Therapy-induced Alterations Research Articles

Abstract A07: Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns

Abstract Acute myeloid leukemia (AML) represents a heterogeneous hematopoietic disorder characterized by accumulation of immature hematopoietic precursors with differentiation block. FLT3 internal tandem duplications (FLT3-ITD) are commonly occurring genetic alterations in AML and are associated with poor prognosis. Preclinical studies showed combining FLT3 inhibitor with MDM2 inhibitor, which is a negative regulator of p53, was synergistic in FLT3-ITD/TP53 wild type (WT) AML. We performed single-cell proteomic evaluation of leukemia landscape in FLT3-ITD patients treated with FLT3i and MDM2i to assess proteomic profiles associated with response and resistance. We performed CyTOF analysis of leukemia cells in serially collected samples from six FLT3-ITD and TP53 WT AML patients following treatment with FLT3i+MDM2i treatment. Using 51 features assessed in CyTOF, we first performed UMAP dimension reduction and clustering to identify distinct cells in leukemia compartment. Notably, the frequencies of blasts identified through CyTOF data analysis were compatible with clinical lab reports. In line with previous reports, we also detected that NPM mutant AML cells did not express CD34. Of note, CD34+ and CD34- leukemia cells were clustered together in NPM1 mut patients, indicating that they have overlapping proteomic profiles. Interestingly, the CD34+ leukemia cells were eliminated at the early time points in CR patients while the CD34- leukemia cells were still detectable after two months. On the other hand, CD34+ leukemia cells in nonresponders(NR) persisted despite therapy. These findings indicate CD34+ leukemia cells were more sensitive to the treatment compared to NPM1 mutant CD34- leukemia cells. Next, we interrogated leukemia proteomic landscape in serial samples, evaluated the therapy-induced alterations in proteomic profiles and sought to identify potential adaptive mechanisms. To this end, we performed differential expression analysis and observed that signaling pathways (p-4EBP1, p-GSK3, p-MEK1/2, p-S6) and differentiation markers(CD33, CD68,CXCR4 HLADR) were more enriched on day 8 post treatment in NR patients, revealing that compensatory signaling activity, phenotypic profiles and differentiation status could be associated with therapy response. Moreover, we found that leukemia cells in NR patients had distinct phenotypic profiles(CD11b,CD68,CXCR4), higher levels of anti-apoptotic molecules (BCL2,MCL1) and enriched survival pathways (p-GSK3, YTHDF2) compared to baseline. In contrast, we did not observe rebound increases in CR patients post-therapy. These findings demonstrate high levels of BCL2 and MCL1 and preferential survival of more differentiated cells may be associated with therapy resistance and treatment failure in NR patient treated with MDM2i and FLT3i. In conclusion, multiplexed single-cell proteomic analysis permitted longitudinal monitoring of leukemia landscape and identified proteomic alterations associated with therapy resistance. Citation Format: Li Li, Muharrem Muftuoglu, Mahesh Basyal, Naval Daver, Michael Andreeff. Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A07.

High-Throughput Examination of Therapy-Induced Alterations in Redox Metabolism in Spheroid and Microtumor Models.

The capacity of cancer cells to adjust their metabolism to thrive in new environments and in response to treatments has been implicated in the acquisition of treatment resistance. To optimize therapeutic strategies such as photodynamic therapy (PDT)-based combination treatments, methods to characterize the plasticity of cancer metabolism in response to treatments are required. This protocol provides a method for high-throughput and label-free tracking of metabolic redox states in cancer tissues, leveraging the autofluorescent properties of nicotinamide dinucleotide (NAD(P)H) and oxidized flavoprotein adenine dinucleotide (FAD). The methodology is optimized to be applied to 3D spheroid/microtumor/organoid cultures, regardless of the culture type (e.g., adherent or suspension cultures) and morphology. The exploitation of these methods may elucidate mechanisms of metabolic adaptation and perturbations in redox homeostasis, and chart the overall tumor health in both 3D culture models and ex vivo tissues following cancer therapies, such as PDT.

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome, Creating Novel Antitumor CD8 T Cell Reactivities

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry–based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.

Temozolomide Treatment Increases Fatty Acid Uptake in Glioblastoma Stem Cells.

Simple SummaryPatients diagnosed with glioblastoma (GBM) brain tumors typically survive less than two years, despite aggressive therapy with surgery, radiation, and chemotherapy. A major factor underlying this lethality is the ability of GBM tumors to adapt to stress, including the stress of treatment. The role of metabolism in this process remains incompletely understood. We, therefore, explored the connection between cellular phenotype, chemotherapeutic stress, and metabolism in GBM. We found that inducing changes in GBM phenotypes led to alterations in metabolic behavior. Further, during treatment with chemotherapy, GBM cells that became resistant to therapy increased their fatty acid uptake. These therapy-induced alterations in nutrient uptake may underlie therapy resistance and deadly recurrence.Among all cancers, glioblastoma (GBM) remains one of the least treatable. One key factor in this resistance is a subpopulation of tumor cells termed glioma stem cells (GSCs). These cells are highly resistant to current treatment modalities, possess marked self-renewal capacity, and are considered key drivers of tumor recurrence. Further complicating an understanding of GBM, evidence shows that the GSC population is not a pre-ordained and static group of cells but also includes previously differentiated GBM cells that have attained a GSC state secondary to environmental cues. The metabolic behavior of GBM cells undergoing plasticity remains incompletely understood. To that end, we probed the connection between GSCs, environmental cues, and metabolism. Using patient-derived xenograft cells, mouse models, transcriptomics, and metabolic analyses, we found that cell state changes are accompanied by sharp changes in metabolic phenotype. Further, treatment with temozolomide, the current standard of care drug for GBM, altered the metabolism of GBM cells and increased fatty acid uptake both in vitro and in vivo in the plasticity driven GSC population. These results indicate that temozolomide-induced changes in cell state are accompanied by metabolic shifts—a potentially novel target for enhancing the effectiveness of current treatment modalities.

Abstract 1131: CD8 immuno-PET using a fully human mAb can detect increases in tumor-infiltrating T cells by a T cell activating immunotherapy

Abstract Detection and quantification of immune cell subsets and changes in the tumor immune microenvironment would be a powerful tool to correlated to response in cancer immunotherapy. Currently, biopsies used to monitor the tumor microenvironment suffer from sampling error, restricted sampling over time and to inaccessible tissues/organs, and do not reflect discordance across tumor lesions of the same patient. Immuno-PET detection of CD8-expressing T cells is a promising translational imaging approach to assess CD8+ tumor-infiltrating lymphocytes (TILs) pre- and post-therapy. We have developed a fully human anti-CD8 antibody that was radiolabeled with the positron emitting radionuclide Zirconium-89 (89Zr) using the bifunctional chelator p-SCN-Bn-Desferoxamine (DFO). CD8-genetically humanized immunocompetent mice were used to validate dose dependent targeting of 89Zr-CD8 to lymphoid tissues. Next, two models were developed to monitor the response of the T cell activating bispecific antibody REGN1979 (CD3xCD20). In the first model designed to lack a CD8 antigen sink, Raji lymphoma cells were co-implanted with human peripheral blood mononuclear cells (hPBMCs) in immune deficient NSG mice and treated with REGN1979. Here, 89Zr-CD8 detected a two-fold increase in CD8+ TILs as quantified by flow cytometry compared to control antibody treated mice. To determine if 89Zr-CD8 could detect changes in CD8+ TILs in the presence of increased circulating and splenic T cells, a different model was developed. Raji lymphoma cells were implanted subcutaneously and hPBMCs were implanted intraperitoneally in immune deficient SRG-15 mice (hSIRP KI, Rag2 KO, IL2Rg KO, hIL-15 KI). Due to the humanization of IL-15, T cells from the implanted hPBMCs rapidly expanded and produced a CD8 antigen sink. In the SRG-15 model, total tumor uptake of 89Zr-CD8 was lower and spleen uptake higher than in the NSG model due to the presence of the antigen sink. These SGR-15 mice treated with REGN1979 also demonstrated an increase in CD8+ TILs by flow cytometry that was specifically detected with 89Zr-CD8. This work demonstrates that 89Zr-CD8 can specifically detect CD8+ T cells in lymphoid tissues of CD8 humanized mice and therapy-induced alterations of CD8+ TILs. Importantly, the detection of CD8+ TILs occurs in both the presence and absence of an antigen sink. This work supports the clinical translation of 89Zr-CD8 immuno-PET to investigate its utility for predicting and monitoring response in patients undergoing immunotherapy. Citation Format: Richard Tavare, Makenzie Danton, Jason T. Giurleo, Marcus P. Kelly, Sosina Makonnen, Carlos Hickey, Tomas C. Arnold, Dangshe Ma, William C. Olson, Gavin Thurston, Jessica R. Kirshner. CD8 immuno-PET using a fully human mAb can detect increases in tumor-infiltrating T cells by a T cell activating immunotherapy [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 1131.

Age and the risks of high-efficacy disease modifying drugs in multiple sclerosis.

A variety of high-efficacy disease-modifying therapies (DMTs) are available for the treatment of multiple sclerosis (MS). After evaluation and approval by regulatory agencies, DMTs are likely to be administered to patients whose characteristics differ from those enrolled in clinical trials. This may contribute to the emergence of unexpected adverse events observed in the real-world setting. Higher age may be a relevant factor that could change the benefit-risk balance of DMTs, as it may associate with lower efficiency and higher frequency of adverse events. The absolute and relative number of patients with MS who reach the age of 55 and higher increases. Growing evidence demonstrates lower efficacy of DMTs in older persons with MS. Specific risks during DMTs for MS, such as the risk of developing progressive multifocal leukoencephalopathy (PML) or the outcome following PML, have been associated with age. It is hypothesized that age-related and therapy-induced alterations to the immune system may have (super)additive effects, resulting in an acceleration of physiological immunosenescence and inflamm-aging. In this article, we review the risks of high-efficacy DMTs in MS with a specific focus on age-related efficacy and risks, including opportunistic infections, malignancies, and autoimmune reactions.

Transient alleviation of tumor hypoxia during first days of antiangiogenic therapy as a result of therapy-induced alterations in nutrient supply and tumor metabolism – Analysis by mathematical modeling

A number of experiments on mouse tumor models, as well as certain clinical data, have demonstrated, that antiangiogenic therapy can lead to transient improvement in tumor oxygenation, that allows to increase efficiency of following radiotherapy. In the majority of works, this phenomenon has been explained by enhanced tumor perfusion due to normalization of capillaries’ structure, that results in elevated oxygen inflow in tumor. However, changes in tumor perfusion often haven’t been directly measured in relevant works, moreover, antiangiogenic therapy has been proven to have ambiguous effect on tumor perfusion both in mouse tumor models and in clinics. Herein, we suggest that elevation of blood perfusion may be not the only reason for transient alleviation of tumor hypoxia, and that it may manifest itself even under unchanged tumor blood flow. We propose that it may be as well caused by the decrease in tumor oxygen consumption rate (OCR) due to the reduction of tumor proliferation level, caused by nutrient shortage in result of antiangiogenic treatment. We provide detailed explanation of this hypothesis and visualize it using a specially developed mathematical model, which takes into account basic features of tumor growth and antiangiogenic therapy. We investigate the influence of the model parameters on oxygen dynamics; demonstrate, that transient alleviation of tumor hypoxia occurs in a fairly wide range of physiologically justified values of parameters; and point out the major factors, that determine oxygen dynamics during antiangiogenic therapy.

Age-associated and therapy-induced alterations in the cellular microenvironment of experimental gliomas.

The poor prognosis associated with advanced age in patients with glioblastoma remains poorly understood. Glioblastoma in the elderly has been particularly associated with vascular endothelial growth factor (VEGF)-dependent angiogenesis, and early uncontrolled studies suggested that the anti-angiogenic agent bevacizumab (BEV), an antibody to VEGF, might be preferentially active in this patient population.Accordingly, we explored host age-dependent differences in survival and benefit from radiotherapy (RT) or BEV in syngeneic mouse glioma models. Survival was inferior in older mice in the SMA-540 and and less so in SMA-560, but not in the SMA-497 or GL-261 models. Detailed flow cytometric studies revealed increased myeloid and decreased effector T cell population frequencies in SMA-540 tumors of old compared to young mice, but no such difference in the SMA-497 model. Bone marrow transplantation (BMT) from young to old mice had no effect, whereas survival was reduced with BMT from old to young mice. BEV significantly decreased vessel densities in gliomas of old, but not young mice. Accordingly, old, but not young SMA-540 tumor-bearing mice benefited from BEV alone or in combination with RT. End-stage tumors of old BEV- and BEV/RT-treated mice exhibited increased infiltration of T helper and cytotoxic T cells compared to tumors of young mice.The SMA-540 model may provide a valuable tool to evaluate the influence of host age on glioblastoma progression and treatment response. The biological host factors that modulate glioma growth in old as opposed to young mice remain to be identified.

Abstract 419: Integrative analysis of multi-omics tumor profiles identifies pathways associated with resistance to anti-HER2 therapy in early stage breast cancer

Abstract Background: HER2 positive breast cancers are heterogeneous at both the clinical and molecular levels, with the HER2-enriched subtype exhibiting increased levels of immune infiltration signatures and the highest rate of pathologic complete response (pCR), while the HER2-Basal subtype is resistant to anti-HER2 therapy (Varadan et al, CCR 2016). Here we aim to characterize the molecular underpinnings of response and their interaction with increased immune infiltration across these HER2-subtypes using integrative analyses of genomic and transcriptomic data from two multicenter trials (DFCI 03-311 and BrUOG 211B). Methods: Fresh tumor core biopsies were taken at baseline and a 2-week time point after a single dose of trastuzumab. 80 HER2+ early breast cancer (EBC) patients were enrolled in the 03-311 trial, and 60 patients in the 211B trial. Biopsy samples were profiled for gene expression (Microrrays:03-311; RNAseq:211B), somatic mutations (Whole-exome sequencing: 211B; Targeted Sequencing: 03-311) and somatic copy-number aberrations (SNP-arrays: 03-311; Whole-exome Sequencing: 211B). Subtyping was performed using gene expression data and tumors were classified into HER2-Enriched, HER2-Luminal and HER2-Basal subtypes. Integrative analysis of gene expression and copy-number data to infer signaling network activities per sample, was performed using the recently developed InFlo framework (Dimitrova et al, Oncogene 2016). Results: HER2-Basal tumors exhibited lower average copy number for HER2 and were less likely to have high-level amplifications of co-amplicons (e.g. 11q13, 20q13). In the 211B and 03-311 trials, respectively, 62% and 63% of somatic mutations persisted after one dose of therapy, while 21% and 19% of mutations were undetectable after one dose of therapy. Tumors harboring amplifications in the 8p11 (FGFR1) genomic locus exhibited higher indices immune signatures associated with macrophages (P=0.0073) and T-cells (P = 0.0493) in 211B, but this association did not achieve significance in the 03-311 trial. Integrative InFlo-based analysis of tumor gene expression and copy-number profiles after one dose of trastuzumab in the 211B trial revealed significantly higher activity of signaling pathways associated with CD4+ T-cells in the responders (P=0.008), while higher activity of mTOR pathway was observed in non-responders (P=0.0014). Conclusions: Changes in mutational profiles over time may either be related to therapy-induced alterations of clonal architecture or the consequence of intra-tumor heterogeneity, thus warranting further exploration. Integrative analysis of gene expression and copy-number profiles reveal signaling pathways associated with response and resistance, enabling the discovery of biomarkers of response to anti-HER2 therapy. Citation Format: Salendra Singh, Hannah Gilmore, Maysa Abu-Khalaf, George Somlo, William Sikov, Lyndsay Harris, Vinay Varadan. Integrative analysis of multi-omics tumor profiles identifies pathways associated with resistance to anti-HER2 therapy in early stage breast cancer [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 419. doi:10.1158/1538-7445.AM2017-419

Increased Regenerative Capacity of the Olfactory Epithelium in Niemann-Pick Disease Type C1.

Niemann–Pick disease type C1 (NPC1) is a fatal neurovisceral lysosomal lipid storage disorder. The mutation of the NPC1 protein affects the homeostasis and transport of cholesterol and glycosphingolipids from late endosomes/lysosomes to the endoplasmic reticulum resulting in progressive neurodegeneration. Since olfactory impairment is one of the earliest symptoms in many neurodegenerative disorders, we focused on alterations of the olfactory epithelium in an NPC1 mouse model. Previous findings revealed severe morphological and immunohistochemical alterations in the olfactory system of NPC1−/− mutant mice compared with healthy controls (NPC1+/+). Based on immunohistochemical evaluation of the olfactory epithelium, we analyzed the impact of neurodegeneration in the olfactory epithelium of NPC1−/− mice and observed considerable loss of mature olfactory receptor neurons as well as an increased number of proliferating and apoptotic cells. Additionally, after administration of two different therapy approaches using either a combination of miglustat, 2-hydroxypropyl-β-cyclodextrin (HPβCD) and allopregnanolone or a monotherapy with HPβCD, we recorded a remarkable reduction of morphological damages in NPC1−/− mice and an up to four-fold increase of proliferating cells within the olfactory epithelium. Numbers of mature olfactory receptor neurons doubled after both therapy approaches. Interestingly, we also observed therapy-induced alterations in treated NPC1+/+ controls. Thus, olfactory testing may provide useful information to monitor pharmacologic treatment approaches in human NPC1.

P08.51 Age-associated and therapy-induced alterations in the cellular microenvironment of experimental gliomas

P08.51 Age-associated and therapy-induced alterations in the cellular microenvironment of experimental gliomas

Hyperpolarized 13C Magnetic Resonance Treatment Response Monitoring: A New Paradigm for Multiorgan Metabolic Assessment of Pharmacological Interventions?

Metformin is one of the most used hyperglycemic control therapeutics in patients with diabetes, although the exact mechanism of action is still not fully understood (1,2). Metformin is the preferred antihyperglycemic drug in patients with type 2 diabetes; its use in patients with type 1 diabetes is limited however (3,4). A potential serious side effect of metformin treatment is lactic acidosis, which has reduced the applicability in renal-impaired patients; however, this has been questioned recently (5,6). These together support further investigations of, first, the exact mechanism of action and, second, the noninvasive methods for monitoring the treatment, in particular the organ-specific modulations imposed by metformin and their complex interorgan interactions, which historically have been especially difficult to assess. This is particularly true in diseases where several organs are simultaneously affected, such as the cardio-renal syndrome, where dysfunction of one organ affects the other and vice versa and where the use of pharmacological interventions in the treatment of one organ can have detrimental effects on the other and vice versa (7,8). Thus the understanding of the organ-specific phenotypic characteristics in diabetes and the therapy-induced alterations is essential …

An Effective Immuno-PET Imaging Method to Monitor CD8-Dependent Responses to Immunotherapy

The rapidly advancing field of cancer immunotherapy is currently limited by the scarcity of noninvasive and quantitative technologies capable of monitoring the presence and abundance of CD8(+) T cells and other immune cell subsets. In this study, we describe the generation of (89)Zr-desferrioxamine-labeled anti-CD8 cys-diabody ((89)Zr-malDFO-169 cDb) for noninvasive immuno-PET tracking of endogenous CD8(+) T cells. We demonstrate that anti-CD8 immuno-PET is a sensitive tool for detecting changes in systemic and tumor-infiltrating CD8 expression in preclinical syngeneic tumor immunotherapy models including antigen-specific adoptive T-cell transfer, agonistic antibody therapy (anti-CD137/4-1BB), and checkpoint blockade antibody therapy (anti-PD-L1). The ability of anti-CD8 immuno-PET to provide whole body information regarding therapy-induced alterations of this dynamic T-cell population provides new opportunities to evaluate antitumor immune responses of immunotherapies currently being evaluated in the clinic.

Tumor-associated and immunochemotherapy-dependent long-term alterations of the peripheral blood NK cell compartment in DLBCL patients

Natural Killer (NK) cells are a key component of tumor immunosurveillance and thus play an important role in rituximab-dependent killing of lymphoma cells via an antibody-dependent cellular cytotoxicity (ADCC) mechanism. We evaluated the phenotypic and functional assets of peripheral blood NK cell subsets in 32 newly-diagnosed diffuse large B-cell lymphoma (DLBCL) patients and in 27 healthy controls. We further monitored long-term modifications of patient NK cells for up to 12 months after rituximab-based immunochemotherapy. At diagnosis, patients showed a higher percentage of CD56dim and CD16+ NK cells, and a higher frequency of GrzB+ cells in CD56dim, CD56bright, and CD16+ NK cell subsets than healthy controls. Conversely, DLBCL NK cell killing and interferon γ (IFNγ) production capability were comparable to those derived from healthy subjects. Notably, NK cells from refractory/relapsed patients exhibited a lower “natural” cytotoxicity. A marked and prolonged therapy-induced reduction of both “natural” and CD16-dependent NK cytotoxic activities was accompanied by the down-modulation of CD16 and NKG2D activating receptors, particularly in the CD56dim subset. However, reduced NK cell killing was not associated with defective lytic granule content or IFNγ production capability. This study firstly describes tumor-associated and therapy-induced alterations of the systemic NK cell compartment in DLBCL patients. As these alterations may negatively impact rituximab-based therapy efficacy, our work may provide useful information for improving immunochemotherapeutic strategies.

Whole-tumour CT-perfusion of unresectable lung cancer for the monitoring of anti-angiogenetic chemotherapy effects

To determine whether CT-perfusion (CT-p) can be used to evaluate the effects of chemotherapy and anti-angiogenic treatment in patients with non-small-cell lung carcinoma (NSCLC) and whether CT-p and standard therapeutic response assessment (RECIST) data obtained before and after therapy correlate. 55 patients with unresectable NSCLC underwent CT-p before the beginning of therapy and 50 of them repeated CT-p 90 days after it. Therapeutic protocol included platinum-based doublets plus bevacizumab for non-squamous carcinoma and platinum-based doublets for squamous carcinoma. RECIST measurements and calculations of blood flow (BF), blood volume (BV), time to peak (TTP) and permeability surface (PS) were performed, and baseline and post-treatment measurements were tested for statistically significant differences. Baseline and follow-up perfusion parameters were also compared based on histopathological subclassification (2004 World Health Organization Classification of Tumours) and therapy response assessed by RECIST. Tumour histology was consistent with large cell carcinoma in 14/50 (28%) cases, adenocarcinoma in 22/50 (44%) cases and squamous cell carcinoma in the remaining 14/50 (28%) cases. BF and PS differences for all tumours between baseline and post-therapy measurements were significant (p=0.001); no significant changes were found for BV (p=0.3) and TTP (p=0.1). The highest increase of BV was demonstrated in adenocarcinoma (5.2±34.1%), whereas the highest increase of TTP was shown in large cell carcinoma (6.9±22.4%), and the highest decrease of PS was shown in squamous cell carcinoma (-21.5±18.5%). A significant difference between the three histological subtypes was demonstrated only for BV (p<0.007). On the basis of RECIST criteria, 8 (16%) patients were classified as partial response (PR), 2 (4%) as progressive disease (PD) and the remaining 40 (80%) as stable disease (SD). Among PR, a decrease of both BF (18±9.6%) and BV (12.6±9.2%) were observed; TTP increased in 3 (37.5%) cases, and PS decreased in 6 (75%) cases. SD patients showed an increase of BF, BV, TTP and PS in 6 (15%), 21 (52.5%), 23 (57.5%) and 2 (5%) cases, respectively. PD patients demonstrated an increase of BF (26±0.2%), BV (2.7±0.1%) and TTP (3.1±0.8%) while only PS decreased (23±0.2%). CT-p can adequately evaluate therapy-induced alterations in NSCLC, and perfusion parameters correlate with therapy response assessment performed with RECIST criteria. Evaluating perfusional parameters, CT-p can demonstrate therapy-induced changes in patients with different types of lung cancer and identify response to treatment with excellent agreement to RECIST measurements.

Immunological monitoring of the tumor immunoenvironment for clinical trials.

Monitoring of immunotherapeutic clinical trials has undergone a considerable change in the last decade resulting in a general agreement that immune monitoring should guide the development of cancer vaccines. The emphasis on immune cell functions and quantitation of antigen-specific T cells have been playing a major role in the attempts to establish meaningful correlations between therapy-induced alterations in immune responses and clinical endpoints. However, one significant unresolved issue in modern immunotherapy is that when a tumor-specific cellular immune response is observed following the course of immunotherapy, it does not always lead to clinically proven cancer regression. This disappointing lack of a correlation between the tumor-specific cytotoxic immune responses and the clinical efficacy of immunotherapy may be explained, among other reasons, by the notion that the analysis of any single immunological parameter is not sufficient to provide clinically feasible information about the complex interactions between different cell subsets in the peripheral blood and immune, tumor, and stromal cells in the tumor milieu. By contrast, a systemic approach is required for improving the quality of a serial monitoring to ensure that it adequately and reliably measures potential changes induced in patients by administered vaccines or immunomodulators. Comprehensive evaluation of the balance between the immunostimulatory and immunosuppressive compartments of the immune system could be critical for a better understanding of how a given immunotherapy works or does not work in a particular clinical trial. New approaches to characterize tumor-infiltrating leukocytes, their phenotypic, biochemical, and genetic characteristics within the tumor microenvironment need to be developed and validated and should complement current monitoring techniques. These immune-monitoring assays for the local tumor immunoenvironment should be developed, validated, and standardized for reliability and consistency in order to establish the overall performance standards.

Gliomrezidiv?

Neuroradiologic follow-up suggesting recurrent glioma frequently prompts reoperation. Histological examination mostly confirms recurrent glioma, however, occasionally only reactive or therapy-induced alterations are seen. This report compiles a presentation given at the 16th Heidelberg Grand Round. Topic of that presentation was demonstration of neuropathologic findings related to the often problematic differential diagnosis of recurrent glioma and reactive or therapy-induced alterations.

Imaging Proliferation to Monitor Early Response of Lymphoma to Cytotoxic Treatment

Positron emission tomography with the thymidine analogue 3'-deoxy-3'-[18F]fluorothymidine (FLT) has been reported to closely reflect lymphoma proliferation in vivo. In this preclinical study, we have investigated if FLT can also be utilized for imaging therapy-induced alterations of the nucleoside metabolism and if FLT is a surrogate marker for early response to cytotoxic treatment. Immunodeficient mice bearing high-grade lymphoma xenotransplants were treated with the cytotoxic agent doxorubicin (day 0). In the time course of day +1 to +9, antiproliferative effects were assessed non-invasively with FLT-PET and correlated to changes of the proliferation fraction and induction of apoptosis, as assessed by immunohistochemistry. Tumor growth in untreated animals was significantly higher than in treated animals. In FLT-PET scans, these observations correlated with a significant decrease of tumor-to-background ratio in the therapy group already at day 1. Likewise, median tumor-to-muscle ratio of FLT uptake already declined at day 1. The proliferation fraction assessed by Ki-67 immunohistochemistry decreased after chemotherapy, while activated caspase 3 increased, suggesting both cell cycle arrest and induction of apoptosis as underlying mechanisms of the observed PET-signal alterations. In a lymphoma xenotransplant model, we show that positron emission tomography using the proliferation marker FLT is suitable to detect early response to cytotoxic treatment. A significant decrease of FLT uptake but not tumor growth was detectable already 24 h after therapy and correlated with reduced proliferation and induction of apoptosis. Thus, FLT-PET has a potential for imaging early response to treatment in malignant lymphoma.

Proton and sodium MRI assessment of emerging tumor chemotherapeutic resistance.

The ultimate goal of any cancer therapy is to target the elimination of neoplastic cells. Although newer therapeutic strategies are in constant development, therapeutic assessment has been hampered by the inability to assess, rapidly and quantitatively, efficacy in vivo. Diffusion imaging and, more recently, sodium MRI have demonstrated their distinct abilities to detect therapy-induced alterations in tumor cellularity, which has been demonstrated to be indicative of therapeutic efficacy. More importantly, both imaging modalities detect tumor response much earlier than traditional methodologies that rely on macroscopic volumetric changes. In this study, the correlation between tumor sodium and diffusion was further tested to demonstrate the sensitivity of sodium imaging to gauge tumor response to therapy by using a 9L rat gliosarcoma treated with varying doses of BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea]. This orthotopic model has been demonstrated to display variability in response to BCNU therapy where initial insult has been shown to lead to drug-resistance. In brief, a single 26.6 mg/kg BCNU dose yielded dramatic responses in both diffusion and sodium MRI. However, a second equivalent BCNU dose yielded a much smaller change in diffusion and sodium, suggesting a drop in tumor sensitivity to BCNU. The MRI responses of animals treated with 13.3 mg/kg BCNU were much lower and similar responses were observed after the initial and secondary applications of BCNU. Furthermore, these results were further validated using volumetric measurements of the tumor and also ex vivo determination of tumor sensitivity to BCNU. Overall, these experiments demonstrate the sensitivity and applicability of sodium and diffusion MRI as tools for dynamic assessment of tumor response to therapy.

Diffusion-weighted magnetic resonance imaging for monitoring diffusion changes in rectal carcinoma during combined, preoperative chemoradiation: preliminary results of a prospective study

Purpose: To evaluate the clinical value of diffusion-weighted magnetic resonance imaging (DW-MRI) to monitor response of primary carcinoma of the rectum to preoperative chemoradiation by measuring tumor apparent diffusion coefficient (ADC). Materials and methods: Diffusion data of nine patients undergoing preoperative combined chemoradiation for clinical staged T3, N 0–2, M 0 carcinoma of the rectum were analyzed. Diffusion-weighted echo-planar MR images were obtained prior to and at specified intervals during chemoradiation and ADCs calculated from acquired tumor images. Results: Comparison of mean ADC and cumulative radiation dose showed a significant decrease of mean ADC at the 2nd ( P=0.028), 3rd ( P=0.012), and 4th ( P=0.008) weeks of treatment. Cytotoxic edema and fibrosis were considered as reasons for ADC decrease. Conclusion: This study demonstrated tumor ADC changes via detection of therapy-induced alterations in tumor water mobility. Our results indicate that diffusion-weighted imaging may be a valuable clinical tool to diagnose the early stage of radiation-induced fibrosis.