Thesis advisor: Ellen Mahoney / The diagnosis of a primary malignant brain tumor (PMBT) can be devastating for individuals and their families due to the limited treatment options and poor prognosis. Patients often rely on family members to manage their care; however, many caregivers feel under-prepared and overwhelmed by the experience. Though caring for a person with a PMBT is challenging and complex, little research has addressed the family caregiver's performance. The purpose of this study was to identify the extent to which preparedness and caregiver role strain explained the family caregiver’s performance in symptom management. An adapted theoretical framework, The Theory of Unpleasant Symptoms for Family Caregivers, guided this study. The study employed a descriptive, correlational research design in which the researcher obtained cross-sectional data during one collection period. The participants were adults who identified themselves as family caregivers of persons with a PMBT. Forty caregivers were enrolled in the study at the Brain Tumor Center at Beth Israel Deaconess Medical Center. Results from the regression analyses indicated that caregiver role strain and preparedness explained 31% of the variance (adjusted R2) in perceived psychological symptom distress and 29% (adjusted R2) of the variance in perceived physical symptom distress. Caregiver role strain was the major contributor to psychological (B=.68, p=.000) and physical symptoms (B=.48, p=0.001), indicating that higher levels of caregiver role strain were predictive of higher levels of perceived symptom distress and this relationship was strong. Preparedness contributed less to the explained variance in psychological (B=-.24, p=.20) and physical symptoms (B=-.21, p=.14). The negative beta indicates that higher preparedness was related to lower perceived symptom distress, but this relationship was small when compared with role strain. This study informs clinicians in neuro-oncology that care giver role strain is often high in family caregivers of patients with a PMBT and can have a negative impact on caregiver performance. These findings also support the need for more tailored nursing interventions to assist caregivers with ways to decrease caregiver role strain and improve caregiver preparedness. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.

Foi realizada uma revisão integrativa da literatura com o objetivo de caracterizar as pesquisas na área de enfermagem oncológica. O levantamento bibliográfico abrangeu as publicações nacionais em enfermagem oncológica, de 1980 a 2004, disponível nos bancos de dados Lilacs e BDENF. Foram identificados 91 artigos, dos quais 84 compuseram a amostra final do estudo. A análise dos estudos permitiu identificar que os mesmos foram publicados com maior freqüência na Revista Brasileira de Cancerologia e que as publicações se intensificaram após o ano 2000. Em relação à identificação dos autores, a maioria é docente (41,5%); 85,3% dos pesquisadores são enfermeiros; 33,4% procedem da Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo e da Universidade Federal de São Paulo. Os estudos, em sua maioria, foram desenvolvidos na região Sudeste e 52,4% deles foram localizados na base de dados Lilacs; as palavras-chave mais utilizadas foram enfermagem e câncer (72,6%); 66,7% dos estudos apresentaram os objetivos de forma clara, 23,8% não apresentaram objetivos, 9,5% não apresentaram adequadamente os objetivos e 19,0% eram derivados de dissertação ou tese. Entre os desenhos de pesquisa, 36,9% utilizaram a abordagem metodológica qualitativa e 21,4% a quantitativa; 9,5% realizaram estudos quanti-qualitativos e 32,1% não discriminaram o método utilizado. Em relação à ética, 41,7% dos estudos não fizeram nenhuma referência; 65,5% apresentaram as características da amostra estudada e 34,5% não apresentaram; 45,2% dos estudos investigaram pacientes com diferentes tipos de câncer, sendo a enfermagem em ginecologia a área com maior número de investigações. Em relação à área temática destacam-se os estudos referentes à atuação do enfermeiro com 16,6%; 39,3% apresentavam quadro teórico; 97,6% apresentavam revisão de literatura em todas as etapas do processo de pesquisa; 89,3% apresentavam considerações finais ou conclusões; 65,5% responderam aos objetivos e 71,4% fizeram recomendações para o desenvolvimento da profissão. Identificamos o grande interesse da enfermagem pela abordagem metodológica qualitativa, devido ao grande número de estudos. Entretanto, como os resultados apontaram a falta de esclarecimentos importantes para demonstrar o rigor dos estudos analisados, consideramos que ainda não há um corpus de estudos que possibilite a metanálise ou metassíntese desse conhecimento. Destacamos que esse corpus dificulta a aplicação dos resultados de pesquisa para a prática assistencial de enfermagem em oncologia. Além disso, sugerimos identificar prioridades de pesquisa em enfermagem oncológica; refinar estratégias de síntese de resultados de pesquisa; criar uma cultura de pesquisa dentro das instituições de saúde; desenvolver estudos relacionados aos tipos de câncer mais incidentes; replicar estudos em outros contextos; conduzir com rigor os estudos respeitando-se as etapas do método científico e melhor elaboração dos relatórios encaminhados para publicação, a fim de que a pesquisa em enfermagem oncológica possa promover cuidados efetivos e com qualidade. / An integrative review of the literature was performed with the aim of characterizing the researches produced by the Brazilian oncology nursing. The bibliographic research covered national nursing publications, from 1980 to 2004, on the Lilacs and BDENF data bases; from which 91 articles were identified and, of the total, 84 composed the final sample of the study. The analysis of the studies allowed identify that they have been published most frequently on the Brazilian Journal of Carncerology and that the publications were increased after the year 2000. In relation to the identification of the authors, most are university professors (41.5%), 85.3% of the researchers are nurses, 33.4% are from the Ribeirão Preto School of Nursing of the University of São Paulo and from the Federal University of São Paulo. Most of the studies were developed in the Southeast region of Brazil. Of the total studies, 52.4% were found by using Lilacs; the most used keywords were nursing and cancer (72.6%); 66.7% made a clear statement, 23.8% did not present any goals, and 9.5% did not present their goals appropriately and 19,0% were derived from dissertations or thesis. Among the research schemes, 36.9% used the qualitative methodological approach, 21.4% used the quantitative methodological approach, 9.5% performed quanti-qualitative studies, and 32.1% did not state the used method. In terms of ethics, 41.7% of the studies did not make any statement. Of the studies, 65.5% presented characteristics of the studied samples, and 34.5% did not. A total of 45.2% of the studies investigated patients with different types of cancer, being nursing in gynecology the area with the highest number of investigations. In relation to the thematic area, nursing actuation stands out with a total of 16,6%. Of the total, 39.3% of the studies presented a theoretical background, 97.6% presented literature reviews in all steps of the research process, 89.3% presented final considerations or conclusions, 65.5% comply with the goals, and 71.4% made recommendations for the profession’s development. We identified the great interest of nursing on the qualitative methodological approach, due to the high number of studies. However, since the results showed there is a lack of important elucidations to demonstrate the rigor of the analyzed studies, we consider that there still isn’t a corpus of studies that allow the meta-analysis or metasynthesis of knowledge. We point out that this corpus increases the difficulty of applying the research results to the practice of nursing aid in oncology. In addition, we suggest identifying priorities in research on oncology nursing, refining the strategies of synthesis of research results, creating a research culture within health institutions, developing studies related to the most incident types of cancer, replicate studies in other contexts, conducting studies rigorously by respecting the steps of the scientific method and enhancing the development of reports forwarded for publication, in a way that research on oncology nursing may promote effective and quality care.

The accuracy of dose calculation algorithms used for radiotherapy treatment planning play a significant role in the clinical outcome of various treatment regimens. Heterogeneities in human anatomy such as lung, air cavities, bone, soft tissue and fat present challenges to the dose calculation algorithms as they are prone to disrupt the charged-particle equilibrium. Monte Carlo (MC) based dose calculation algorithms are proven to be superior to all the current analytical algorithms owing to their ability to account for all the physical interactions that are involved in radiation transport. Numerous publications have examined the differences in physical doses calculated by analytical algorithms when compared to MC in dealing with heterogeneities. However, before this work the clinical significance of these differences in physical dose has never been investigated in detail. An EGSnrc, BEAMnrc and DOSXYZnrc based MC dose calculation engine was set up in a parallel computing environment to simulate three-dimensional conformal radiotherapy (3DCRT) and intensity modulated radiation therapy (IMRT). A Varian 2100 C/D accelerator head was modeled and validated to match measurements of open and dynamic wedged fields in a homogeneous water phantom which was found to be in good agreement with measurements within 2%/2mm and 3%/3mm respectively. In addition, MC calculated doses in a heterogeneous lung phantom were compared to radiochromic film measurements. Overall, there was good agreement between the two, although large differences of upto 16% were found in some cases. This dose calculation system was used to perform MC simulations on computed tomography (CT) images. The clinical impact of the differences in absolute doses calculated by various photon dose calculation algorithms for two clinical tumour sites was investigated. The tumour control probability (TCP) and normal tissue complication probability (NTCP) were estimated using well established bio-mathematical radiobiological models. This work includes the analysis of 7 convolution (i.e. pencil-beam) and convolution-superposition (CS) based photon dose algorithms available in commercial treatment planning systems (TPSs) as well as MC, in treatment plans of non-small cell lung carcinoma (NSCLC) and nasopharyngeal carcinoma (NPC). In both NSCLC and NPC, the convolution algorithms overestimate the dose to the tumour and hence overestimate the TCP to up to 45%. Some of the CS algorithms were comparable to MC though others exhibit significant differences. In NSCLC, the absolute differences in the NTCP values with radiation pneumonitis and rib fracture as end points were not as large as the differences found in the TCPs. On the other hand, in NPC, the overestimation of probability of occurrence of xerostomia by some TPS algorithms may be preventing dose escalation. Parameters for the TCP model were derived by fitting the TCP predictions to published outcome for four widely varying dose-fractionation regimens for a patient cohort undergoing radical radiotherapy treatment for NSCLC. The derived parameter sets strongly depend on the accuracy of the dose calculation algorithm involved. Parameters derived based on dose-distribution data sets obtained using one particular dose calculation algorithm may not hold good when evaluating treatment plans calculated with a different algorithm. In this sub-study, the influence of dose calculation algorithms on TCP model parameters was evaluated. Significant differences were found in TCPs when calculated with inconsistent parameters. Hence, the choice of dose calculation algorithm is crucial and although some algorithms generally perform close to MC in handling inhomogeneities, it is necessary to understand how the underlying differences affect the predicted clinical outcome.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the five leading causes of cancer-related deaths in the West, and this, largely, is due to metastatic disease. In order to better understand PDAC metastatic spread and identify novel therapeutic targets, we analysed the proteome of primary tumours and matched lymph node (LN) metastases. As frozen specimens of metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded (FFPE) tissues. Whilst such tissue is in routine diagnostic use, the cross-linkages induced by fixation have, in the past, precluded proteomic investigation for research purposes. Recent technological advances have, however, overcome this technical limitation. Using laser capture microdissection (P.A.L.M system), we isolated malignant epithelia from seven FFPE primary PDAC tumours and matched LN metastases. Following dissection, samples were analysed in duplicate using Multidimensional Protein Identification Technology (MudPIT); this resulted in the identification of 1504 proteins, 854 of which were common to all samples analysed. Comparison of the obtained proteins with data from previous proteomics studies on pancreatic tissue, pancreatic juice, serum and urine resulted in a less than 30 % overlap, indicating that our study has expanded the current database of proteins expressed in this malignancy substantially. Statistical analysis further showed that 115/854 proteins (13.5%) were significantly differentially expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly significant g-values were confirmed to be significantly differentially expressed (S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) 4 in a larger series of 55 cases of matched primary PDAC and LN metastases using immunohistochemistry. We chose to investigate further the roles of S100P in lymphatic invasion in vitro and in vivo. By co-culturing a Panc1 S100P-overexpressing clone (S5L), or a vector control clone (V3L), with human dermal lymphatic endothelial cells (HDLEC), we were able to show that different receptors mediate S5L adhesion to resting and activated HDLEC as opposed to V3L; and that the presence of S5L cells in these co-cultures significantly increased permeability at one (p = 0.02), four (p = 0.002) and eight (p = 0.007) hours post-seeding, and significantly increased translymphatic endothelial migration at 72 hours (p = 0.006). Using the V3L and S5L cell lines, which were transduced to express luciferase, we also created an orthotopic mouse model of PDAC, as well as experimental metastatic mouse models, in CD1 nude mice. These models were used to evaluate the effects of S100P on primary tumour growth, metastasis and site-specific growth. S100P was only found to significantly increase primary tumour growth in this model (n = 10 animals/group), both by bioluminescence (p = 0.002) and tumour weight (p = 0.01). No metastases (spontaneous and/or experimental) were seen however. Thus, this model can be used to evaluate the anti-tumour efficacy of novel therapies to S100P in the future.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and the 5th most common cause of cancer death in the UK. One of the peculiarities of this malignancy is its ability to invade nerves, a process called perineural invasion (PNI). PNI is found in almost 100% of PDAC, and is associated with poor prognosis, tumour recurrence and generation of pain. However, the molecular bases of PNI remain largely unknown. We investigated the molecular alterations underlying the neuro-epithelial interactions in PNI using one and two dimensional liquid chromatography – mass spectrometry (1D and 2D LC-MS) of laser microdissected PNI and non-PNI cancer from formalin fixed, paraffin embedded PDAC tissues. We also performed 1D LC-MS analysis of invaded and non-invaded nerves from the same cases. In addition, we developed an in vitro model of PNI using a co-culture system comprising PC12 cells, a rat pheochromocytoma cell line, as the neuronal element and PDAC cell lines. The overall proteomic profiles of PNI and non-PNI cancer appeared largely similar; of very few deregulated proteins, we have validated the up-regulation of antiapoptotic protein Olfactomedin 4 in PNI cancer using immunohistochemistry. In contrast, nerve samples demonstrated widespread molecular alterations characteristic of neuronal plasticity upon invasion by cancer cells. Immunohistochemistry confirmed the up-regulation of VGF in nerves from PDAC and chronic pancreatitis (CP) specimens compared to normal pancreas, as well as in invaded compared to non-invaded nerves in PDAC tissues. Furthermore, VGF expression strongly correlated with pain in CP; similar analysis in PDAC cases is still pending. Using the in vitro co-culture model, several PDAC cell lines were able to induce PC12 cells neuronal plasticity including survival, neurite extension as well as VGF expression, recapitulating thus the changes observed in human tissues. PDAC-induced PC12 plasticity was not mediated via NGF, a neurotrophin acting upstream of VGF and thought to be involved in the neuro-epithelial interactions. The induction of VGF expression was shown not to be necessary for PC12 cell survival, however, it contributed to the neurite extension induced by PDAC cell lines. In summary, based on proteomics analysis and in vitro modelling, we show the complex and intricate involvement and crosstalk of both tumoral and neural elements that are activated during perineural invasion in pancreatic cancer.

Replication-selective oncolytic adenoviruses represent a promising anticancer approach with proven efficacy in cancer cell lines and tumour xenografts in vivo. Anti-tumour efficacy, both in preclinical studies and clinical trials, was significantly improved in combination with chemotherapeutics in numerous cancers, including prostate cancer. It has been established that expression of the viral E1A gene is essential for the enhancement of cell killing in combination with cytotoxic drugs. The overall goal of this project is to identify specific E1A gene regions involved in the sensitization to the cytotoxic drugs mitoxantrone and docetaxel, the current standard of care for late stage prostate cancers, to enable the development of improved anti-cancer therapies. Specific regions in the E1A proteins bind to numerous cellular factors to regulate the host cell function and the viral life cycle, including the p300, p400 and pRb family proteins. This work was aimed at determining the mechanisms involved in the synergistic cell killing in prostate cancer cells in response to the combination of the replication-selective (oncolytic) mutant AdΔΔ with cytotoxic drugs. Previous findings suggested an enhancement of drug-induced apoptosis. I found that the small E1A12S protein, unable to induce viral replication, is sufficient to sensitize the prostate cancer cells, 22Rv-1 (AR+), and PC-3 and DU145 (AR-), to drugs. The non-replicating AdE1A12S-mutant AdE1A1104 (defective in p300-binding) could not sensitize the cells while mutants with intact E1A-p300 binding (AdE1A12S, AdE1A1102, AdE1A1108) and defective in p400- (AdE1A1102) or pRb-binding (AdE1A1108) potently sensitized all tested cell lines. In fact, all mutants except AdE1A1104 potently synergised with mitoxantrone and docetaxel to kill the prostate cancer cells. When comparing the non-replicating E1A12S mutants with the corresponding replicating E1A-deletion mutants (expressing E1A12S and 13S) synergy was demonstrated with all replicating mutants except dl1104, which caused an additive effect with mitoxantrone. We hypothesised that the synergistic cell killing is the result of pathway convergence through E1A-p300 and mitoxantrone-activated DNA-damage/apoptosis events. To address this I employed an extensive miRNA array screen to identify potential pathways. Several miRNAs were found to be differentially regulated in response to the combination of AdE1A12S with mitoxantrone compared to each single agent treatment. The majority of these miRNAs are reported to be part of cell death and survival pathways (e.g. apoptosis and autophagy) and to be differentially regulated in prostate cancer. To further investigate the role of these pathways, I determined changes in expression levels of key proteins that had previously been suggested to be targeted by the identified miRNAs, thereby preventing translation of the respective mRNAs. The greatest changes in protein levels in response to AdE1A12S and mitoxantrone were observed for Bcl-2, p-Akt, LC3BII and p62. Finally, I verified similar mechanisms of action when the oncolytic AdΔΔ was combined with mitoxantrone under synergistic conditions. These findings will direct future investigations aimed at dissecting the mechanisms of action for virus-induced sensitization to cytotoxic drugs and may aid in the development of improved therapies for prostate cancer by design of novel oncolytic mutants and combination strategies and/or identification of targets for small molecules inhibitors.

Androgen receptor (AR) cell signalling is active in most castration-resistant prostate cancer (PCa) tumours and suppression is hypothesized to impede cell proliferation. Hey1, a corepressor of AR is being investigated as a therapeutic transgene for late-stage PCa. A replication-defective recombinant adenovirus deleted for E1 and E3 and expressing Hey1 under a CMV promoter was constructed (Ad5Hey1). A dual luciferase reporter system demonstrated that Ad5Hey1 repressed AR activity in a dose dependent manner in miboleronestimulated 22Rv1 cells. Ad5Hey1 was cytotoxic in both AR-positive 22Rv1 and LNCaP and AR-negative DU145 cells. The doses required to kill 50% of cells (EC50) were comparable to those of AdE1A12S expressing the cytotoxic E1A12S gene from an identical vector. The mechanisms of Ad5Hey1-induced cell killing were investigated in 22Rv1 and DU145 cells. Using RNA interference towards AR or p53 in 22Rv1 cells we concluded both proteins were required for optimal cell killing by Ad5Hey1. In DU145 cells, with non-functional p53, Ad5Hey1 decreased levels of phospho- STAT3 and total STAT3 suggesting Ad5Hey1 might inhibit STAT3 signalling while the JAK1/2 inhibitor, AZD1480 was ineffective at sensitising DU145 cells to Ad5Hey1. Preliminary data therefore suggests Ad5Hey1 may interfere with JAK/STAT signalling in these cells. Cell-killing efficacy with Ad5Hey1 in combination with cytotoxic drugs currently used in the clinic for the treatment of late-stage PCa, mitoxantrone and docetaxel, resulted in a synergistic enhancement of cell death in 22Rv1 and DU145 cells. LNCaP cells were also sensitised to the drugs. Characterisation of the mode of cell killing demonstrated augmented mitochondrial membrane depolarisation and caspase-3 activation when combined with docetaxel in all cell lines and with mitoxantrone in 22Rv1 and LNCaP cells, typical of apoptotic death. In DU145 cells, the combination of Ad5Hey1 with mitoxantrone decreased the proportion of apoptotic cells suggesting cells are dying by alternative cell death mechanisms. In this thesis I have demonstrated that Ad5Hey1 potently eliminates PCa cells both in the presence and absence of functional AR or p53, and that cell killing is 6 improved in combination with cytotoxic drugs. I demonstrate that the mechanisms by which Ad5Hey1 acts as a cell death enhancer is mainly through cooperation with drugs on apoptotic pathways while other factors such as inhibition of survival are also involved. In conclusion, these data suggest that it is feasible to develop a future replication-selective adenovirus expressing Hey1 as a cytotoxic transgene to improve antitumour efficacy in vitro and in vivo, especially in combination with apoptosis-inducing drugs.

CLL T cells exhibit functional defects and alterations in gene expression that show similarities to exhausted T cells in chronic viral infections. It is unclear whether CLL T cells are truly exhausted, or whether these defects are restricted to expanded populations of CMV specific cells. The phenotype and function of T cells from CLL patients was compared to age- and CMV-serostatus-matched controls. There were an increased proportion of effector T cells in CLL patients and CMV-seropositive individuals. CD8+ and CD4+ T cells from CLL patients had increased expression of exhaustion markers CD160 and CD244 irrespective of CMV-serostatus, whereas increased PD1 expression on CD8+ T cells was limited to CMV-seronegative patients. CLL CD8+ T cells also showed defects in proliferation and cytotoxicity irrespective of CMV-serostatus, with the cytolytic defect caused by impaired granzyme packaging into vesicles and non-polarized degranulation. In contrast to virally-induced exhaustion, CLL T cells had increased expression of TBET and increased interferon-γ production, but normal IL-2 production. As lenalidomide repairs the functional and phenotypic defects associated with T-cell exhaustion, the effect of this agent on the gene expression profiles of lymphocyte subsets from CLL patients and healthy controls was investigated. Lenalidomide induced the expression of genes involved in cytoskeletal signalling, lymphocyte activation, and proliferation. In particular, lenalidomide up-regulated the expression of several genes involved in tight junction signalling, a pathway that is potentially involved in lymphocyte motility, immune synapse formation, and transendothelial migration. This pathway was down-regulated in T cells from CLL patients, but, intriguingly, was up-regulated in CLL cells compared with healthy B cells. This pathway is known to be negatively regulated by a phosphatase, PP2A. Treatment of CLL cells and T cells with the PP2A inhibitor okadaic acid mimicked the effect of lenalidomide. In conclusion, CLL T cells exhibit features of pseudo-exhaustion irrespective of CMV serostatus. Lenalidomide up-regulates tight junction signalling, which is down-regulated in CLL T cells. Inhibition of PP2A is implicated in the mechanism of action of lenalidomide on T cells.

In the last decade unprecedented improvement in cure rates and overall survival was achieved in diffuse large B-cell Lymphoma (DLBCL) through the introduction of rituximab and anthracyclin-based chemotherapy (R-CHOP) as first line treatment. However, 40% of patients are refractory or relapse after R-CHOP and are hardly salvaged. To date, only age, International Prognostic Index (IPI) stratification and genetic aberrations defining gray-zone lymphomas have been used in clinical trials to select high-risk patients for more aggressive regimens. However, these prognostic features do not take into account the full biological heterogeneity of DLBCL. This reflects our limited knowledge on comprehensive prognostication in this group of disorders and supports our choice to investigate old and new prognostic factors for DLBCL in this thesis. Molecular characterization is generating opportunities for personalized therapy in poor-risk DLBCL. In order for targeted therapies to succeed in this disease, reliable and reproducible strategies that adequately segregate patients into distinct molecular groups are needed. While gene expression profiling (GEP) is the gold standard method, there is presently a lack of standardized methodology for array analysis, which can lead to variable results. The lack of a routine methodology for GEP has led investigators to develop immunohistochemistry (IHC) based approaches for the molecular classification in DLBCL. In fact, the Hans algorithm is being used to identify non-GCB DLBCLs in clinical trials offering NF-kB targeting agents to patients with this subtype. By performing a systematic comparison of nine IHC algorithms for molecular classification in a new large dataset of diagnostic DLBCL, we document an extremely low concordance across all classifiers (<21%) when classifying each individual patient, and a lack of outcome impact of all strategies, demonstrating that IHC is not a reliable alternative to molecular-based methods to be used for clinical decisions in DLBCL. GEP studies also suggested that the microenvironment could provide prognostic biomarkers in DLBCL in the R-CHOP era. Most authors have focused on the use of IHC to enumerate and functionally characterize the microenvironment in DLBCL. In our second study, by comparing two methods of semi-automated analysis for IHC staining Abstract 6 of the microenvironment, we demonstrate that the computerized results are highly reproducible, add the required robustness to IHC studies and should be used in the future instead of manual analysis. By applying comprehensive statistical analysis we propose that CD3 and FoxP3 should be validated as predictors of response to R-CHOP in clinical trials. Whereas a number of mechanisms by which cancer cells influence macrophage function have been described, currently there is very limited understanding of the macrophage polarisation status and effector function in human DLBCL. In our third study we analysed the GEP of macrophages sorted from human DLBCL samples. Unsupervised hierarchical clustering does not resolve DLBCL macrophage samples from reactive macrophage samples, indicating that macrophage heterogeneity in DLBCL should be considered. 202 genes are differentially expressed in DLBCL relative to controls. Functional annotation supports that these genes are macrophage-specific. We demonstrate that DLBCL macrophages have a bidirectional M1 and M2 functional activation, challenging the concept, widespread in the literature, that macrophages in tumours have a predominant M2 transcriptome. In our fifth study we used a two-cell co-culture model in an attempt to demonstrate that DLBCL cells influence macrophage transcriptome and proteome. The heterogeneity of the results, which precludes the confirmation of our hypothesis, is fully discussed. In our last study we tease out the DLBCL macrophage GEP heterogeneity and propose IFN- as a culprit B-cell derived molecule influencing macrophage activation status. Finally, using immunofluorescence we demonstrate that both M1 and M2 proteins are expressed in DLBCL macrophages.

Bortezomib-based induction therapies have shown to increase complete response rates and are used as an upfront therapy for newly diagnosed multiple myeloma patients. The standard treatment uses twice a week bortezomib at 1.3 mg/m^2 with dexamethasone PO on the day of and day after bortezomib, however, peripheral neuropathy is often a dose-limiting factor. For elderly patients with multiple co-morbidities and polypharmacy, we propose an alternate schedule of once a week bortezomib IV at 1.6 mg/m^2 with dexamethasone PO on the day of and day after bortezomib. In this phase II, open-labeled, multi-site study, we hypothesize that patients receiving weekly bortezomib will have comparable efficacy as the standard twice a week schedule with increased convenience and lower toxicity profile, especially related to peripheral neuropathy. METHODS: 50 patients with newly diagnosed symptomatic multiple myeloma who were ineligible for transplant or postponed transplant were enrolled from 12 Veterans Affairs hospitals. One cycle consisted of once a week 1.6 mg/m2 bortezomib IV (days 1, 8, 15, 22) plus dexamethasone PO on the day of and after bortezomib (days 1, 2, 8, 9, 15, 16, 22, 23) for 4 weeks, with the 5th week off of treatment. Responding patients could receive up to 6 cycles. RESULTS: The median age of patients was 71 ± 1.46 years (range: 50-89) with β-2 microglobulin of 5.80 ± 0.46 mg/L and c-reactive protein of 10.61 ± 5.54 mg/L. Patients also had multiple co-morbidities including cardiovascular disease (76%) renal insufficiency (54%) and pulmonary problems (36%) and were receiving a median of 13 concurrent medications at baseline. Of the fifty patients, 43 patients were evaluable for response. Seven patients received <1 cycle or died before response could be evaluated. An objective response rate of 79% was observed in 43 evaluable patients with 14% achieving nCR/CR, and at least VGPR in 44% of patients. The median progression-free survival was 9.6 months and overall survival was 46.5 months. The most common toxicity of all grades was thrombocytopenia (42%), lymphopenia (46%), asthenia (48%), and constipation (38%). Peripheral neuropathy occurred in 24% with grade 3 neuropathy occurring only in 6% of patients. In conclusion, a weekly bortezomib plus dexamethasone regimen is efficacious and safe, with lower neurotoxicity in elderly patients with newly diagnosed multiple myeloma complicated by extensive co-morbidities and polypharmacy.