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An 18-year-old woman with an established history of neurofibromatosis type 1 (NF-1) presented for her 1-year dermatologic follow-up. Physical examination revealed two subcutaneous nodules on her right arm, axillary freckling, scattered café-au-lait macules (CALMs) on the trunk, and a 12 cm × 17 cm hyperpigmented rectangular region on her right flank (Figure 1). The pigmented patch contained numerous new CALMs that were morphologically consistent with CALMs identified on prior examinations; neither the patch nor the CALMs within it were present at prior examinations. Interestingly, the appearance of the patch and associated CALMs was preceded by a rectangular-shaped, second-degree thermal burn. On further questioning, the patient revealed that she had burned herself with hot water 4 months prior to her presentation in clinic, and noted the development of multiple CALMs within the skin area of her prior burn approximately 4 weeks after the incident. Of note, her left flank had sparsely scattered CALMs, which was consistent with her prior skin examinations (Figure 2). A depigmenting cream was to be applied to the rectangular pigmented patch; unfortunately, post-inflammatory hyperpigmentation from the burn and the adjoining lesions resulting from the Koebner phenomenon continue to be refractory to treatment.

Backgrounds Folate Hydrolase‐1 (FOLH1; PSMA) is a type II transmembrane protein, luminally expressed by solid tumour neo‐vasculature. Monoclonal antibody (mAb), J591, is a vehicle for mAb‐based brachytherapy in FOLH1+ cancers. Brachytherapy is a form of radiotherapy that involves placing a radioactive material a short distance from the target tissue (e.g., on the skin or internally); brachytherapy is commonly accomplished with the use of catheters, needles, metal seeds and antibody or small peptide conjugates. Herein, FOLH1 expression in primary (p) and metastatic (m) Merkel cell carcinoma (MCC) is characterized to determine its targeting potential for J591‐brachytherapy. Materials & Methods Paraffin sections from pMCC and mMCC were evaluated by immunohistochemistry for FOLH1. Monte Carlo simulation was performed using the physical properties of conjugated radioisotope lutetium‐177. Kaplan–Meier survival curves were calculated based on patient outcome data and FOLH1 expression. Results Eighty‐one MCC tumours were evaluated. 67% (54/81) of all cases, 77% (24/31) pMCC and 60% (30/50) mMCC tumours were FOLH1+. Monte Carlo simulation showed highly localized ionizing tracks of electrons emitted from the targeted neo‐vessel. 42% (34/81) of patients with FOLH1+/− MCC had available survival data for analysis. No significant differences in our limited data set were detected based on FOLH1 status (p = 0.4718; p = 0.6470), staining intensity score (p = 0.6966; p = 0.9841) or by grouping staining intensity scores (− and + vs. ++, +++, +++) (p = 0.8022; p = 0.8496) for MCC‐specific survival or recurrence free survival, respectively. Conclusions We report the first evidence of prevalent FOLH1 expression within MCC‐associated neo‐vessels, in 60‐77% of patients in a large MCC cohort. Given this data, and the need for alternatives to immune therapies it is appropriate to explore the safety and efficacy of FOLH1‐targeted brachytherapy for MCC. What's already known about this topic? We report the first evidence of prevalent folate hydrolase‐1 (FOLH1; also known as prostate‐specific membrane antigen) expression within MCC‐associated neovessels. What does this study add? Herein, FOLH1 expression in Merkel cell carcinoma neovasculature is validated, and the therapeutic mechanism of specific, systemic targeting of disseminated disease with antibody‐based brachytherapy, is defined.

Dermatofibrosarcoma protuberans (DFSP) is a rare, infiltrative, soft tissue tumor. It has a propensity for deep invasion but a low risk for distant metastasis. The classic presentation is a slowly progressive, painless, and erythematous to purpuric patch on the trunk or arms. A deep, subcutaneous punch biopsy or incisional biopsy should be performed for diagnosis in all suspected cases; wide undermining of the skin is to be avoided for minimizing the risk of tumor seeding and for retaining the feasibility of histopathologic examination of re-excisions. Histopathologic distinction of DFSP from dermatofibroma requires immunohistochemical assessment for CD34, factor XIIIa, nestin, apolipoprotein D, and cathepsin K. Management of this cutaneous sarcoma involves a multidisciplinary oncologic approach. Surgical excision is usually the first step in management. DFSP has a high propensity for local recurrence, even when surgical margins are negative; therefore, radiation therapy or rarely systemic therapy is recommended, especially for locally advanced or metastatic cases. The indolent nature of DFSP requires lifelong surveillance for recurrence; however, most recurrences occur within 3 years of the primary excision. The median time for the development of a local recurrence is estimated to be 32 months. An emerging theragnostic transmembrane receptor target, folate hydrolase-1 (FOLH1; prostate-specific membrane antigen), has been expressed in benign dermatofibromas and in high-grade sarcomatous phenotypes. These findings suggest that DFSP may also express FOLH1, which could allow for surveillance with FOLH1 PET/CT and antibody-mediated brachytherapy.

Treatment of refractory palmar-plantar vitiligo is particularly challenging because the skin in these regions has a limited supply of follicle-derived melanocytic stem cells. Autologous hair transplantation monotherapy is effective in some forms of vitiligo through the provision of melanocytic stem cells. CO2 laser followed by exposure to light (i.e., sunlight or narrow-band ultraviolet-B [nbUVB]) has independently shown to be an effective treatment strategy. Recently, it was found that the combination of hair transplantation and CO2 laser followed by nbUVB exposure had superior efficacy to either modality as monotherapy. Similar to CO2 laser, microneedling produces skin cell proliferation and releases pro-pigmentary cytokines. Given the important role of the cytokines in vitiliginous skin, microneedling may also be an effective therapeutic modality for refractory vitiligo. Herein, we conducted a pilot study to evaluate the efficacy of hair transplantation and CO2 laser or microneedling followed by nbUVB. Microneedling and fractional CO2 laser in combination with hair transplantation and nbUVB both demonstrated utility in the induction of repigmentation in refractory palmar-plantar vitiligo; however, a larger trial would be needed to determine a difference in treatment efficacy. Nonetheless, microneedling is cost-effective and requires minimal training; therefore, microneedling can be easily incorporated into standard dermatological practice.

Psychological morbidity, sexuality, and health/system information have been identified as the highest areas of support needs in patients undergoing management of their prostate cancer (PCa). Management of a patient's sexual function prior to, during and after PCa radiotherapy requires multidisciplinary coordination of care between radiation oncologists, urologists, dermatologists, pharmacists, and psychiatrists. The finale of this three-part review provides a framework for clinicians to better understand the role of mental healthcare providers in the management of sexual toxicities associated with prostatic radiotherapy. The authors recommend that patients be referred for psychological evaluation and possibly to individual, couples or group general or cognitive behavioral sex therapy at the time of their PCa diagnosis, for a more specialized focus on management of sexual toxicities and sexual recovery. The importance and implications of the masculine self-esteem, sexual orientation, gender identification, cultural expectations, relationship status and patient education are reviewed. Well-informed patients tend to have a better quality of life outcomes compared to patients that take on a passive role in their cancer management.

Prostate cancer is the most common malignancy and the second leading cause of cancer-related death in men. Radiotherapy is a curative option that is administered via external beam radiation, brachytherapy, or in combination. Erectile, ejaculatory and orgasm dysfunction(s) is/are known potential and common toxicities associated with prostate radiotherapy. Our multidisciplinary team of physicians and/or scientists have written a three (3) part comprehensive review of the pathogenesis and management radiation-induced sexual dysfunction. Part I reviews pertinent anatomy associated with normal sexual function and then considers the pathogenesis of prostate radiation-induced sexual toxicities. Next, our team considers the associated radiobiological (including the effects of time, dose and fractionation) and physical (treatment planning and defining a novel Organ at Risk (OAR)) components that should be minded in the context of safe radiation treatment planning. The authors identify an OAR (i.e., the prostatic plexus) and provide suggestions on how to minimize injury to said OAR during the radiation treatment planning process.

Prostate cancer is the most common malignancy in men and the second leading cause of cancer-related death in men. Radiotherapy is a curative option that is administered via external beam radiation, brachytherapy, or in combination. Sexual dysfunction is a common toxicity following radiotherapy, similar to men undergoing radical prostatectomy, but the etiology is different. The pathophysiology of radiation-induced sexual dysfunction is multi-factorial, and the toxicity is a major cause of impaired quality of life among long-term prostate cancer survivors. Management of a patient's sexual function during and after radiotherapy requires multidisciplinary coordination of care between radiation oncology, urology, psychiatry, pharmacy, and dermatology. This review provides a framework for clinicians to better understand prostatic radiotherapy-induced sexual dysfunction diagnosis, evaluation, and a patient-centered approach to toxicity preventive strategies and management.

Purpose Infection 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. Materials and methods The 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. Conclusions In 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.

Introduction: Cancer cells can escape tumor-specific T cell responses via engagement of inhibitory immune checkpoints. PD-L1/PD-1 interaction is one of the major checkpoints that limit effector T cell function against cancer cells, and monoclonal antibodies that block this interaction have been approved as therapies in multiple tumor indications. As a next generation therapy, small molecule inhibitors of PD-L1 potentially have advantageous properties compared to approved monoclonal antibodies, such as better penetration into solid tumors, reduced immunogenicity, lack of Fc-mediated side effects, convenience of oral administration, and lower cost of goods. We therefore embarked on an effort to identify and develop an orally available small molecule capable of targeting PD-L1/PD-1 interactions. Methods: Inhibition of the PD-L1/PD-1 interaction was measured using a binding assay, followed by a cell culture system assessing PD-1 inhibition of T cell receptor (TCR) activation. Human T cell responses were assessed in vitro using the mixed lymphocyte reaction (MLR) assay, and a human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. For in vivo studies CCX559 was dosed orally in a syngeneic tumor model and in a human tumor cell/PBMC co-implantation model in immune deficient mice. Results: Using structural information and focused medicinal chemistry, we identified CCX559 as a potent inhibitor of PD-L1 interaction with PD-1. CCX559 prevented PD-L1/PD-1 inhibition of TCR signaling in a cell-based reporter assay, increased IFNγ secretion in allogeneic MLR assays, and increased tumor cell killing by human PBMCs. We demonstrated that CCX559 potentially employs multiple mechanisms to inhibit PD-L1, which are distinct from those published for human anti-PD-L1 antibodies. In murine tumor models, orally administered CCX559 reduced tumor growth similarly to a clinically-approved anti-human PD-L1 antibody. Summary: CCX559 is a highly potent, small molecule PD-L1 inhibitor that can be orally administered. CCX559 enhanced primary human T cell activity in vitro and demonstrated anti-tumor efficacy in two murine tumor models. Based on its unique mechanism of PD-L1 inhibition, strong anti-tumor activity, desirable drug properties, and good safety profile, we plan to advance CCX559 into clinical development in the first half of 2021. Citation Format: Chris Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Scamp, Vicky Chhina, Alice Kumamoto, Ryan Ong, Ton Dang, Ashton Easterday, Niky Zhao, Shirley Liu, Rajinder Singh, Israel Charo, Kathleen Sullivan, Thomas J. Schall, Penglie Zhang. CCX559 is a potent orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1274.

Introduction: Antibody-based therapies targeting the Programmed cell Death-1/ Programmed Death-Ligand 1 (PD-1/PD-L1) immune checkpoint axis have achieved great success in cancer immunotherapy in recent years. As a next generation therapy, small molecule inhibitors of PD-1/PD-L1 offer the potential for increased tumor penetration, shorter half-life (to better manage immune related adverse events), and lower cost. We therefore embarked on an effort to identify and develop orally available small molecules capable of targeting PD-L1. Methods: We designed and optimized a number of small molecule PD-L1 inhibitors which potently disrupted the interaction of PD-1 with PD-L1. Active compounds were first profiled by an ELISA assay measuring inhibition of the PD-1/PD-L1 interaction, followed by a functional cell-based reporter assay, mixed lymphocyte reaction (MLR) assay, and human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. Targeted medicinal chemistry efforts were employed to improve potency and oral bioavailability, and candidate compounds were then evaluated in murine tumor models. Due to the specific reactivity of these compounds to human PD-L1 (but not murine PD-L1), a syngeneic tumor model with murine MC-38 colon tumor cells expressing human PD-L1 (MC38-hPD-L1 tumor model) was used. Results: The optimized PD-L1 inhibitors were highly potent in cell-based reporter assay, the MLR assay and PBMC-mediated tumor cell killing assays. These compounds also possess high oral bioavailability and desirable safety profiles. In the MC38-hPD-L1 tumor model, Lead compounds potently reduced tumor growth similarly to an anti-human PD-L1 antibody, which was used as a positive control for the experiments. The tumor microenvironment analysis by flow cytometry demonstrated that these compounds almost completely occupied human PD-L1 on the tumor cells in vivo, and thus could potently block the interaction of PD-1/PD-L1 and enhance the immune responses against tumor. Summary: We have identified and advanced unique small molecule inhibitors of human PD-L1 by rational design and optimization. Molecules resulting from these efforts exhibited marked inhibition of the PD-1/PD-L1 interaction and signaling in vitro, and potent anti-tumor effects in an animal model. Citation Format: Shijie "Chris" Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Ong, Vicky Chhina, Alice Kumamoto, Simon Yau, Ton Dang, Ashton Easterday, Shirley Liu, Rajinder Singh, Israel Charo, Thomas J. Schall, Penglie Zhang. Anti-tumor effect of orally available small molecule PD-L1 inhibitors in a murine model of colon adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5693.

Cutaneous injury can ignite excessive fibroproliferative growth that results in keloid formation. Keloids are associated with significant morbidity related to disfigurement and/or symptoms (e.g., pain and pruritus). First-line treatment of formed keloids involves topical or intralesional steroids. Recurrent or resistant keloids are managed by surgical excision or cryotherapy, followed by steroidal application or adjuvant irradiation. Although adjuvant irradiation appears to be most efficacious, alternative therapeutic options are needed for patients without access to radiation centers. Botulinum Toxin A (BTA) appears to have similar inhibitory effects to irradiation on the cell cycle via downregulation of pathogenic cytokines. Herein, we conducted a study to compare the efficacy of intralesional triamcinolone used alone, or in combination with BTA, in the treatment of formed keloid scars. Twenty patients with a cumulative of 40 keloids completed the study. There was no significant difference between treatment arms with respect to height vascularization, pliability, and pigmentation scores. The addition of BTA resulted in significant symptomatic improvement of pain and pruritus as compared to intralesional triamcinolone alone (p < 0.001). Irradiation is only effective when administered in the adjuvant setting where inhibitory effects on cell cycle and migration are optimized. Future studies with intralesional triamcinolone and BTA should be performed adjuvantly.

Purpose/Objective(s): TTFields are FDA-approved for the treatment of recurrent and newly diagnosed Glioblastoma (GBM) in the supratentorial brain. The EF-14 trial showed that combining TTFields with adjuvant chemo-radiation leads to a significant increase in overall survival compared to adjuvant chemo-radiation alone. High-grade gliomas also occur in the infratentorium and brainstem of pediatric and adult patients. Brainstem gliomas have a median survival of 9 to 11 months, necessitating the exploration of novel treatment combinations, such as delivering TTFields to the infratentorial brain. We recently showed in a simulation-based study that TTFields can be successfully delivered to the infratentorium. Effective delivery was achieved by placement of the arrays on the vertex, bilateral posterolateral occiput, and superior-posterior neck; the array placement results in TTFields at therapeutic intensities throughout the brainstem and cerebellum of realistic computational head models. The previous simulation-based study was performed using realistic computational models of healthy individuals; it did not provide detailed insight into field distributions within tumor tissue located in the infratentorium. Here, we aim to expand the results of this previous study, by simulating the delivery of TTFields to the infratentorium of adult and pediatric patients with high-grade brainstem gliomas. Materials/Methods: A realistic computational model was created using MRI data of adult and pediatric patients with high-grade brainstem gliomas. Transducer arrays were placed on the model and the delivery of TTFields to the infratentorial brain was then simulated using a commercial numerical solver. The electric field distribution in the supratentorium, infratentorium, and within the tumor were derived from the simulations, and the dose in the tumor analyzed. Results: The distribution of calculated isofield lines demonstrates effective delivery of TTFields to infratentorial brain tumors. Conclusion: Our results provides rationale for clinically investigating the utility of TTFields in treating infratentorial high-grade gliomas. Citation Format: Marigdalia K. Ramirez-Fort, Brittany Cross, Ariel Naveh, Shearwood McClelland III, Melissa Mendez, Roberto Santiago, Sean S. Mahase, Jaime Matta, Ze'ev Bomzon, Christopher S. Lange. Simulating delivery of TTFields to the infratentorium in patients with brainstem gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4430.

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]. First{\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]. This article is protected by copyright. All rights reserved.

To use the Surveillance, Epidemiology, and End Results (SEER) Database to analyze the impact of postoperative radiation after limb sparing surgery for high-grade extremity soft tissue sarcomas (STS).We identified patients, aged 20 to 79, who were diagnosed between 1988 and 2006 with high-grade STS of the extremities and underwent radical limb sparing surgery with or without postoperative external beam radiation. Kaplan-Meier and Cox regression analyses were performed to evaluate the effect of postoperative external beam radiation therapy on overall survival (OS) and disease-specific survival (DSS).A total of 983 patients met the selection criteria: 788 (80.2%) received postoperative radiation and 195 (19.8%) underwent surgery alone. For the whole cohort, there were no differences between the groups in OS (P=0.06) or DSS (P=0.20). On subgroup analysis, for tumors ≤5 cm there remained no significant differences in OS (P=0.8) or DSS (P=0.93). However, for tumors5 cm the 3-year OS improved with the addition of postoperative radiation from 55.6% to 73.4% (P0.001). Similarly, the 3-year DSS improved from 68.1% to 80.6% (P=0.005).Because of the retrospective nature of this study and inherent limitations of the SEER database, a large prospective study is needed to further elucidate the relationship between postoperative radiation and survival. However, these data do support the use of adjuvant radiation for patients with high-grade extremity STS measuring5 cm.

Purpose/Objective(s) Prostate-specific membrane antigen (PSMA), also known as folate hydrolase 1 (FOLH1), is a highly specific biomarker and therapeutic target for prostate cancer (PC). J591, a monoclonal antibody specific to PSMA, is the primary method used to detect PSMA expression. It is currently under investigation as a vehicle to deliver targeted therapies to PC cells. Androgen deprivation therapy (ADT) upregulates PSMA. Preclinical models of androgen resistant (AR) xenografts demonstrate PSMA upregulation by ADT increases the therapeutic index of J591-drug conjugates, suggestive of enhanced drug uptake. We sought to evaluate the effect of external beam radiation (EBRT) on PSMA expression in PC cells ± ADT. Methods Androgen sensitive LNCaP and AR CWR22Rv1 PC cell lines were cultured in standard and charcoal-stripped media for two weeks. Both cell lines were grown in both media conditions, and received either no EBRT, EBRT in 2 Gray (Gy) daily fractions to a maximum of 10 Gy, or 7.5 Gy in 1 fraction. Cell surface PSMA expression was measured by flow cytometry as mean fluorescence intensity 24 hours after each 2 Gy fraction, or on days 1, 3, 5, 7, and 9 after 7.5 Gy. Results LNCaP in standard media upregulated PSMA by 2.97-fold (SEM ± 0.72) with fractionated EBRT to a cumulative dose of 4 Gy, peaking on days 2 and 3 after 4 Gy and 6 Gy cumulative doses, respectively. CWR22Rv1 in standard medium, upregulated PSMA expression by 3.21-fold (SEM ± 0.44) after a cumulative dose of 8 Gy, peaking on day 4. Under ADT (charcoal-stripped media), EBRT did not further increase LNCaP cell PSMA expression (maximal fold-change of 1.01 (SEM ± 0.02)) relative to ADT alone. Compared to CWR22Rv1 with ADT alone, ADT plus EBRT upregulated PSMA by 3.73-fold (SEM ± 0.44) after a cumulative fractionated dose of 8 Gy, and peaking on days 4 and 5. However, 7.5 Gy in one fraction did not significantly increase PSMA expression of LNCaP or CWR22Rv1 in standard media. LNCaP showed a maximal fold-change of 1.14 (SEM ± 0.29) on day 9. CWR22Rv1 showed a maximal fold-change of 1.27 (SEM ± 0.02) on day 1. Conclusions We show PSMA expression is independently enhanced by ADT and by low-dose fractionated EBRT, but not with single-fraction ablative EBRT. These observations warrant additional studies using in vitro and in vivo PC models to validate our findings. PSMA upregulation may aid in optimizing J591 targeting for treatment of local and disseminated or micrometastatic disease. Validation of PSMA upregulation by ADT and EBRT supports use of J591-drug conjugates as an adjunct treatment to these standard therapies for PC. Furthermore, if fractionated EBRT upregulates PSMA, then targeted brachytherapy using appropriately selected J591-radionucleotide conjugates may increase its own therapeutic index in a time-dependent manner as a result of regional low-dose rate beta or alpha emission. Citation Format: Marigdalia K. Ramirez-Fort, Sean S. Mahase, M. Junaid Niaz, He Liu, Vincent Navarro, Goran Rac, Harry S. Clarke, Scott T. Tagawa, Joseph M. Jenrette, Neil H. Bander, Christopher S. Lange. External beam radiation therapy and androgen deprivation therapy increase PSMA expression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1774.

Folate hydrolase-1 (FOLH1) is a type II transmembrane protein. Oncologically, FOLH1 is upregulated throughout prostate cancer cells; it is also luminally expressed by the neovasculature of most solid tumors but not by normal vessels. J591, a monoclonal antibody (AB), is specific to, and is effectively endocytosed after extracellular binding to, FOLH1. J591 is presently being developed in clinical trials as a vehicle for AB-based brachytherapy in FOLH1+ cancers. Merkel cell carcinoma (MCC) is a rare, neuroendocrine tumor; metastatic (m) MCC is associated with poor survival. We characterized FOLH1 expression in MCC to determine its target potential for J591-brachytherapy. Paraffin sections from primary (p) and mMCC were deparaffinized and rehydrated. Samples were stained with 3E6 (DAKO), a mouse IgG1 monoclonal anti-human FOLH1. Mouse IgG1 (10 ug/mL in 1% bovine serum albumin) was used as an isotype-matched negative control. Anti-CD31 (IgG1) was used as a positive control. Kaplan-Meier survival curves were calculated based on patient outcome data and FOLH1 expression. 81 MCC tumors were evaluated. 67% (54/81) of all cases with 77% (24/31) of pMCC and 60% (30/50) of mMCC tumors demonstrated FOLH1+ neovessels. No cellular staining of tumor cells was identified. 34 patients with FOLH1 +/- MCC were demographically homogeneous in terms of sex, age, immunosuppression status, prior therapies, stage at diagnosis, and local or distant recurrences. No significant differences were detected based on FOLH1 status, in regards to MCC specific survival (P=0.905), or overall survival (P=0.687), as measured from time of diagnosis. FOLH1 is expressed in the majority of pMCC and mMCC cases and is nonprognostic. Our findings support further investigation of targeted therapy with J591-brachytherapy for the management of MCC. Citation Format: Barbara Meier, Marigdalia K. Ramirez-Fort, Kristina Lachance, Candice D. Church, Sean S. Mahase, Joseph M. Jenrette, Christopher S. Lange, Lars E. French, Paul Nghiem, Neil H. Bander. Folate hydrolase-1 is a novel target for J591-brachytherapy in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2518.

Purpose: The 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. Methods: Dosimetry 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 ( V 30 ) , 50 ( V 50 ) , and 70 Gy ( V 70 ) , dose to 30% of rectum and bladder, minimum and maximum point dose to CDPTV, and prescription dose covering 95% of CDPTV ( D 95 ) . Results: When the data for all patients were combined, mean dose to prostate and CDPTV was higher with 3D than IMRT plans ( P 0.01 ) . Mean D 95 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 ) . V 30 was less ( P 0.01 ) , V 50 was the same ( P > 0.2 ) , and V 70 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 ) . V 30 for bladder with 3D plans was less than that of IMRT plans ( P 0.01 ) . V 50 and V 70 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 cm 3 (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). Conclusions: Volumes 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 cm 3 , respectively, would be suitable for 3D-CRT. Patients with prostate and prostate + SV volumes > 65 and 85 cm 3 , respectively, might get benefit from IMRT.

Background Patients 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. Methods Using 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. Results A 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 Conclusions The 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.

Purpose: The purpose of this study was to analyze the relationship between prostate, bladder, and rectum volumes on treatment planningCT day and prostate shifts in the X Y Z directions on treatment days. Methods: Prostate, seminal vesicles, bladder, and rectum were contoured on CTimages obtained in supine position. Intensity modulated radiation therapy plans was prepared. Contours were exported to BAT-ultrasound imaging system. Patients were positioned on the couch using skin marks. An ultrasound probe was used to obtain ultrasoundimages of prostate, bladder, and rectum, which were aligned with CTimages. Couch shifts in the X Y Z directions as recommended by BAT system were made and recorded. 4698 couch shifts for 42 patients were analyzed to study the correlations between interfraction prostate shifts vs bladder, rectum, and prostate volumes on planning CT. Results: Mean and range of volumes (cc): Bladder: 179 (42–582), rectum: 108 (28–223), and prostate: 55 (21–154). Mean systematic prostate shifts were (cm, ±SD) right and left lateral: − 0.047 ± 0.16 (−0.361–0.251), anterior and posterior: 0.14 ± 0.3 (−0.466–0.669), and superior and inferior: 0.19 ± 0.26 (−0.342–0.633). Bladder volume was not correlated with lateral, anterior/posterior, and superior/inferior prostate shifts ( P > 0.2 ) . Rectal volume was correlated with anterior/posterior ( P 0.001 ) but not with lateral and superior/inferior prostate shifts ( P > 0.2 ) . The smaller the rectal volume or cross sectional area, the larger was the prostate shift anteriorly and vice versa ( P 0.001 ) . Prostate volume was correlated with superior/inferior ( P 0.05 ) but not with lateral and anterior/posterior prostate shifts ( P > 0.2 ) . The smaller the prostate volume, the larger was prostate shift superiorly and vice versa ( P 0.05 ) . Conclusions: Prostate and rectal volumes, but not bladder volumes, on treatment planningCT influenced prostate position on treatment fractions. Daily image-guided adoptive radiotherapy would be required for patients with distended or empty rectum on planning CT to reduce rectal toxicity in the case of empty rectum and to minimize geometric miss of prostate.

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

We tested the Cancer Stem Cell (CSC) hypothesis using the patented Hybrid Spheroid (HS) Assay (HSA) and by applying Koch's postulates to test its validity. The HSA is an in vitro assay that enables one to take a viable sample of an individual patient's 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 Koch's postulates: (1) Does the patient's 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 Koch's 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.

We found no significant decrease in the colony forming ability (clonogenicity) of either HeLa or S91/6 melanoma cells in hybrid spheroids incubated for 24 hr at 37°C with up to 30 μM chloroquine, nor for incubations of up to 9 hr at either 37 or 41°C with 30 μM chloroquine. Incubation for 24 hr at 41°C with chloroquine caused spheroids to disintegrate. When heating was applied to hybrid spheroids intermittently for 3 hr at the beginning and at the end of a 24 hr period of incubation with 30 μM chloroquine, there was no effect on HeLa cells but there was a highly significant decrease in the clonogenicity of S91/6 melanoma cells. Short-term treatment (3 hr) with a combination of mild hyperthermia and 30 μM chloroquine which is non-toxic to unirradiated cells in hybrid spheroids is, nevertheless, effective in decreasing the clonogenicity of irradiated cells of both lines. A similar increase in radiation lethality [radiation potentiating factor (RPF) of about 1.6] was obtained in both HeLa and in S91/6 cells. RPFs did not change significantly after correction of survival curves for cellular multiplicity, nor were they different from those previously reported by us for these cell lines in two versions of dense suspension cultures. Since many cells from biopsies will not grow in conventional culture systems but will do so in hybrid spheroids, the finding of similar RPF values in both systems for cell lines which will grow under both conditions suggests that hybrid spheroids may be useful for radiopotentiation studies. © 1995 Wiley-Liss, Inc.

Background Rodent studies show that neurogenesis is necessary for mediating the salutary effects of antidepressants. Nonhuman primate (NHP) studies may bridge important rodent findings to the clinical realm since NHP-depression shares significant homology with human depression and kinetics of primate neurogenesis differ from those in rodents. After demonstrating that antidepressants can stimulate neurogenesis in NHPs, our present study examines whether neurogenesis is required for antidepressant efficacy in NHPs. Materials/Methodology Adult female bonnets were randomized to three social pens (N = 6 each). Pen-1 subjects were exposed to control-conditions for 15 weeks with half receiving the antidepressant fluoxetine and the rest receiving saline-placebo. Pen-2 subjects were exposed to 15 weeks of separation-stress with half receiving fluoxetine and half receiving placebo. Pen-3 subjects 2 weeks of irradiation (N = 4) or sham-irradiation (N = 2) and then exposed to 15 weeks of stress and fluoxetine. Dependent measures were weekly behavioral observations and postmortem neurogenesis levels. Results Exposing NHPs to repeated separation stress resulted in depression-like behaviors (anhedonia and subordinance) accompanied by reduced hippocampal neurogenesis. Treatment with fluoxetine stimulated neurogenesis and prevented the emergence of depression-like behaviors. Ablation of neurogenesis with irradiation abolished the therapeutic effects of fluoxetine. Non-stressed controls had normative behaviors although the fluoxetine-treated controls had higher neurogenesis rates. Across all groups, depression-like behaviors were associated with decreased rates of neurogenesis but this inverse correlation was only significant for new neurons in the anterior dentate gyrus that were at the threshold of completing maturation. Conclusion We provide evidence that induction of neurogenesis is integral to the therapeutic effects of fluoxetine in NHPs. Given the similarity between monkeys and humans, hippocampal neurogenesis likely plays a similar role in the treatment of clinical depression. Future studies will examine several outstanding questions such as whether neuro-suppression is sufficient for producing depression and whether therapeutic neuroplastic effects of fluoxetine are specific to antidepressants.

Murine L517BY (LY) lymphoma sublines, LY-R (X-radiation resistant) and LY-S (X-radiation sensitive) displayed a difference in susceptibility to camptothecin: susceptibility of LY-S cells to the alkaloid was shifted towards higher concentrations as compared to LY-R cells. A similar difference was observed at the level of genomic DNA when a number of DNA-protein cross-links was determined or single-strand breaks were revealed by the fluorescent nucleoid halo assay. Activities of topoisomerases I and II were the same in both sublines. In turn, a higher resistance to camptothecin was found for the isolated LY-S topoisomerase I in the DNA cleavage test, suggesting that an altered enzyme was responsible for the susceptibility difference observed at the cellular level. In the relaxation test the enzymes from the two sublines showed a different sensitivity to β-lapachone, an activator of topoisomerase I, but were similarly sensitive to all inhibitors, except camptothecin.

The 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.Dosimetry 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).When the data for all patients were combined, mean dose to prostate and CDPTV was higher with 3D than IMRT plans (P0.01). Mean D95 to CDPTV was the same for 3D and IMRT plans (P0.2). 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 (P0.01). Mean dose to 30%, rectum with 3D and IMRT plans was comparable (P0.1). V30 was less (P0.01), V50 was the same (P0.2), and V70 was more (P0.01) for rectum with 3D than IMRT plans. Mean dose to bladder was less with 3D than IMRT plans (P0.01). V30 for bladder with 3D plans was less than that of IMRT plans (P0.01). V50 and V70 for 3D plans were the same for 3D and IMRT plans (P0.2). Mean dose to femurs was more with 3D than IMRT plans (P0.01). 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 volumes65 (38/48) and 85 cm3 (39/48), respectively (P0.01). The larger the dose to rectum or bladder with 3D plans, the larger also was the dose to these structures with IMRT (P0.001). For both 3D and IMRT plans, dose to rectum and bladder increased with the increase in the volumes of prostate and seminal vesicles (P0.02 to 0.001).Volumes 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 volumes65 and 85 cm3, respectively, would be suitable for 3D-CRT. Patients with prostate and prostate+SV volumes65 and 85 cm3, respectively, might get benefit from IMRT.

The purpose of this study was to analyze the relationship between prostate, bladder, and rectum volumes on treatment planning CT day and prostate shifts in the XYZ directions on treatment days.Prostate, seminal vesicles, bladder, and rectum were contoured on CT images obtained in supine position. Intensity modulated radiation therapy plans was prepared. Contours were exported to BAT-ultrasound imaging system. Patients were positioned on the couch using skin marks. An ultrasound probe was used to obtain ultrasound images of prostate, bladder, and rectum, which were aligned with CT images. Couch shifts in the XYZ directions as recommended by BAT system were made and recorded. 4698 couch shifts for 42 patients were analyzed to study the correlations between interfraction prostate shifts vs bladder, rectum, and prostate volumes on planning CT.Mean and range of volumes (cc): Bladder: 179 (42-582), rectum: 108 (28-223), and prostate: 55 (21-154). Mean systematic prostate shifts were (cm, +/-SD) right and left lateral: -0.047 +/- 0.16 (-0.361-0.251), anterior and posterior: 0.14 0.3 (-0.466-0.669), and superior and inferior: 0.19 +/- 0.26 (-0.342-0.633). Bladder volume was not correlated with lateral, anterior/posterior, and superior/inferior prostate shifts (P0.2). Rectal volume was correlated with anterior/posterior (P0.001) but not with lateral and superior/inferior prostate shifts (P0.2). The smaller the rectal volume or cross sectional area, the larger was the prostate shift anteriorly and vice versa (P0.001). Prostate volume was correlated with superior/inferior (P0.05) but not with lateral and anterior/posterior prostate shifts (P0.2). The smaller the prostate volume, the larger was prostate shift superiorly and vice versa (P0.05).Prostate and rectal volumes, but not bladder volumes, on treatment planning CT influenced prostate position on treatment fractions. Daily image-guided adoptive radiotherapy would be required for patients with distended or empty rectum on planning CT to reduce rectal toxicity in the case of empty rectum and to minimize geometric miss of prostate.

We examined, by the fluorescent halo assay, alterations in the nucleoid structure (structure formed from cells under mild lysis conditions: in non-ionic detergent Triton X-100, 0.0005% and 1.5 mol/l NaCl) of L5178Y (LY) cell sublines which had been untreated, treated with reducing/chelating agents (beta-mercaptoethanol or sodium diethyl dithiocarbamate (DDTC(Na))) or X-irradiated. These sublines differ in radiation sensitivity: LY-R is more resistant (D0 = 1.1 Gy) and LY-S more sensitive (D0 = 0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI) (0.5 to 50 micrograms/ml) at pH 6.9 or 9. The maximal DNA unwinding in PI was obtained at 7.5 micrograms/ml PI, at both pH 6.9 and 9 in both sublines; the maximal halo diameter was larger in LY-S than in LY-R cells. In nucleoids from both sublines DNA could be rewound at higher (10-50 micrograms/ml) PI concentrations both at pH 6.9 and 9. This ability was impaired by mercaptoethanol or DDTC(Na) (at pH 9) or by X-irradiation, indicating damage and/or alteration in the DNA superhelical structure. The susceptibility to reducing/chelating agents was greater in LY-S than in LY-R nucleoids, pointing to differences in chromatin structure between these sublines. The amount of X-ray-inflicted damage was higher, when measured at pH 9 than at pH 6.9 and was about twice larger in LY-S than in LY-R nucleoids, when the cells were irradiated with the same X-ray dose.(ABSTRACT TRUNCATED AT 250 WORDS)

In breast cancer (BC) patients the brachial plexus syndrome (BPS) has been reported to be due to loco-regional metastases or radiation plexopathy. Associated arm edema is considered more suggestive of the latter. Radiation therapy is the only effective treatment for BPS reported. The charts of all BC patients who presented to our clinic from 1982 to 2006 with homolateral arm pain and neurological deficits, without humerus, cervical spine, or brain metastases, were reviewed. There were 28 patients fulfilling these criteria for BPS. Supraclavicular, axillary or chest wall metastases developed synchronously with the BPS in 26 patients; in 21 they were recurrences, found 6–94 months (median 34 months) after primary BC treatment, while in 5 others they were progressing inoperable primary tumors and nodes. Arm edema first occurred at the same time as loco-regional metastases in 19 patients. Treatment for the BPS was administered to 22 patients; it was directed at their locoregional metastases. The BPS was initially treated with radiation (8 patients) or chemo- or endocrine therapy (14 patients); 19 (86%) had partial or complete remission of pain and neurologic deficits, with an 8-month median duration. The BPS in BC patients is due to loco-regional metastases and is often associated with arm edema. Chemoor endocrine therapy induced the remission of pain and deficits as frequently as radiation therapy.

A new in vitro method for determining the clonogenicity of mammalian cells in culture is described. The method is based on packaging clonogens into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates, termed hybrid spheroids, provide an in vivo-like environment for entrapped test cells, offering a realistic system for prospective tumor control studies. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of non-colony forming spheroids, the average number of clonogens per spheroid can be calculated, and the survival of irradiated test cells determined. In this fashion survival curves were obtained for HeLa, B-16 and HEp3 cells which corresponded to survival curves obtained in the conventional manner. The clonogenicity of cells, derived from a human maxillary melanoma surgical specimen was also determined by the hybrid spheroid method. With this method, plating efficiency increased in those cells which normally plate poorly, such as tumor cells, thus enabling survival measurements when this is not practical using conventional methods.

Full-size linear chromosomes were prepared from mycoplasmas by using gamma-irradiation to introduce one (on average) double-strand break in their circular chromosomes. Chromosome sizes were estimated by pulsed-field gel electrophoresis (PFGE) from the mobilities of these full-length molecules relative to DNA size references. Sizes estimated for Ureaplasma urealyticum T960 and 16 Mycoplasma species ranged from 684 kbp (M. hominis) to 1315 kbp (M. iowae). Using this sample, we found no correlation between the mobility of the full-size linear chromosomes and their G + C content. Sizes for A. laidlawii and A. hippikon were within the range expected from renaturation kinetics. PFGE size estimates are in good agreement with sizes determined by other methods, including electron microscopy, an ordered clone library, and summation of restriction fragments. Our estimates also agree with those from renaturation kinetics for both the largest and some of the smallest chromosomes, but in the intermediate size range, renaturation kinetics consistently provides lower values than PFGE or electron microscopy. Our PFGE estimates show that mycoplasma chromosomes span a continual range of sizes, with several intermediate values falling between the previously recognized large and small chromosome size clusters.

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.

Optimization in radiotherapy may be conceivably achieved by individualized treatment regimens. For this, the radiosensitivity of the tumor cells to be treated must be known. A method is presented to show that the effect of radiation on tumor cells in spheroids can be quantitatively evaluated without complicated cell determinations of spheroid composition. This evaluation is based on the dynamics of inactivation of the colony forming ability of whole spheroids composed chiefly of non-transformed diploid fibroblasts and a minority of HeLa "test" cells. Here, spheroids of identical composition, but of different sizes are inactivated proportional to their sizes, thus obviating the need for tedious single cell procedures. The use of spheroids of different sizes permits the deduction of dose-effect relationships, and the innate radiosensitivity of tumors cells. This is a novel method for measuring the radio and chemosensitivity of tumors in primary culture, i.e. cells directly isolated from tumors.

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

The clonogenic hybrid spheroid assay has been used to determine the toxicity of 5-fluorouracil (5-FU), alone or in combination with indomethacin, in LoVo cells (a human colon adenocarcinoma line). The principal finding was that 5-FU toxicity, determined as loss of colony-forming ability, increased as a function of dose (concentration x duration of exposure). and that indomethacin causes a generalized alleviation of 5-FU toxicity, but only if given concurrently with 5-FU. The implications of these findings in the control of cancer cells by 5-FU are discussed.

We have measured survival after successive 2-Gy doses of X rays in HeLa cells and 1-Gy doses in cells of the nonimmortalized human fibroblast cell line AG15-22 under conditions where any effect of cell proliferation during multifraction X irradiation has been factored out. When HeLa cells in parallel series of (pseudo)hybrid spheroids (i.e. in agglomerates consisting of a mixture of supralethally irradiated HeLa feeder and viable HeLa cells) were exposed to n daily radiation doses and then trypsinized and exposed to the last dose, the surviving fraction at 2 Gy (SF2) declined exponentially from 0.55 +/- 0.01 to 0.31 +/- 0.01 after the fifth fraction. In monolayer HeLa cell cultures, the decline in SF2 was smaller but significant and was not influenced by the presence of feeder cells. Pure spheroids, composed entirely of viable HeLa cells, showed the same decline in SF2 as did monolayer cells. The cumulative-effect curve (i.e. the product of SF2 values) was linear-quadratic with the quadratic term increasing in the order monolayer, pure spheroids, pseudohybrid spheroids. SF2n and D0Eff (deduced from the initial SF2) severely underestimated cumulative radiosensitivity. This cumulative effect is clearly associated with the proximity of lethally irradiated cells and might be explained by differential population shifts in the course of the multifraction regimen. Similarly, AG15-22 cells irradiated with daily 1-Gy doses of X rays showed a larger increase in radiosensitivity when in hybrid spheroids than when in pure spheroids. However, for the AG15-22 cells, SF1 was twofold lower for the former than for the latter condition and remained constant for both conditions rather than decreasing with increasing fraction number. This large radiosensitizing effect remains unexplained.

Modulation of radiosensitivity by components of the extracellular matrix (ECM) and cytoskeletal elements has not been adequately studied. Although differences in the radiosensitivities of cells grown as monolayers, as spheroids, or grown in vitro in animal models are known, explanations have in the past neglected possible influences by the ECM and cytoskeleton. Using collagen gel cultures, it is shown that the fibrillar component of the ECM (which is responsible for cell anchorage) induces shifts in radiosensitivity. The effect is critically dependent on the affinity of the cell type towards collagen. The shifts in radiosensitivity induced by ECM alteration are manifested as changed Dq values. By applying four specific cytoskeletal poisons which either stabilize or destabilize specific cytoskeletal elements, the involvement of microfilaments and microtubuli was qualitatively appraised. Cytochalasin B, which destabilizes microfilaments (by preventing polymerization), caused a significant rise in radioresistance. This rise was due to increased D0. Although the cellular morphological change accompanying cytochalasin B treatment was essentially similar to that obtained with trypsin, the respective shifts in radioresponses were qualitatively different and opposite, suggesting differences in mechanism of action.

Cells from the L5178Y murine lymphoma subline LY-R are twice as resistant to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by H2O2, the effect being more pronounced at 37 degrees C than 0 degree C. Initial DNA damage after H2O2 treatment (both temperatures, 5 min) has been estimated by the 'comet' assay (single-cell gel electrophoresis) and fluorescent halo technique. According to both methods, the initial damage is significantly higher in LY-R cells, particularly that inflicted at 0 degree C. Differences between DNA unwinding and rewinding abilities at pH 9 and 6.9 (estimated by the fluorescent halo technique) point to a considerable difference in pH-9-labile damage between the sublines, as observed previously for x-irradiated cells (Kapiszewska et al. 1992). In contrast to findings with x-irradiated cells, however, after H2O2 treatment this damage is more extensive in LY-R cells than in LY-S cells. Thus, the initial pH-9-labile damage corresponds to the pattern of sensitivity to H2O2 and x-rays. We suggest that this is caused by different proportions of cuprous and ferric ions found in the nuclei of LY sublines and by the different ability of these ions to react with H2O2 and water radiolysis products. The copper/iron ratio in the nucleus is 1.31 in LY-R cells and 4.84 in LY-S cells.

Our analysis of the data of van den Engh, Sachs, and Trask (Science 257, 1410 (1992)), for the dependence of the mean square distance between pairs of hybridization sites (L2n, micron 2) on the known genomic distance (n, bp) separating these sites on chromosome number 4 in G1 human fibroblast nuclei, shows thatL2nis proportional to n2v with v = 3/5 for n1 Mbp. The v-value of 3/5 is characteristic of flexible polymer chains with excluded volume effects in dilute good solutions. Since the DNA concentration in nuclei is very high (ca. 1-10 mg/ml), and theory (Flory, J. Chem. Phys. 17, 303, 1949) predicts v = 1/2 for overlapping polymers, the finding of v = 3/5 means that the chromatin fibers do not overlap in interphase nuclei. The dependence ofL2non n for n4 Mbp is consistent with the model of large (approximately 6 Mbp, 3 microns diameter) loops of interphase chromatin attached to nuclear membrane sites. Using the constant (e.g., Widom, Ann. Rev. Biophys. Biophys. Chem. 18, 365 (1989)) and variable (WilliamsLangmore, Biophys. J. 59, 606 (1991)) diameter fiber models, the Kuhn statistical segment of the 30 nm chromatin fiber was estimated to have a length of 196-272 nm with a corresponding DNA content of 21-37 kbp.(ABSTRACT TRUNCATED AT 250 WORDS)