Mihalcik, Stephen A.

ABSTRACT Pediatric radiation therapy presents unique challenges compared to adult treatments, including those of immobilization, potential need for sedation, and the critical importance of accurate, reproducible positioning. Additionally, heightened attention to imaging doses is necessary to minimize long‐term toxicity in survivors. Many of these challenges must be addressed during the process of simulation. This report provides guidelines for pediatric radiotherapy simulation from the Children's Oncology Group. These guidelines cover all anatomic sites and include recommendations for photon and proton therapies, as well as specialized procedures such as stereotactic body radiation therapy, brachytherapy, and total body irradiation. These guidelines are presented as recommendations rather than prescriptive standards as the practice will vary depending on patient age, diagnosis, clinical judgment, and institutional resources.

Abstract Purpose/Objective(s) To report outcomes for disseminated Medulloblastoma (stage M1-M3) who received radiation (RT) on the Pediatric Proton/Photon Consortium Registry (PPCR). Materials/Methods 71 patients were included with either M1 (6), M2 (21), or M3 (44) Medulloblastoma on PPCR. Survival (OS, Overall; EFS, Event Free) analyses were performed using the Kaplan Meier method and log rank p-values from RT start. Median follow up was calculated using the reverse Kaplan Meier method. Results Median age at RT was 7.5 years (range 2.4-21.89) and the median follow up was 4.3 years. Histology was classic (52), diffuse anaplasia (12), focal anaplasia (5), desmoplastic nodular (1), or other (1). Treatment included surgery, chemotherapy with or without stem cell support, and RT. 54 patients had gross total resection (GTR) or near total resection (NTR); 14 had subtotal resection (STR). Pre-RT chemotherapy was given in 69 (97.1%) patients; 23 had myeloablative chemotherapy with stem cell support. The 5 and 10-year OS of the entire cohort were 73% and 65%, and 5 and 10-year EFS were 65% and 59%. Patients with progression prior to RT (10 patients) had inferior OS (5-year OS 44%) compared to patients without progression pre-RT (5-year OS 78%, p = 0.045). There was a trend for better OS (p = 0.07) and improved EFS (p = 0.043) for M2 stage. GTR/NTR did not impact OS (p = 0.75) or EFS (p = 0.97) compared to STR. Craniospinal (CSI) radiation (70 patients, 99%) doses were: £ 23.4Gy (11.4%), 23.5Gy-36Gy (11.4%), or ³ 36Gy (74.3%). Patients receiving CSI ³ 36Gy had superior OS (p = 0.043), with 5 and 10-year OS of 82% and 71% compared to 5 and 10-year OS of 52% and 52% for lower dose CSI. Conclusion Full dose CSI (36Gy) for M1-M3 medulloblastoma was associated with excellent long-term survival this series, whereas lower dose CSI and progression prior to RT had inferior outcomes.

ABSTRACTIntroductionProton beam therapy (PT) offers a targeted approach to treating pediatric tumors by minimizing radiation exposure to surrounding healthy tissue. However, the specific toxicity profile associated with it remains underexplored. This study aims to investigate the incidence and risk factors of acute and late treatment‐related toxicities following PT in pediatric patients with pelvic tumors.MethodsUsing data from the Pediatric Proton Consortium Registry (PPCR), we analyzed 126 pediatric patients who underwent PT for pelvic tumors. We assessed the incidence of gastrointestinal (GI), genitourinary (GU), and musculoskeletal (MSK) toxicities at baseline, during treatment, and across follow‐up intervals.ResultsAcute toxicities were observed in 61.9% of patients posttreatment, with new GI, GU, and MSK toxicities occurring in 54.3%, 9.7%, and 7.0% of baseline toxicity‐free patients, respectively. Follow‐up indicated that 20.6%, 22.2%, and 13.5% of patients developed late GI, GU, and MSK toxicities, respectively. Use of passive scattering proton therapy and radiation to the abdomen were significant predictors of GI toxicity development, while higher radiation dose was associated with MSK toxicity development.ConclusionOur findings demonstrate the safety profile of PT among pediatric patients while identifying specific risk factors, such as passive scattering proton therapy and abdominal irradiation, that contribute to increased GI toxicity. While proton therapy appears to be a well‐tolerated treatment option, the observed toxicity rates highlight the need for continued monitoring and long‐term follow‐up.

Abstract BACKGROUND Standard of care treatment for recurrent malignant glioma after prior radiotherapy (RT) is not well established. Proton therapy (PT) is increasingly used for reirradiation (ReRT) at the time of recurrence; however, treatment outcomes, toxicity, and prognostic factors for patients treated with PT-ReRT remain poorly defined. METHODS The prospective, multi-institutional Proton Collaborative Group (PCG) registry was queried for patients with malignant glioma who previously received RT and underwent PT-ReRT between 7/2011-12/2023. Overall survival (OS) and progression-free survival (PFS) were assessed using the Kaplan-Meier method, and Cox proportional hazards regression was used for uni- and multivariate analysis (UVA and MVA). Dose was calculated using equivalent dose in 2-Gy fractions (EQD2). RESULTS 143 consecutive patients were identified. Median follow-up was 11.2 months and median time interval (TI) from prior RT (median 58.5 Gy) to PT-ReRT (median 44.6 Gy) was 42.4 months. Primary histology was glioblastoma (n=74), astrocytoma (n=35), and oligodendroglioma (n=34). Median PFS and OS were 8.1 and 11.2 months, respectively. On UVA, improved OS was associated with oligodendroglioma (HR 0.37) and astrocytoma histology (HR 0.53) compared to glioblastoma, TI >60 months (HR 0.2), CNS WHO Grade 2 compared to 4 (HR 0.42), and ECOG performance status 0 (HR 0.47). On MVA, improved OS remained associated only with oligodendroglioma (HR 0.42) and TI >60 months (HR 0.49). Acute and Late Grade 3 toxicity occurred in 7% and 4% of patients. Acute Grade 3 toxicity was associated with poor performance status (HR 16.15). CONCLUSIONS In the largest series of glioma PT-ReRT reported to date, retreatment appears well tolerated with variable outcomes based on clinical prognostic factors. Toxicity rates were acceptable compared to historical photon-based literature despite a high median prescription dose.

Abstract Throughout differentiation, mature B cells express distinct combinations of the BAFF and APRIL receptors, BAFF-R, TACI, and BCMA. The patterns of B lineage cell receptor expression reflect their stage of differentiation and impart the ability to respond to ligands, in some cases delivering a powerful anti-apoptotic signal. B cell malignancies arise from each stage of differentiation, typically exhibit the patterns of receptor expression that reflect their cell of origin, and have been shown to exploit the generally anti-apoptotic effects of BAFF and/or APRIL. For example, there is evidence for a role for BAFF in mature B cell cancers, including B cell chronic lymphocytic leukemia (B-CLL). As the majority of circulating CLL B cells are quiescent cells, prolonged survival is a significant hallmark, a trait that signals through BAFF-R could initiate or reinforce. Therapeutic strategies designed to interrupt this pro-survival pathway have thus far primarily focused on blocking ligand binding. Therapeutic modalities impacting receptor expression may be similarly effective. However, despite the apparent precise activation stage-dependent orchestration of B cell BAFF-R, TACI, and BCMA expression, the genetic mechanisms regulating expression of the three receptors remain undefined, and the question of whether each receptor governs expression of the other two remains unanswered. In agreement with previous studies in our own lab and others, analyses of the receptor profiles of CLL B cells continue to show BAFF-R surface expression, albeit at lower levels than seen on normal peripheral B cells, and the curious variable presence of BCMA and TACI. Similarly, multiple myeloma (MM) plasma cells (PCs), like normal PCs, uniformly lack BAFF-R expression, express BCMA, and variably express TACI. Our current study explores the mechanisms of receptor regulation in B cells, with an emphasis on BAFF-R, the receptor that is most consistently expressed on the CLL B cell population and that has the most clearly defined survival function. We began by analyzing the BAFF-R gene’s genomic context. We identified a possible regulatory element 2 kb upstream of the first exon with significant homology across seven mammalian species that overlapped a cluster of B cell lineage transcription factor binding sites, and, thus, we called the 2.5 kb directly upstream of the gene the putative BAFF-R promoter. We cloned the region and created promoter-reporter vectors in which the full-length promoter and 0.5 kb 5’ truncations thereof drive firefly luciferase production. While studies of primary B cells continue, studies with malignant B cell lines suggest that we have successfully cloned a powerful positive regulatory region. Specifically, B cell lines that express surface BAFF-R show positive inductions of firefly to control renilla luciferase activity in all of the promoter constructs over the empty construct with the greatest promoter activity in the longest constructs: 6-, 18-, and 3-fold inductions with the 2.5 kb promoter in RAJI, Loukes, and MEC-1 cells, respectively. To further test the promoter specificity, we transfected malignant PC lines, ALMC-1, ALMC-2, and KAS-6/1, which are negative by RT-PCR and surface staining for BAFF-R. These lines showed little promoter activity over baseline, with fold inductions between 0.5 and 2.5 for all of the promoter constructs. These results suggest that the MM lines no longer express the transcription factors required to drive BAFF-R expression and underscore our conclusion that we have identified the BAFF-R promoter. At the same time, investigations into epigenetic modification may reveal a crucial level of control. The transcriptional start site of the BAFF-R gene falls within a region of high CG content, and may be a possible CpG island. Upon treatment with the methyltransferase inhibitor, 5-azacytidine, primary blood B cells and MM cells showed no change in receptor expression. However, in CLL B cells, treatment of cultured cells caused a slight (9%) decrease in BAFF-R expression and prevented TACI upregulation in cells stimulated with CpG (a 76% increase fell to 36%). This evidence suggests that methylation indirectly suppresses expression of BAFF-R and TACI. It is essential to understand the regulation of survival receptors critical to normal B lineage cell survival, which may also be crucial for their malignant counterparts, in order to target those mechanisms as therapy.

Abstract B cell-activating factor (BAFF) is known to regulate normal B cell development and homeostasis primarily by signaling through the high affinity receptor, BAFF-R, one of three BAFF binding receptors (BBRs). BAFF also binds two other receptors, BCMA and TACI with lesser affinity. We have recently shown that normal peripheral blood (PB) B cells express high levels of prebound soluble BAFF, which is lost upon B cell activation. Because of BAFF’s activity on normal B cells, we have been interested in the roles of BAFF and BBRs in B cell chronic lymphocytic leukemia (B-CLL). We and others have demonstrated that BAFF promotes primary CLL B cell survival and that serum BAFF levels are elevated in some patients. Although CLL B cells are known to express BBRs, a comprehensive and quantitative analysis of BBR levels and CLL B cell capacity to bind BAFF has not yet been done. We began this study by characterizing the level of soluble BAFF bound to freshly isolated CLL B cells, measured by both western blot analysis and flow cytometry. To assess receptor occupancy, cells were incubated with or without exogenous BAFF before assessing anti-BAFF reactivity and changes in median fluorescence intensity (ΔMFI; defined by dividing the MFI of the anti-BAFF antibody by the MFI of the isotype matched control antibody) were calculated. Normal B cells have higher detectable levels of bound BAFF with a ΔMFI ranging from 16 to 35 (mean=22.2). Upon addition of exogenous BAFF, the ΔMFI range increased to 27–96.6 (mean=49.1; n=8). Thus, despite evidence of prebound BAFF, clearly not all BBRs were occupied on normal PB B cells. By contrast, the levels of prebound BAFF on CLL B cells were significantly lower with a ΔMFI ranging from 1 to 13.1 (mean=2.7; n=36). Of note, 10/36 patients did not exhibit increased anti-BAFF reactivity upon incubation with exogenous BAFF (mean fold induction=0.8) whereas 26/36 patients displayed a mean fold induction of anti-BAFF reactivity of 3.5. These observations prompted us to next quantitate CLL B cell BBR expression. All patient CLL B cells expressed BAFF-R but at significantly lower levels than observed in normal B cells (p=0.0009). When CLL patients were categorized into IGHV mutated (M; n=22) and unmutated (UM; n=24), UM patients were observed to express higher levels of BAFF-R (ΔMFI =8.9) than M patients (ΔMFI =5.24). Regarding TACI, we previously demonstrated that normal memory B cells uniformly express TACI (ΔMFI =12.7; n=10) and there is a small population of activated naïve B cells that express TACI at lower levels (ΔMFI =8.3; n=10). In our CLL cohort, 14/22 M patients were TACI+ (ΔMFI =7.0) and 19/24 UM patients were TACI+ (ΔMFI =4.7). Finally, whereas normal PB B cells completely lack BCMA expression, 7/22 M and 4/22 UM patients expressed BCMA. Thus, using the BBR profile and analysis of expression levels relative to normal PB B cells, the following subgroups of B-CLL can be defined: BAFF-R+; BAFF-R/TACI+; BAFF-R/BCMA+; BAFF-R/TACI/BCMA+. It remains to be determined if these BBR profiles correlate with aspects of clinical disease. In addition, given the putative importance of BAFF in this disease, it is interesting to note that in general, CLL B cells display overall lower levels of prebound BAFF. Current studies are focused on determining whether this reflects CLL B cell activation status, increased competition for BAFF, and/or reduced levels of BBR expression.

Abstract Abstract 92 BACKGROUND: All somatic cell types, including all B lineage cells, constitutively express a number of DNA repair proteins to maintain genomic stability and thwart tumorigenesis in the face of ongoing constitutive levels of DNA damage. Compromises in expression of these essential DNA repair factors have been shown to readily induce the development of various cancers including B cell lymphomas, suggesting expression levels are tightly regulated and limited in quantity. In germinal center (GC) B cells, however, additional DNA repair capacity is likely required to counterbalance the heightened level of mutagenic activity owing to the induced expression of activation-induced cytidine deaminase (AID) during somatic hypermutation (SHM). AID is a DNA editing enzyme which introduces somatic mutations into immunoglobulin (Ig) V regions at an estimated rate which is almost a million-fold higher than the spontaneous rate in somatic cells. HYPOTHESIS: We hypothesize that to maintain the genomic wellness of GC B cells which are undergoing SHM, there is a need for induction of an accompanying robust DNA repair system. We further hypothesize that inefficient induction of these repair genes may predispose to malignant transformation. METHODS: Using tonsillar tissue sections and purified tonsillar B cell subpopulations, we compared expression levels of various DNA repair genes and related these levels to AID expression across the subsets using immunohistochemistry, real-time RT-PCR, and Western blot analysis. To characterize the nature of signals capable of inducing expression levels of AID and/or DNA repair proteins, peripheral blood B cells were activated in vitro using a panel of stimuli, including coculture with activated CD4 T cells. As a surrogate measure of mismatch repair (MMR) activity in the relative absence of T cell help, we quantitated the number of somatic hypermutations at A/T sites in the Ig heavy chain variable (IGHV) region genes in a collection of IGHV sequences obtained from normal B cells and HIV-related lymphoma cells. RESULTS: Using immunohistochemistry, we observed that, similar to the expression of AID, DNA MMR genes are significantly induced in tonsillar GC B cells. These results were further validated using a more sensitive real-time RT-PCR assay and analysis by Western blotting. By expanding our DNA repair gene panel, we observed that proteins of homologous recombination, base excision repair and DNA single strand break signalling pathway are also similarly induced in GC B cells at RNA, protein, and functional levels compared to their expression in naïve and memory B cells. By contrast, expression of non-homologous end joining and DNA double strand break signalling molecules are unchanged. We have termed this selective induction of repair mechanisms in GC B cells as somatic hyperrepair (SHR). To identify pathways that lead to the activation of AID and SHR, we used an in vitro system and a variety of stimuli and we discovered that multiple B cell stimuli including CpG, CD40L, and anti-BCR could each independently induce the expression of AID while SHR induction strictly required the engagement of CD4+ T cells. This provocative observation suggests a novel role for CD4+ T cells in mitigating tumorigenesis of post-GC B cells through their ability to induce the SHR pathway in cells that have been induced to undergo SHM. To demonstrate the possible role of SHR in lymphomagenesis, we analysed the mutation pattern of IGHV genes from a panel of B cell lymphomas obtained from HIV infected (CD4+ T cell suppressed) patients. We found that HIV-related lymphoma cells displayed a significantly lower frequency of SHM at A/T positions relative to normal memory B cells, indicative of compromised MMR of their precursor cells during GC transit. Our findings resolve the lingering paradox that B cell malignancies are overwhelmingly prevalent under T cell suppression conditions such as HIV infection, post-organ transplant, and aging. Finally, our results also suggest for the first time that mounting efficient tumor suppression for some cells may depend on signals transmitted by neighboring cells and the specific microenvironment. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2400 Poster Board II-377 Tumor necrosis factor superfamily member BAFF-R has been shown to transduce a powerful survival signal in B lineage cells upon ligation by BAFF, its soluble high affinity ligand. The availability of BAFF in lymphoid organs and its detectable levels in the blood suggest that it is the control of receptor expression by the B cell rather than ligand production by supportive cells that limits the activity of this survival signal. The presence of BAFF-R on the surface of B cells from the transitional stages of development through maturity mirrors the expression of BAFF-R on the malignancies thought to arise from these various developmental stages and may enable B lineage malignancies to receive an anti-apoptotic signal from BAFF that reinforces and supports the expansion of the malignant cells. This additional survival signal may be crucial in indolent malignancies like chronic lymphocytic leukemia (CLL), a cancer characterized more by the prolonged survival of the cancerous cells than by their abundant proliferation. While there are ongoing efforts to thwart this survival pathway with antibody-based therapeutics directed toward BAFF and BAFF-R, understanding the regulation of BAFF-R expression may open a more proximal, potent, and novel therapeutic avenue. Rel/NF-kB family member c-Rel is an appealing candidate BAFF-R regulator, since its nuclear localization and expression within B lineage cells roughly correlate with BAFF-R expression, predominating at nuclear kB sites as a dimer with p50 in mature B cells and declining in plasma cells. Recently published evidence has shown that B cell receptor (BCR)-induced c-Rel expression can modulate BAFF-R in murine transitional B cells, providing a critical increase in BAFF-R expression and thus, BAFF-responsiveness, during B cell development (Castro, I. J Immunol. 2009; 182(12): 7729). A role for the BCR is especially provocative in the context of CLL, which is commonly characterized by lower levels of surface BAFF-R despite a generally activated phenotype and, in the unmutated subtype, typically increased expression of genes downstream of the BCR. In related studies, we have analyzed BAFF-R promoter activity in BAFF-R expressing B cells vs. myeloma cell lines, which generally lack expression of this receptor, and have indeed demonstrated differential promoter activity of the region directly upstream of the BAFF-R gene. The goal of our current study was to focus on the role of the transcription factor, c-Rel, in regulating BAFF-R expression. While in silico tools identifying transcription factor binding sites in the three kilobases upstream of the BAFF-R gene found numerous possible c-Rel binding sites, only three c-Rel sites remained under the most stringent search conditions. An initial chromatin immunoprecipitation (ChIP) experiment with an anti-c-Rel antibody suggested that the binding site within the first five hundred bases upstream of the transcriptional start site may be bound by c-Rel: ChIP with RAMOS B cells showed a 6-fold increase in the precipitated DNA of the region over the isotype-matched antibody control. Electrophoretic mobility shift assays (EMSAs) using peripheral blood (PB) B cell nuclear extracts both from normal donors and CLL patients as well as malignant cell lines, demonstrate the different levels of binding to the putative c-Rel sites identified in silico and support the hypothesis that c-Rel directly regulates BAFF-R expression in these cells. These assays, which demonstrated the affinity of c-Rel for the putative promoter sites upstream of the BAFF-R gene, led to the transfection of new luciferase reporter constructs into malignant B and plasma cell lines, which demonstrate the effects both of amplifying and of mutating the c-Rel binding sites in our reporter assays. We supplemented these assays with experiments measuring the production of c-Rel in these cells both by quantitative RT-PCR, which demonstrated a twenty-fold decrease in plasma cell line ALMC-1 and a two-fold decrease in CLL cell c-Rel mRNA as compared to normal PB B cells, and by Western blot. Finally, we used a c-Rel expression vector to directly increase BAFF-R expression in the malignant cell lines. By identifying c-Rel expression and activation as a possible mechanism of direct control of BAFF-R expression in human B cells, we reveal a critical new rationale for the use of NF-kB inhibitors as chemotherapeutic adjuvants in B cell cancers. Disclosures: No relevant conflicts of interest to declare.

Abstract B lymphocyte stimulator (BLyS) is a well-known direct costimulator of adaptive immune cells, particularly B lineage cells. However, we have reported recently that BLyS is also able to activate monocytes. Other innate immune cells, such as dendritic cells (DCs), play a key role in the initiation of adaptive immune responses and the purpose of the current study was to assess whether there is a direct role for BLyS in modulating human DC functions. In this study, we show that BLyS induces DC activation and maturation. Thus, BLyS strongly induced up-regulation of surface costimulatory molecule expression and secretion of specific cytokines and chemokines in DCs. BLyS-stimulated DCs (BLyS-DCs) were also able to augment allogeneic CD4 T cell proliferation to a greater extent than control DCs. BLyS-DCs secreted elevated levels of the major Th1-polarizing cytokine, IL-12p70, and they promoted naive CD4 T cell differentiation into Th1 T cells. Regarding BLyS receptor expression, DCs primarily express cytoplasmic transmembrane activator and CAML interactor; however, low levels of cell surface transmembrane activator and CAML interactor are expressed as well. Collectively, our data suggest that BLyS may modulate adaptive immune cells indirectly by inducing DC maturation.