidin, a natural toxin from blister beetles, used as medicinal agent since antiquity, has been described to modulate the HLF competitor NFIL3 and is under clinical evaluation as an antitumor and antimetastatic agent. The molecule is not a substrate for multidrug resistant pumps and does not cause myelosuppression, and therefore it represents a promising compound for selective ablation of LSCs. Cantharidin and norcantharidin, a derivative with reduced toxicity, decreased HLF protein levels and induced apoptosis in the AML cell line MV4-11 by modulating the expression of several molecules that govern survival pathway, including HLF, SLUG, NFIL3 and c-myc, thereby inducing p53 and the mitochondrial caspase cascade. In vitro, cantharidin readily targeted primary AML stem and progenitor cells in contrast to conventional chemotherapeutic agents, such as Ara-C and daunorubicin, that mainly targeted more differentiated leukemic cells. In vitro the compound did not exhibit a therapeutic window, being equally toxic to normal HSCs and LSCs. In vivo cantharidin did not produce myelosuppression. Because of dose-limiting toxicity in vivo, neither cantharidin nor norcantharidin proved therapeutical benefit in AML xenograft models as a single agent. However, its potent in vitro LSC activity and pathway targeting may still be exploited clinically with a new generation of cantharidin derivatives or formulations and with appropriate drug combinations.

Quantification of the CD8+ T cell response against a mucin epitope in patients with breast cancer.

INTRODUCTION: Mucin 1, encoded by the MUC1 gene, is a tumor-associated antigen expressed on the surface of breast cancer cells. It would be of interest to see whether there is a naturally existing T cell immune response against mucin epitopes in cancer patients. MATERIALS AND METHODS: Using tetramer and interferon-gamma assays, the immune response to one MUC1 peptide epitope in the peripheral blood of breast cancer patients was quantified. The data were compared with the clinical course of the patients. RESULTS: CD8(+) T cells capable of recognizing the HLA-A*0201-restricted STAPPVHNV epitope were detected in 9 of 19 patients with a frequency ranging 0.01-0.082%. No significant difference was found between the occurrence of epitope-specific CD8(+) T cells of patients with progressive disease and disease-free patients. However, all patients with stable disease showed a specific immune response, including both patients with the highest frequency. CONCLUSIONS: The results of this study provide further evidence that a natural specific cellular immune response against this mucin epitope exists in breast cancer patients.

Structural polarity and dynamics of male germline stem cells in an insect (milkweed bug Oncopeltus fasciatus).

Knowing the structure opens a door for a better understanding of function because there is no function without structure. Male germline stem cells (GSCs) of the milkweed bug (Oncopeltus fasciatus) exhibit a very extraordinary structure and a very special relationship with their niche, the apical cells. This structural relationship is strikingly different from that known in the fruit fly (Drosophila melanogaster) -- the most successful model system, which allowed deep insights into the signaling interactions between GSCs and niche. The complex structural polarity of male GSCs in the milkweed bug combined with their astonishing dynamics suggest that cell morphology and dynamics are causally related with the most important regulatory processes that take place between GSCs and niche and ensure maintenance, proliferation, and differentiation of GSCs in accordance with the temporal need of mature sperm. The intricate structure of the GSCs of the milkweed bug (and probably of some other insects, i.e., moths) is only accessible by electron microscopy. But, studying singular sections through the apical complex (i.e., GSCs and apical cells) is not sufficient to obtain a full picture of the GSCs; especially, the segregation of projection terminals is not tangible. Only serial sections and their overlay can establish whether membrane ingrowths merely constrict projections or whether a projection terminal is completely cut off. To sequence the GSC dynamics, it is necessary to include juvenile stages, when the processes start and the GSCs occur in small numbers. The fine structural analysis of segregating projection terminals suggests that these terminals undergo autophagocytosis. Autophagosomes can be labeled by markers. We demonstrated acid phosphatase and thiamine pyrophosphatase (TPPase). Both together are thought to identify autophagosomes. Using the appropriate substrate of the enzymes and cerium chloride, the precipitation of electron-dense cerium phosphate granules indicates the presence of enzymes and their location. Because the granules are very fine, they can be easily assigned to distinct cell organelles as the autophagosomes. Two methods, electron microscopy and immunocytochemistry, have pointed out a structural polarity and dynamics that are unprecedented for stem cells. We propose that these dynamics indicate a novel type of signal exchange and transduction between stem cells and their niche.

Cellular and humoral immunogenicity of hamster polyomavirus-derived virus-like particles harboring a mucin 1 cytotoxic T-cell epitope.

In this study, we examined hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) as a carrier for a human tumor-associated cytotoxic T lymphocyte (CTL) epitope. The VP1 tolerated the insertion of an HLA-*A2-restricted CTL epitope from human mucin 1 (MUC1) into two sites independently and simultaneously, without interfering with assembly of chimeric VLPs. Chimeric VLPs did not differ in the entry pathway or maturation potential of human dendritic cells (hDCs) compared to unmodified VLPs. Recently we demonstrated that immunization of BALB/c mice with chimeric VLPs harboring two MUC1 insertions resulted in the generation of MUC1-specific monoclonal antibodies. Here we demonstrate that the monoclonal antibodies generated react specifically with human tumor cells. Co-cultivation of chimeric VLP-primed hDCs with autologous peripheral blood leukocytes resulted in the activation of MUC1 epitope-specific CD8(+) T cells. This was evidenced by IFN-gamma secretion of an expanded MUC1-specific CD8(+) T-cell pool. The induction of epitope-specific T cells in a human in vitro model and of murine MUC1-reactive antibodies in vivo indicate the potential of chimeric HaPyV VP1-derived VLPs as a delivery vehicle for immunotherapeutic targets.

Constitutive activation of Flt3 and STAT5A enhances self-renewal and alters differentiation of hematopoietic stem cells.

OBJECTIVE: To model human leukemogenesis by transduction of human hematopoietic stem cells (HSC) with genes associated with leukemia and expressed in leukemic stem cells. METHODS: Constitutive activation of Flt3 (Flt3-ITD) has been reported in 25 to 30% of patients with acute myeloid leukemia (AML). Retroviral vectors expressing constitutively activated Flt3 and STAT5A were used to transduce human cord blood CD34(+) cells and HSC cell self-renewal and differentiation were evaluated. RESULTS: We have demonstrated that retroviral transduction of Flt3 mutations into CD34(+) cells enhanced HSC self-renewal as measured in vitro in competitive stromal coculture and limiting-dilution week-2 cobblestone (CAFC) assays. Enhanced erythropoiesis and decreased myelopoiesis were noted together with strong activation of STAT5A. Consequently, transduction studies were undertaken with a constitutively active mutant of STAT5A (STAT5A[1( *)6]) and here also a marked, selective expansion of transduced CD34(+) cells was noted, with a massive increase in self-renewing CAFC detectable at both 2 and 5 weeks of stromal coculture. Differentiation was biased to erythropoiesis, including erythropoietin independence, with myeloid maturation inhibition. The observed phenotypic changes correlated with differential gene expression, with a number of genes differentially regulated by both the Flt3 and STAT5A mutants. These included upregulation of genes involved in erythropoiesis and downregulation of genes involved in myelopoiesis. The phenotype of week-2 self-renewing CAFC also characterized primary Flt3-ITD(+) AML bone marrow samples. Isolation of leukemic stem cells (LSC) with a CD34(+), CD38(-), HLA-DR(-) phenotype was undertaken with Flt3-ITD(+) AML samples resulting in co-purification of early CAFC. Gene expression of LSC relative to the bulk leukemic population revealed upregulation of homeobox genes (HOXA9, HOXA5) implicated in leukemogenesis, and hepatic leukemia factor (HLF) involved in stem cell proliferation. CONCLUSION: Myeloid leukemogenesis is a multi-stage process that can involve constitutively activated receptors and downstream pathways involving STAT5, HOX genes, and HLF.

Apoptosis of male germ-line stem cells after laser ablation of their niche.

Male germ-line stem cells (GSCs) and their niche-the apical cells or hub cells-display a unique feature at the apices of insect testicular follicles. In the locust, Locusta migratoria, the niche consists of only one large apical cell surrounded by about 60 GSCs. The apical cell can be readily identified in the intact follicle. Using laser ablation it is feasible to destroy the apical cell exclusively without injuring neighboring GSCs or any other cells. The most immediate effect on GSCs is the loss of their structural polarity. Beginning about 3 h after laser treatment chromatin starts to clump and condense in individual GSCs, and some show the first signs of cellular breakdown. These symptoms intensify during the 96-h observation period after laser ablation of the apical cell. TUNEL staining and electron microscopic observations confirm an apoptotic cell death of the GSCs. Laser ablation of individual GSCs had no effect on neighboring GSCs or the apical cell. Destroyed apical cells were not replaced during the observation period. Mitotic divisions of GSCs ceased after about 24 h after apical cell ablation. It is speculated that it might be a general principle in stem cell-niche relationships that stem cells undergo apoptosis when the niche is dysfunctional. This could be a control mechanism to prevent tumor growth of orphaned GSCs.