Acid Sphingomyelinase-Ceramide Induced Vascular Injury Determines Colorectal Cancer Stem Cell Fate.

BACKGROUND/AIMS: It is unknown whether cancer stem cells respond differentially to treatment compared with progeny, potentially providing therapeutic vulnerabilities. Our program pioneered use of ultra-high single dose radiotherapy, which cures diverse metastatic diseases at a higher rate (90-95%) than conventional fractionation (~65%). Single dose radiotherapy engages a distinct biology involving microvascular acid sphingomyelinase/ceramide signaling, which, via NADPH oxidase-2-dependent perfusion defects, initiates an adaptive tumor SUMO Stress Response that globally-inactivates homologous recombination repair of double stand breaks, conferring cure. Accumulating data show diverse stem cells display heightened-dependence on homologous recombination repair to repair resolve double stand breaks. METHODS: Here we use colorectal cancer patient-derived xenografts containing logarithmically-increased Lgr5+ stem cells to explore whether optimizing engagement of this acid sphingomyelinase dependent biology enhances stem cell dependent tumor cure. RESULTS: We show radioresistant colorectal cancer patient-derived xenograft CLR27-2 contains radioresistant microvasculature and stem cells, whereas radiosensitive colorectal cancer patient-derived xenograft CLR1-1 contains radiosensitive microvasculature and stem cells. Pharmacologic or gene therapy enhancement of single dose radiotherapy-induced acid sphingomyelinase/ceramide-mediated microvascular dysfunction dramatically sensitizes CLR27-2 homologous recombination repair inactivation, converting Lgr5+ cells from the most resistant to most sensitive patient-derived xenograft population, yielding tumor cure. CONCLUSION: We posit homologous recombination repair represents a vulnerability determining colorectal cancer stem cell fate, approachable therapeutically using single dose radiotherapy.

Enhancement of Soft Tissue Sarcoma Response to Gemcitabine through Timed Administration of a Short-Acting Anti-Angiogenic Agent.

BACKGROUND/AIMS: Despite enormous effort, anti-angiogenic drugs have not lived up to the promise of globally-enhancing anti-cancer therapies. Clinically, anti-angiogenic drugs have been used to persistently suppress vascular endothelial growth factor (VEGF) in order to "normalize" dysfunctional neo-angiogenic microvasculature and prevent recruitment of endothelial progenitors. Recently, we showed that a 1h pre-treatment with anti-angiogenic drugs prior to ultra-high single dose radiotherapy and specific chemotherapies transiently de-represses acid sphingomyelinase (ASMase), leading to enhanced cancer therapy-induced, ceramide-mediated vascular injury and tumor response. Here we formally decipher parameters of chemotherapy induction of endothelial sphingolipid signaling events and define principles for optimizing anti-angiogenic chemosensitization. METHODS: These studies examine the antimetabolite chemotherapeutic gemcitabine in soft tissue sarcoma (STS), a clinically-relevant combination. RESULTS: Initial studies address the theoretic problem that anti-angiogenic drugs such as bevacizumab, an IgG with a 3-week half-life, have the potential for accumulating during the 3-week chemotherapeutic cycles currently standard-of-care for STS treatment. We show that anti-angiogenic ASMase-dependent enhancement of the response of MCA/129 fibrosarcomas in sv129/BL6 mice to gemcitabine progressively diminishes as the level of the VEGFR2 inhibitor DC101, an IgG, accumulates, suggesting a short-acting anti-angiogenic drug might be preferable in multi-cycle chemotherapeutic regimens. Further, we show lenvatinib, a VEGFR2 tyrosine kinase inhibitor with a short half-life, to be superior to DC101, enhancing gemcitabine-induced endothelial cell apoptosis and tumor response in a multi-cycle treatment schedule. CONCLUSION: We posit that a single delivery of a short-acting anti-angiogenic agent at 1h preceding each dose of gemcitabine and other chemotherapies may be more efficacious for repeated sensitization of the ASMase pathway in multi-cycle chemotherapy regimens than current treatment strategies.

Long-Term Implications of a Positive Posttreatment Biopsy in Patients Treated with External Beam Radiotherapy for Clinically Localized Prostate Cancer.

PURPOSE: We determined the prognostic importance of a positive posttreatment biopsy after prostate radiotherapy. MATERIALS AND METHODS: A total of 382 patients underwent a posttreatment biopsy after external beam radiotherapy for clinically localized prostate cancer. Posttreatment biopsies were classified as positive (prostatic adenocarcinoma without typical radiation induced changes), negative (no evidence of carcinoma) or adenocarcinoma with a severe treatment effect. Median followup in survivors was 9 years. Competing risks regression was used to assess relationships between prognostic predictors and cause specific mortality, distant metastasis and prostate specific antigen failure. RESULTS: The prevalence of positive biopsy, treatment effect and negative biopsy was 30%, 22% and 48%, respectively. Androgen deprivation therapy omission and high risk disease were associated with a 2.6 and 1.8-fold increase, respectively, in the odds of positive posttreatment biopsy. The 15-year PSA relapse rate associated with negative, severe treatment effect and positive posttreatment biopsies was 34%, 36% and 79%, respectively (p <0.001). After controlling for known predictors the risk of distant metastasis was 2.6-fold higher in patients with a positive biopsy (p <0.001) and cause specific mortality was twice as high in patients with a positive biopsy compared to those with negative and severe treatment effect biopsy outcomes (HR 2.00, p = 0.022). CONCLUSIONS: A positive posttreatment biopsy after external beam radiotherapy was associated with a higher risk of distant metastasis and prostate cancer related death. Patients with severe treatment effect classified biopsies have biological characteristics more like patients with a negative biopsy than a positive biopsy. Posttreatment biopsies were more often positive in the setting of external beam radiotherapy alone without androgen deprivation therapy or in the presence of high risk disease.

A CERTain role for ceramide in taxane-induced cell death.

An unexpected benefit of functional genomic screens is that at times they answer questions that they were not designed to ask. A siRNA screen reported by Swanton et al. in this issue of Cancer Cell reveals that silencing of spindle assembly checkpoint genes facilitates mitotic slippage, resulting in escape from taxane-induced cell death, aneuploidy, and chromosomal instability, hallmarks of taxane resistance. Unexpectedly, the screen disclosed that the sphingolipid ceramide is a key regulator of the taxane-mediated spindle assembly checkpoint and taxane-induced cell death. Ceramide metabolism thus serves as a legitimate target for modulation of taxane effect on tumors.

Spinal metastases: From conventional fractionated radiotherapy to single-dose SBRT.

AIM: To review the recent evolution of spine SBRT with emphasis on single dose treatments. BACKGROUND: Radiation treatment of spine metastases represents a challenging problem in clinical oncology, because of the high risk of inflicting damage to the spinal cord. While conventional fractionated radiation therapy still constitutes the most commonly used modality for palliative treatment, notwithstanding its efficacy in terms of palliation of pain, local tumor control has been approximately 60%. This limited effectiveness is due to previous lack of technology to precisely target the tumor while avoiding the radiosensitive spinal cord, which constitutes a dose-limiting barrier to tumor cure. MATERIALS AND METHODS: A thorough review of the available literature on spine SBRT has been carried out and critically assessed. RESULTS: Stereotactic body radiotherapy (SBRT) emerges as an alternative, non-invasive high-precision approach, which allows escalation of tumor dose, while effectively sparing adjacent uninvolved organs at risk. Engaging technological advances, such as on-line Cone Beam Computed Tomography (CBCT), coupled with Dynamic Multi-Leaf Collimation (DMLC) and rapid intensity-modulated (IMRT) beam delivery, have promoted an interactive image-guided (IGRT) approach that precisely conforms treatment onto a defined target volume with a rapid dose fall-off to collateral non-target tissues, such as the spinal cord. Recent technological developments allow the use of the high-dose per fraction mode of hypofractionated SBRT for spinal oligometastatic cancer, even if only a few millimeters away from the tumor. CONCLUSION: Single-dose spine SBRT, now increasingly implemented, yields unprecedented outcomes of local tumor ablation and safety, provided that advanced technology is employed.

Crypt base columnar stem cells in small intestines of mice are radioresistant.

BACKGROUND & AIMS: Adult stem cells have been proposed to be quiescent and radiation resistant, repairing DNA double-strand breaks by nonhomologous end joining. However, the population of putative small intestinal stem cells (ISCs) at position +4 from the crypt base contradicts this model, in that they are highly radiosensitive. Cycling crypt base columnar cells (CBCs) at crypt positions +1-3 recently were defined as an alternative population of ISCs. Little is known about the sensitivity of this stem cell population to radiation. METHODS: Radiation-induced lethality of CBCs was quantified kinetically in Lgr5-lacZ transgenic mice. γ-H2AX, BRCA1, RAD51, and DNA-PKcs foci were used as DNA repair surrogates to investigate the inherent ability of CBCs to recognize and repair double-strand breaks. 5-ethynyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine incorporation assays were used to study patterns of CBC growth arrest and re-initiation of cell cycling. Apoptosis was evaluated by caspase-3 staining. RESULTS: CBCs are relatively radioresistant, repairing DNA by homologous recombination significantly more efficiently than transit amplifying progenitors or villus cells. CBCs undergo apoptosis less than 24 hours after irradiation (32% ± 2% of total lethality) or mitotic death at 24-48 hours. Survival of CBCs at 2 days predicts crypt regeneration at 3.5 days and lethality from gastrointestinal syndrome. Crypt repopulation originates from CBCs that survive irradiation. CONCLUSIONS: Adult ISCs in mice can cycle rapidly yet still be radioresistant. Importantly, homologous recombination can protect adult stem cell populations from genotoxic stress. These findings broaden and refine concepts of the phenotype of adult stem cells.

Engaging the vascular component of the tumor response.

Recent research has shed new light on the critical role of tissue microvasculature in regulating the tumor response to radiation and drugs. In this issue of Cancer Cell, Moeller et al.(2005) demonstrate that HIF-1 activation during the course of fractionated radiotherapy initiates pleiotropic adaptive responses in both tumor cells and the microvascular network, radiosensitizing tumor cells but concomitantly conferring tumor radioresistance due to protection of the microvascular endothelium. HIF-1 thus serves as a legitimate target for differential modulation of tissue response to radiation.

Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation.

c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs.

Anti-ceramide Single-Chain Variable Fragment Mitigates Gastrointestinal-Acute Radiation Syndrome and Improves Marrow Reconstitution, Rendering Near-Normal 90-Day Autopsies.

PURPOSE: After September 11, 2001, nuclear threat prompted government agencies to develop medical countermeasures to mitigate two syndromes, the hematopoietic-acute radiation syndrome (H-ARS) and the higher-dose gastrointestinal-acute radiation syndrome (GI-ARS), both lethal within weeks. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS, no mitigator potentially deliverable under mass casualty conditions preserves the GI tract. We recently reported that anti-ceramide single-chain variable fragment (scFv) mitigates GI-ARS lethality, abrogating ongoing small intestinal endothelial apoptosis to rescue Lgr5+ stem cells. Here, we examine long-term consequences of prevention of acute GI-ARS lethality. METHODS AND MATERIALS: For these studies, C57BL/6J male mice were treated with 15 Gy whole body irradiation, the 90% GI-ARS lethal dose for this mouse strain. RESULTS: Mice irradiated with 15 Gy alone or with 15 Gy + bone marrow transplantation (BMT) or anti-ceramide scFv, succumb to an ARS within 8 to 10 days. Autopsies reveal only mice receiving anti-ceramide scFv at 24 hours post-whole body irradiation display small intestinal rescue. No marrow reconstitution occurs in any group with attendant undetectable circulating blood elements. Mice receiving 15 Gy + BMT + scFv, however, normalize blood counts by day 12, suggesting that scFv also improves marrow reconstitution, a concept for which we provide experimental support. We show that at 14 Gy, the upper limit dose for H-ARS lethality before transition to GI-ARS lethality, anti-ceramide scFv markedly improves marrow take, reducing the quantity of marrow-conferring survival by more than 3-fold. Consistent with these findings, mice receiving 15 Gy + BMT + scFv exhibit prolonged survival. At day 90, before sacrifice, they display normal appearance, behavior, and serum biochemistries, and surprisingly, at full autopsy, near-normal physiology in all 42 tissues examined. CONCLUSIONS: Anti-ceramide scFv mitigates GI-ARS lethality and improves marrow reconstitution rendering prolonged survival with near normal autopsies.

Radiation-induced gastrointestinal (GI) syndrome as a function of age.

Previous studies show increased sensitivity of older mice (28-29 months) compared with young adult mice (3 months, possessing a mature immune system) to radiation-induced GI lethality. Age-dependent lethality was associated with higher levels of apoptotic stem cells in small intestinal crypts that correlated with sphingomyelinase activity, a source of pro-apoptotic ceramide. The objective of this study is to determine whether the cycling crypt base columnar cells (CBCs) in aging animals are specifically more sensitive to radiation effects than the CBCs in young adult mice, and to identify factors that contribute to increased radiosensitivity. Mortality induced by subtotal body radiation was assessed at different doses (13 Gy, 14 Gy, and 15 Gy) in young adult mice versus older mice. Each dose was evaluated for the occurrence of lethal GI syndrome. A higher death rate due to radiation-induced GI syndrome was observed in older mice as compared with young adult mice: 30 vs. 0% at 13 Gy, 90 vs. 40% at 14 Gy, and 100 vs. 60% at 15 Gy. Radiation-induced damage to crypts was determined by measuring crypt regeneration (H&E staining, Ki67 expression), CBC biomarkers (lgr5 and ascl2), premature senescence (SA-ß-gal activity), and apoptosis of CBCs. At all three doses, crypt microcolony survival assays showed that the older mice had fewer regenerating crypts at 3.5 days post-radiation treatment. Furthermore, in the older animals, baseline CBCs numbers per circumference were significantly decreased, correlating with an elevated apoptotic index. Analysis of tissue damage showed an increased number of senescent CBCs per crypt circumference in older mice relative to younger mice, where the latter was not significantly affected by radiation treatment. It is concluded that enhanced sensitivity to radiation-induced GI syndrome and higher mortality in older mice can be attributed to a decreased capacity to regenerate crypts, presumably due to increased apoptosis and senescence of CBCs.

ATM regulates target switching to escalating doses of radiation in the intestines.

Although stem cells succumbing to reproductive death are assumed to be the single relevant targets in radiation tissue damage, recent studies showed intestinal stem cell damage is conditionally linked to crypt endothelial apoptosis, defining a two-target model. Here we report that when mouse intestines were protected against microvascular apoptosis, radiation switched as the dose escalated to a previously unrecognized crypt stem cell target, activating ceramide synthase-mediated apoptosis to initiate intestinal damage. Whereas ataxia telangiectasia-mutated (ATM) kinase normally represses ceramide synthase, its derepression in Atm(-/-) mice increased crypt stem cell radiosensitivity 3.7-fold without sensitizing the microvascular response. Discovery of this intestinal radiosensitivity mechanism allowed design of an antisense Atm oligonucleotide treatment which phenocopied the Atm(-/-) mouse, reordering ceramide synthase-mediated stem cell death to become the first-line gastrointestinal response of wild-type littermates. These experiments indicate that tissues operate multiple potential targets activated consecutively according to their inherent radiosensitivities that may be reordered therapeutically to control radiation tissue responses.

Early PSA density kinetics predicts biochemical and local failure following extreme hypofractionated radiotherapy in intermediate-risk prostate cancer.

PURPOSE: The present study explores PSA density (PSA-D) as predictor of biochemical and local failure in organ-confined prostate cancer at 3-6 months after hypofractionated stereotactic ablative radiotherapy (SABR). METHODS AND MATERIALS: A cohort of 219, hormone-naïve, NCCN intermediate-risk prostate cancer patients were derived from a phase 2 study of 5 × 9 Gy prostate cancer SABR. PSA-D was calculated at 3 and 6 months by dividing serum PSA by the MR-derived prostate CTV, while the slope of the 3-6 months curve was used to express the kinetics of PSA-D decay. RESULTS: The median follow-up was 60.3 (range 46-76) months, and the actuarial 7-year bRFS was 98.0% for intermediate-risk favorable (FIR) patients versus 84.5% for the unfavorable (UIR) subgroup (P = 0.02). Fourteen patients developed a Phoenix-defined biochemical PSA relapse (bRFS) at a median of 34.2 months, 11 confirmed with 68Ga-PSMA PET/CT scan that revealed tracer uptake at the site of dominant intraprostatic pretreatment lesion in 8 patients. The 3-month PSA-D concertation (cut-off 0.08 ng/ml2) and 3-6 months decay slope (cut-off -0.45) values were predictive of long-term bRFS. A dual adverse PSA-D permutation was detected in 25/148 UIR patients, exhibiting 47.5% 7-year bRFS compared with 94.1% for the remaining 123 UIR patients with favorable PSA-D kinetics (P = 0.0006). Intraprostatic local relapse in patients with a 3-month PSA-D > 0.080 ng/ml2 was 11.0% vs. 1.7% for patients with PSA-D ≤ 0.080 ng/ml2 (P = 0.01) and 2.3% vs. 4.3%, respectively, for nodal progression (P = 0.68). CONCLUSIONS: Early post-treatment PSA-D kinetics transcends pre-treatment risk stratification of tumor relapse and adds a nuance in the biological characterization of intermediate-risk prostate cancer phenotypes. The dual adverse PSA-D algorithm may serve as a tool to validate current search of classifiers of radioresistance in prostate cancer with therapeutic implications.

Health-related quality of life of salvage prostate reirradiation using stereotactic ablative radiotherapy with urethral-sparing.

Purpose: To explore whether prostate motion mitigation using the rectal distension-mediated technique is safe and effective in stereotactic ablative radiation therapy (SABR) salvage treatment of intraprostatic cancer recurrences following initial radiotherapy for primary prostate cancer. Materials and methods: Between July 2013 and December 2020, 30 patients received salvage SABR for 68Ga- PSMA-11 PET/CT-detected intra-prostatic relapses. Median time from primary RT to salvage reirradiation was 70.2 (IQR, 51.3-116.0) months. Median PSA at retreatment was 3.6 ng/mL (IQR, 1.9-6.2). Rectal distension-mediated SABR was achieved with a 150-cm3 air-inflated endorectal balloon and a Foley catheter loaded with 3 beacon transponders was used for urethra visualization and on-line tracking. MRI-based planning employed a 2-mm expansion around the planned target volume (PTV), reduced to 0-mm at the interface with critical organs at risk (OARs). Volumetric Modulated Arc Therapy (VMAT) permitted a 20% dose reduction of the urethra. VMAT simultaneous integrated boost (SIB) of the dominant intraprostatic lesion was deployed when indicated. Median SABR dose was 35 Gy (7 Gy per fraction over 5 consecutive days; range 35-40 Gy). Toxicity assessment used CTCAE v.4 criteria. Results: Median follow-up was 44 months (IQR, 18-60). The actuarial 3- and 4-year biochemical relapse free survival was 53.4% and 47.5%, respectively. Intraprostatic post-salvage relapse by PSMA PET/CT was 53.3%. Acute grade 2 and 3 genitourinary (GU) toxicities were 20% and 0%, respectively. There were no instances of acute grade ≥2 rectal (GI) toxicity. Late grade 2 and 3 GU toxicities occurred in 13.3% and 0% of patients, respectively. There were no instances of grade ≥2 late rectal toxicity. Patient-reported QOL measures showed an acute transient deterioration in the urinary domain 1 month after treatment but returned to baseline values at 3 months. The median IPSS scores rose over baseline (≥5 points in 53% of patients) between month 6 and 12 post-treatment as a result of urinary symptoms flare, eventually receding at 18 months. The bowel domain metrics had no appreciable changes over time. Conclusion: Pursuit of local control in intraprostatic failures is feasible and can be achieved with an acceptably low toxicity profile associated with effective OAR sparing.

Urethra Sparing With Target Motion Mitigation in Dose-Escalated Extreme Hypofractionated Prostate Cancer Radiotherapy: 7-Year Results From a Phase II Study.

Purpose: To explore whether the rectal distension-mediated technique, harnessing human physiology to achieve intrafractional prostate motion mitigation, enables urethra sparing by inverse dose painting, thus promoting dose escalation with extreme hypofractionated stereotactic ablative radiotherapy (SABR) in prostate cancer. Materials and Methods: Between June 2013 and December 2018, 444 patients received 5 × 9 Gy SABR over 5 consecutive days. Rectal distension-mediated SABR was employed via insertion of a 150-cm3 air-inflated endorectal balloon. A Foley catheter loaded with 3 beacon transponders was used for urethra visualization and online tracking. MRI-based planning using Volumetric Modulated Arc Therapy - Image Guided Radiotherapy (VMAT-IGRT) with inverse dose painting was employed in delivering the planning target volume (PTV) dose and in sculpting exposure of organs at risk (OARs). A 2-mm margin was used for PTV expansion, reduced to 0 mm at the interface with critical OARs. All plans fulfilled Dmean ≥45 Gy. Target motion ≥2 mm/5 s motions mandated treatment interruption and target realignment prior to completion of the planned dose delivery. Results: Patient compliance to the rectal distension-mediated immobilization protocol was excellent, achieving reproducible daily prostate localization at a patient-specific retropubic niche. Online tracking recorded ≤1-mm intrafractional target deviations in 95% of treatment sessions, while target realignment in ≥2-mm deviations enabled treatment completion as scheduled in all cases. The cumulative incidence rates of late grade ≥2 genitourinary (GU) and gastrointestinal (GI) toxicities were 5.3% and 1.1%, respectively. The favorable toxicity profile was corroborated by patient-reported quality of life (QOL) outcomes. Median prostate-specific antigen (PSA) nadir by 5 years was 0.19 ng/ml. The cumulative incidence rate of biochemical failure using the Phoenix definition was 2%, 16.6%, and 27.2% for the combined low/favorable-intermediate, unfavorable intermediate, and high-risk categories, respectively. Patients with a PSA failure underwent a 68Ga-labeled prostate-specific membrane antigen (68Ga-PSMA) scan showing a 20.2% cumulative incidence of intraprostatic relapses in biopsy International Society of Urological Pathology (ISUP) grade ≥3. Conclusion: The rectal distension-mediated technique is feasible and well tolerated. Dose escalation to 45 Gy with urethra-sparing results in excellent toxicity profiles and PSA relapse rates similar to those reported by other dose-escalated regimens. The existence of intraprostatic recurrences in patients with high-risk features confirms the notion of a high α/ß ratio in these phenotypes resulting in diminished effectiveness with hypofractionated dose escalation.

Disruption of the crypt niche promotes outgrowth of mutated colorectal tumor stem cells.

Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5-positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane-dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury-induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.

Positron Emission Tomography-Derived Metrics Predict the Probability of Local Relapse After Oligometastasis-Directed Ablative Radiation Therapy.

PURPOSE: Early positron emission tomography-derived metrics post-oligometastasis radioablation may predict impending local relapses (LRs), providing a basis for a timely ablation. METHODS AND MATERIALS: Positron emission tomography data of 623 lesions treated with either 24 Gy single-dose radiation therapy (SDRT) (n = 475) or 3 ×  9 Gy stereotactic body radiation therapy (SBRT) (n = 148) were analyzed in a training data set (n = 246) to obtain optimal cutoffs for pretreatment maximum standardized uptake value (SUVmax) and its 3-month posttreatment decline (ΔSUVmax) in predicting LR risk, validated in a data set unseen to testing (n = 377). RESULTS: At a median of 21.7 months, 91 lesions developed LRs: 39 of 475 (8.2%) after SDRT and 52 of 148 (35.1%) after SBRT. The optimal cutoff values were 12 for SUVmax and -75% for ΔSUVmax. Bivariate SUVmax/ΔSUVmax permutations rendered a 3-tiered LR risk stratification of dual-favorable (low risk), 1 adverse (intermediate risk) and dual-adverse (high risk). Actuarial 5-year local relapse-free survival rates were 93.9% versus 89.6% versus 57.1% (P < .0001) and 76.1% versus 48.3% versus 8.2% (P < .0001) for SDRT and SBRT, respectively. The SBRT area under the ROC curve was 0.71 (95% CI, 0.61-0.79) and the high-risk subgroup yielded a 76.5% true positive LR prediction rate. CONCLUSIONS: The SBRT dual-adverse SUVmax/ΔSUVmax category LR prediction power provides a basis for prospective studies testing whether a timely ablation of impending LRs affects oligometastasis outcomes.

Colorectal Cancer Develops Inherent Radiosensitivity That Can Be Predicted Using Patient-Derived Organoids.

Identifying colorectal cancer patient populations responsive to chemotherapy or chemoradiation therapy before surgery remains a challenge. Recently validated mouse protocols for organoid irradiation employ the single hit multi-target (SHMT) algorithm, which yields a single value, the D0, as a measure of inherent tissue radiosensitivity. Here, we translate these protocols to human tissue to evaluate radioresponsiveness of patient-derived organoids (PDO) generated from normal human intestines and rectal tumors of patients undergoing neoadjuvant therapy. While PDOs from adenomas with a logarithmically expanded Lgr5+ intestinal stem cell population retain the radioresistant phenotype of normal colorectal PDOs, malignant transformation yields PDOs from a large patient subpopulation displaying marked radiosensitivity due to reduced homologous recombination-mediated DNA repair. A proof-of-principle pilot clinical trial demonstrated that rectal cancer patient responses to neoadjuvant chemoradiation, including complete response, correlate closely with their PDO D0 values. Overall, upon transformation to colorectal adenocarcinoma, broad radiation sensitivity occurs in a large subset of patients that can be identified using SHMT analysis of PDO radiation responses. SIGNIFICANCE: Analysis of inherent tissue radiosensitivity of patient-derived organoids may provide a readout predictive of neoadjuvant therapy response to radiation in rectal cancer, potentially allowing pretreatment stratification of patients likely to benefit from this approach.

Cytolytic T cells induce ceramide-rich platforms in target cell membranes to initiate graft-versus-host disease.

Alloreactive donor cytolytic T lymphocytes play a critical role in pathophysiology of acute graft-versus-host disease (GVHD). As GVHD progression involves tumor necrosis factor superfamily receptor activation, and as apoptotic signaling for some tumor necrosis factor superfamily receptors might involve acid sphingomyelinase (ASMase)-mediated ceramide generation, we hypothesized that ASMase deletion would ameliorate GVHD. Using clinically relevant mouse models of acute GVHD in which allogeneic bone marrow and T cells were transplanted into asmase+/+ and asmase(-/-) hosts, we identify host ASMase as critical for full-blown GVHD. Lack of host ASMase reduced the acute inflammatory phase of GVHD, attenuating cytokine storm, CD8+ T-cell proliferation/activation, and apoptosis of relevant graft-versus-host target cells (hepatocytes, intestinal, and skin cells). Organ injury was diminished in asmase(-/-) hosts, and morbidity and mortality improved at 90 days after transplantation. Resistance to cytolytic T lymphocyte-induced apoptosis was found at the target cell membrane if hepatocytes lack ASMase, as hepatocyte apoptosis required target cell ceramide generation for formation of ceramide-rich macrodomains, sites concentrating proapoptotic Fas. These studies indicate a requirement for target cell ASMase in evolution of GVHD in liver, small intestines, and skin and provide potential new targets for disease management.

Pharmacologic inactivation of kinase suppressor of ras-1 abrogates Ras-mediated pancreatic cancer.

Inhibition of the kinase suppressor of ras-1 (KSR1) gene by continuous infusion of phosphorothioate antisense oligonucleotides (ODNs) prevented growth of K-Ras-dependent human PANC-1 pancreatic and A549 non-small-cell lung carcinoma xenografts in nude mice, effected regression of established PANC-1 tumors and inhibited A549 lung metastases, all without apparent toxicity. These studies suggest KSR1 antisense ODNs as a treatment for Ras-dependent human malignancies, in particular pancreatic cancer, which lacks effective curative therapy.

Conformal Avoidance of Normal Organs at Risk by Perfusion-Modulated Dose Sculpting in Tumor Single-Dose Radiation Therapy.

PURPOSE: Although 24 Gy single-dose radiation therapy (SDRT) renders >90% 5-year local relapse-free survival in human solid tumor lesions, SDRT delivery is not feasible in ∼50% of oligometastatic lesions owing to interference by dose/volume constraints of a serial organ at risk (OAR). Conformal OAR avoidance is based on a hypothetical model positing that the recently described SDRT biology specifically permits volumetric subdivision of the SDRT dose, such that high-intensity vascular drivers of SDRT lethality, generated within a major tumor subvolume exposed to a high 24 Gy dose (high-dose planning target volume [PTVHD]), would equilibrate SDRT signaling intensity throughout the tumor interstitial space, rendering bystander radiosensitization of a minor subvolume (perfusion-modulated dose sculpting PTV [PTVPMDS]), dose-sculpted to meet a serial OAR dose/volume constraint. An engineered PTVPMDS may thus yield tumor ablation despite PMDS dose reduction and conformally avoiding OAR exposure to a toxic dose. METHODS AND MATERIALS: Dose fall-off within the PTVPMDS penumbra of oligometastatic lesions was planned and delivered by intensity modulated inverse dose painting. SDRT- and SDRT-PMDS-treated lesions were followed with periodic positron emission tomography/computed tomography imaging to assess local tumor control. RESULTS: Cumulative baseline 5-year local relapse rates of oligometastases treated with 24 Gy SDRT alone (8% relapses, n = 292) were similar in moderate PTVPMDS dose-sculpted (23-18 Gy, n = 76, 11% relapses, P = .36) and extreme dose-sculpted (<18 Gy, n = 61, 14% relapses, P = .29) lesions, provided the major 24 Gy PTVHD constituted ≥60% of the total PTV. In contrast, 28% of local relapses occurred in 26 extreme dose-sculpted PTVPMDS lesions when PTVHD constituted <60% of the total PTV (P = .004), suggesting a threshold for the PTVPMDS bystander effect. CONCLUSION: The study provides compelling clinical support for the bystander radiosensitization hypothesis, rendering local cure of tumor lesions despite a ≥25% PTVPMDS dose reduction of the 24 Gy PTVHD dose, adapted to conformally meet OAR dose/volume constraints. The SDRT-PMDS approach thus provides a therapeutic resolution to otherwise radioablation-intractable oligometastatic disease.