Christopher S. Lange

Cell-cell interactions in spheroids maintained in suspension

We have developed a system of mixed aggregates of cultured cells, to model in situ cell interactions. This three-dimensional (3D) system of floating cell aggregates, termed spheroids for their round shape, enables one to monitor their growth in both size and number of constituent clonogens and to measure survival curves for cells having 3D cell-cell interactions. This system was used to measure the three-dimensional cell-cell interactions on growth, and clonogenicity of either AG1522 fibroblasts, or HeLa cervical cancer cells (pure spheroids, or if both feeder and test cells are the same type, pseudohybrid spheroids), and/or of mixtures of both (hybrid spheroids). By following the increase or decrease in size of, or number of clonogens per, spheroid over time, one obtains growth or inhibition curves. By relating these clonogen numbers, one obtains, after a suitable growth period, relative survival. The system allows one to score the effects of irradiation and of other treatments, as well as the effect of interaction of the constituent cells on their survival. Floating pure, or pseudohybrid (composed of 10% live fibroblasts and 90% supralethally irradiated fibroblast feeder cells) spheroids, shrank to about 10–20% of their volume in three days and then remained at that size for up to six days. In contrast, pure spheroids composed of live HeLa cells increased their volume by an order of magnitude over the same period. Survival of cells in spheroids was measured by the ability of individual spheroids to grow beyond a size implying a ten-fold increase. A caveat to be observed is to correct survival for cellular multiplicity, i.e. reduce survival values to compensate for more than one colony former at the time of irradiation. The system of spheroids floating and growing in nutrient medium provides a selective system for evaluating growth of HeLa, and by implication, other neoplastic cells, without interference from (overgrowth by) normal fibroblasts. Thus it is possible to discriminate between normal and neoplastic cells by virtue of whether or not cells grow in suspension. Such a system seems ideal for testing novel strategies (radiation in combination with chemicals), in an in vivo-like environment.

Survival Following Sublobar Resection for Early-Stage Non-Small Cell Lung Cancer With or Without Adjuvant External Beam Radiation Therapy: A Population-Based Study

BACKGROUNDnPatients undergoing sublobar resection for early-stage non-small cell lung cancer may receive adjuvant radiation therapy in an effort to improve outcomes despite limited data regarding its efficacy.nnnMETHODSnUsing the Surveillance, Epidemiology, and End-Results (SEER) registry we identified patients diagnosed with stage I non-small cell lung cancer between 1988 and 2003 who were definitively treated with sublobar surgical resection with or without adjuvant external beam radiation therapy. Kaplan-Meier, Cox regression, and propensity-score-matched survival analyses were performed to evaluate the effect of adjuvant external beam radiation therapy on survival.nnnRESULTSnA total of 5,908 eligible cases were identified: 493 received external beam radiation therapy and 5,415 received no additional local-regional treatment. The use of external beam radiation therapy was associated with significantly worse median overall and disease-specific survival compared with no additional local-regional therapy: 31 and 45 months vs 51 and 98 months, respectively (P < .001). On multivariate analysis, the most significant predictor of death was the use of adjuvant radiation therapy (hazard ratio 1.505; 95% CI, 1.318-1.717; P < .001). The survival detriment associated with external beam radiation therapy remained after propensity-score-matched analysis.nnnCONCLUSIONSnThe use of adjuvant external beam radiation therapy is associated with a significant decrease in overall and disease-specific survival for patients with T1-2N0M0 non-small cell lung cancer treated with sublobar resection. Although this finding may be related to covariables not reported in SEER, such as margin status, chemotherapy use, radiation dose, and portal, alternative radiation treatment strategies should be explored.

ANAL CANCER: RADIATION AND CONCOMITANT CONTINUOUS INFUSION CHEMOTHERAPY

Abstract Carcinoma of anus, Chemo-radiation therapy, 5-fluorouracil, 5FU, Mitomycin C, COncomitant infusion, Local control and survival, Clinical results, Hybrid spheroid assay.

Prostate and seminal vesicle volume based consideration of prostate cancer patients for treatment with 3D‐conformal or intensity‐modulated radiation therapya)

PURPOSEnThe purpose of this article was to determine the suitability of the prostate and seminal vesicle volumes as factors to consider patients for treatment with image-guided 3D-conformal radiation therapy (3D-CRT) or intensity-modulated radiation therapy (IMRT), using common dosimetry parameters as comparison tools.nnnMETHODSnDosimetry of 3D and IMRT plans for 48 patients was compared. Volumes of prostate, SV, rectum, and bladder, and prescriptions were the same for both plans. For both 3D and IMRT plans, expansion margins to prostate+SV (CTV) and prostate were 0.5 cm posterior and superior and 1 cm in other dimensions to create PTV and CDPTV, respectively. Six-field 3D plans were prepared retrospectively. For 3D plans, an additional 0.5 cm margin was added to PTV and CDPTV. Prescription for both 3D and IMRT plans was the same: 45 Gy to CTV followed by a 36 Gy boost to prostate. Dosimetry parameters common to 3D and IMRT plans were used for comparison: Mean doses to prostate, CDPTV, SV, rectum, bladder, and femurs; percent volume of rectum and bladder receiving 30 (V30), 50 (V50), and 70 Gy (V70), dose to 30% of rectum and bladder, minimum and maximum point dose to CDPTV, and prescription dose covering 95% of CDPTV (D95).nnnRESULTSnWhen the data for all patients were combined, mean dose to prostate and CDPTV was higher with 3D than IMRT plans (P < 0.01). Mean D95 to CDPTV was the same for 3D and IMRT plans (P > 0.2). On average, among all cases, the minimum point dose was less for 3D-CRT plans and the maximum point dose was greater for 3D-CRT than for IMRT (P < 0.01). Mean dose to 30%, rectum with 3D and IMRT plans was comparable (P > 0.1). V30 was less (P < 0.01), V50 was the same (P > 0.2), and V70 was more (P < 0.01) for rectum with 3D than IMRT plans. Mean dose to bladder was less with 3D than IMRT plans (P < 0.01). V30 for bladder with 3D plans was less than that of IMRT plans (P < 0.01). V50 and V70 for 3D plans were the same for 3D and IMRT plans (P > 0.2). Mean dose to femurs was more with 3D than IMRT plans (P < 0.01). For a given patient, mean dose and dose to 30% rectum and bladder were less with 3D than IMRT plans for prostate or prostate+SV volumes <65 (38/48) and 85 cm3 (39/48), respectively (P < 0.01). The larger the dose to rectum or bladder with 3D plans, the larger also was the dose to these structures with IMRT (P < 0.001). For both 3D and IMRT plans, dose to rectum and bladder increased with the increase in the volumes of prostate and seminal vesicles (P < 0.02 to 0.001).nnnCONCLUSIONSnVolumes of prostate and seminal vesicles provide a reproducible and consistent basis for considering patients for treatment with image-guided 3D or IMRT plans. Patients with prostate and prostate+SV volumes <65 and 85 cm3, respectively, would be suitable for 3D-CRT. Patients with prostate and prostate+SV volumes >65 and 85 cm3, respectively, might get benefit from IMRT.

Adjuvant irradiation to prevent keloidal fibroproliferative growth should be standard of care

After cutaneous injury, cytokine mediators recruit an inflammatory infiltrate that stimulates migration of keratinocytes and fibroblasts; subsequent proliferation of fibroblasts and keratinocytes begins 4 to 5 days later [1]. The formation of a keloid is dependent upon fibroblast migration, proliferation and type III collagen production [1]. n nFirst{hyphen}line treatment of keloids involves topical or intralesional steroids. Recurrent or resistant keloids are managed by surgical excision or cryotherapy, followed by steroidal application, that presumably limits inflammation and cellular migration. Adjuvant irradiation to surgical excision is an alternative but underutilized modality that has demonstrated superior patient satisfaction and local control compared to post{hyphen}cryotherapy or resection intralesional steroids, respectively [2, 3]. n nThis article is protected by copyright. All rights reserved.

Rejoining of X-Ray-Induced DNA Double-Strand Breaks Declines in Unstimulated Human Lymphocytes Aging in Vivo

If DNA repair plays a major role in aging (Gensler and Bernstein, 1981; Hart et al., 1979; Little, 1976; Yielding, 1974; recently reviewed in Warner et al., 1987), within a species one would expect to see decreased efficacy of repair processes in older animals. Some data support this prediction, whereas others do not (recently reviewed in Tice and Setlow, 1985). In general, as studied in nonhuman animals, in vivo age-related DNA repair capacities differ by type of damage, by types of repair, by species, and by strain as well as by organ or tissue (Licastro and Walford, 1985; Niedermuller et al., 1985; Su et al., 1984; Tice and Setlow, 1985; Vijg, 1987).

Radiotherapy-induced reactivation of neurotrophic human herpes viruses: Overview and management

PURPOSEnInfection by Human Herpes Viruses (HHV) types 1-3, are prevalent throughout the world. It is known that radiotherapy can reactivate HHVs, but it is unclear how and to what extent reactivations can interact with or affect radiotherapeutic efficacy, patient outcomes and mortality risk. Herein, we aim to summarize what is known about Herpes Simplex Virus (HSV)-1,2 and Varicella Zoster Virus (VZV) pathophysiology as it relates to tumor biology, radiotherapy, chemo-radiotherapy, diagnosis and management so as to optimize cancer treatment in the setting of active HHV infection. Our secondary aim is to emphasize the need for further research to elucidate the potential adverse effects of active HHV infection in irradiated tumor tissue and to design optimal management strategies to incorporate into cancer management guidelines.nnnMATERIALS AND METHODSnThe literature regarding herpetic infection, herpetic reactivation, and recurrence occurring during radiotherapy and that regarding treatment guidelines for herpetic infections are reviewed. We aim to provide the oncologist with a reference for the infectious dangers of herpetic reactivation in patients under their care and well established methods for prevention, diagnosis, and treatment of such infections. Pain management is also considered.nnnCONCLUSIONSnIn the radiotherapeutic setting, serologic assays for HSV-1 and HSV-2 are feasible and can alert the clinician to patients at risk for viral reactivation. RT-PCR is specific in identifying the exact viral culprit and is the preferred diagnostic method to measure interventional efficacy. It can also differentiate between herpetic infection and radionecrosis. The MicroTrak® HSV1/HSV2/VZV staining kit has high sensitivity and specificity in acute lesions, is also the most rapid means to confirm diagnosis. Herpetic reactivation and recurrences during radiotherapy can cause interruptions, cessations, or prolongations of the radiotherapeutic course, thus decreasing the biologically effective dose, to sub-therapeutic levels. Active HHV infection within the treatment volume results in increased tumor radio-resistance and potentially sub-therapeutic care if left untreated. Visceral reactivations may result in fatality and therefore, a high index of suspicion is important to identify these active infections. The fact that such infections may be mistaken for acute and/or late radiation effects, leading to less than optimal treatment decisions, makes knowledge of this problem even more relevant. To minimize the risk of these sequelae, prompt anti-viral therapy is recommended, lasting the course of radiotherapy.

Is HPV vaccination of pregnant women really safe

More than 72 million girls and women have been vaccinated with human papillomavirus (HPV) vaccines worldwide but data on safety of vaccination during pregnancy are limited. HPV vaccination is not recommended during pregnancy, but inadvertent vaccination of women with unrecognized pregnancies was still of concern (Scheller, Pasternak, Mølgaard-Nielsen, Svanstr€ om, & Hviid, 2017; Lawton et al., 2018). To this end, Scheller et al. conducted an analysis to demonstrate that administration of the Quadrivalent HPV vaccine (QHPVV) during pregnancy is not associated with a significant risk of adverse birthing outcomes (Scheller et al., 2017). However, active HPV infection was not determined in either cohort, which may have led to confounding results. Ambuhl showed that HPV infection, alone, is significantly associated with, and may cause, spontaneous abortions and preterm deliveries (Ambuhl, 2016); these findings are supported by Niyibizi, Zanr e, Mayrand, & Trottier (2017) and Cho [2013]. Mosbah, Barakat, Nabiel, & Barakat (2018) found an association between active HPV infection and spontaneous preterm labor, and showed a direct correlation between viral load and preterm labor. Presumably, QHPVV decreases the prevalence of the most commonly encountered high-risk HPV infections in the vaccinated cohort and, therefore, decreases the risk of HPV-associated adverse birthing outcomes. Given the strong association of HPV infection with adverse birthing outcomes, to appropriately measure the risk of QHPVV on birthing outcomes, one should correct and test for active HPV infection between the cohorts.

Abstract 1905: Possible cancer stem cells: Folate hydrolase-1 is expressed in a subset of Oct4-positive melanoma cells

Background: Folate hydrolase-1 (FOLH1; NAALADase; PSMA) is a type II transmembrane protein that binds substrates with terminal glutamates. The MXXXL motif on the cytoplasmic N-terminal domain of FOLH1 interacts with clathrin and caveolin-1 to facilitate constitutive internalization upon substrate binding. J591, an established monoclonal antibody (AB) to FOLH1, is highly specific to and is effectively endocytosed after binding to the extracellular domain of FOLH1; J591 is presently being developed in the clinical trial setting, as a vehicle for AB-based brachytherapy in cancers that express FOLH1. Physiological FOLH1 is expressed in cellular regions that are protected by tight-junctions or the blood-brain-barrier, and are therefore not targeted by circulating J591. Functionally, FOLH1 is responsible for cerebral glutamate production. In the oncological setting, FOLH1 is upregulated throughout prostate cancer cellular membranes, and is luminally expressed by cancer neovessels. We have characterized neovascular FOLH1 expression in malignant melanoma (MM), a solid tumor of neural crest (NC) origin. Comparative RTqPCR analysis of normal skin, primary (p), and metastatic (m) MM revealed respective 10.64 and 18.21 -fold increases in FOLH1 gene-expression in pMM (p=0.0041) and mMM (p=0.042) samples as compared to normal skin. Immunohistochemistry of paraffin-embedded MM tumors revealed FOLH1 protein expression in the neovasculature of 4/11 (36%) of pMM and 9/14 (64%) of mMM cases; we noted a subset of keratinocytic and melanocytic FOLH1 positive cells. These results, and the known functional role of glutamate production in a subset of NC-derived tissue, suggest that MM cells may also express cellular FOLH1. Methods: Six (3 BRAF+; 3 BRAF-) MM cell lines were evaluated for cellular FOLH1 and Oct4 expression with J591-based and anti-Oct4 AB immunofluorescence (IF). Oct4 is a homeodomain binding protein encoded by POU5f1, widely accepted as a stem cell marker. FOLH1+ cell lines with permeablized and non-permeablized membranes were incubated with J591. The effect of γ-irradiation (0, 4.5, or 9 Gy) was also tested in FOLH1+ cell lines with harvesting 6 or 24 hours post-irradiation. Results: Ten to 40% of MM cells demonstrated intracellular FOLH1 expression in 2 BRAF-/6 MM cell lines, with a predominant perinuclear and terminal dendritic distribution (e.g., axonal morphology). IF co-labeling with J591 (cytoplasmic) and antibodies to Oct4 (nuclear) identified possible stemness of FOLH1+ / Oct4+ MM cells. RT-PCR analysis of the irradiated cell lines demonstrated ~2-fold increased FOLH1 mRNA levels in 1/2 MM cell lines. Conclusions: Herein is the first demonstration that MM cells express FOLH1. Given that FOLH1 glutamate production is closely linked to NC-originating glial cells, it can be reasonably postulated that this newly identified subset of FOLH1+/Oct4+ MM cells could be NC precursors, or cancer stem cells, for MM. Further, radiation-induced FOLH1 upregulation may increase the therapeutic ratio of AB-based brachytherapy. Citation Format: Marigdalia K. Ramirez-Fort, He Liu, Vicente Navarro, Barbara Meier, Mitch Levesque, Jonathan Moy, Jessica Vissicchio, Emmanuel Contassot, Sae Kim, Talal Syed, Michael Zhang, Vincent Tem, Paul J. Christos, Scott T. Tagawa, Neil H. Bander, Christopher S. Lange, Lars E. French. Possible cancer stem cells: Folate hydrolase-1 is expressed in a subset of Oct4-positive melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1905. doi:10.1158/1538-7445.AM2017-1905

Abstract 3346: Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates

We tested the Cancer Stem Cell (CSC) hypothesis using the patented Hybrid Spheroid (HS) Assay (HSA) and by applying Koch9s postulates to test its validity. The HSA is an in vitro assay that enables one to take a viable sample of an individual patient9s tumor, make a single cell suspension, mix it in known proportions with human fibroblasts (AG1522) and dispense a known number of cells of the mixture into each well of Ultra Low Attachment (ULA) 96-well plates to agglomerate into 1 HS/well, each containing an average of Applying Koch9s postulates: (1) Does the patient9s tumor contain cells with CSC properties? Yes: (a) The HSs grow to a size consistent with containing a CSC, (b) those that grew contain cells expressing ≥ 1 OKSM factor(s), and (c) such HSs contain differentiated progeny of the initial CSC. (2) Can we isolate and propagate these cells? In progress: testing HS serial growth, dissociation and reculture. (3) Can these cells induce the tumor in vivo? In progress: testing by xenografting individual CSC-containing HSs in NSG mice. (4) Do these cells contain and express specific gene products that give them CSC properties? Yes: Demonstrated for Oct4 and Nanog. (5) If we disrupt these genes, do the cells lose their CSC properties? In progress: (shRNA). (6) If we eliminate the CSCs do we eliminate the cancer? Yes: tested by (a) mathematical modeling of single CSC growth, differentiation of some progeny into ATCs capable of a limited number of divisions, that then become terminal non-proliferative tumor cells in the HSA; if a single CSC survives/remains, the HS will grow, i.e., the cancer will recur (63% for an average of 1 CSC in a HS, with a Poisson distribution, assuming that niche sites remain available), and (b) by measuring surviving fractions after exposure to radiation and/or chemotherapy agents (where sterilization of the CSC prevents the HS from growing ≥ 10 divisions. The HSA was applied to tumor samples taken from individual endometrial adenocarcinoma patients and correctly predicted, based on CSC radioresistance, which patients would fail their standard-of-care treatments. Conclusion: The CSC hypothesis is validated in the HSA. Citation Format: Christopher S. Lange, Talal Syed, Mike Zhang, Christian Sabalvaro, Hana I. Lim, Ghadir Salame, Bhargava Chitti, Ifeanyi Ilonzo, Arsalaan Ansari, Michelle Chan, Catherine Li, Jing Xu, Xiaotong Chen. Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch9s postulates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3346.