Aniruddha Choudhury

... Aniruddha Choudhury,1 Katja Derkow,1 Amir Hossein Daneshmanesh,1 Eva Mikaelsson,1 Shahryar Kiaii,1 Parviz Kokhaei,1 Anders O ... Daneshmanesh, AH, Mikaelsson, E., Jeddi-Tehrani, M., Bayat, AA, Ghods, R., Ostadkarampour, M., Akhondi, M., Lagercrantz, S., Larsson, C ...

Her-2/neu (HER2) is a protooncogene that is known to be amplified in a proportion of many different types of carcinomas. Overexpression of HER2 is generally associated with poor prognosis and resistance to chemo and radiotherapy. There exist many approaches that may be potentially utilized for therapy of HER2 overexpressing tumors. Understanding the biochemical and physiological function of HER2 in oncogenesis as well as its role in facilitating immune escape of tumor cells is critical for the improvement of existing strategies as well as developing new therapeutic candidates targeting HER2.

Lung cancer is globally the second most common form of cancer in both men and women and the single largest cause of cancer-related mortality in the world. It is a disease with poor prognosis and the survival statistics have scarcely improved since historical times. The advent of targeted therapies has caused an incremental increase in survival figures. Nevertheless, significant progress in treatment outcomes need to be achieved before any perceptible improvement in overall survival of lung cancer patients becomes evident. The use of active-specific immunotherapy or cancer vaccines for the treatment of lung cancer is still in its infancy. Nevertheless several cancer vaccines have demonstrated clinical effects and improvements in overall survival in phase II and phase III trials and several more clinical trials are currently ongoing. This review summarizes the recent developments in NSCLC vaccines.

Immune checkpoint receptors are crucial molecules for fine-tuning immune responses. Checkpoint signaling dampens T cell activation to avoid autoimmunity and the destructive effects of an excessive inflammatory response. It is well established that tumors use several mechanisms to avoid elimination by the immune system, and one involves hijacking these checkpoint pathways. Checkpoint blockade therapy utilizes monoclonal antibodies to release the brakes from suppressed T cells, allowing them to be activated and recover their antitumor activity. This therapeutic approach has revolutionized cancer immunotherapy, and extraordinary increases in overall survival were noted, first with anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) and subsequently with anti-PD-1 (programmed cell death receptor-1) in melanoma and other malignancies.

Human leukemia-derived dendritic cells show potential as tools for therapy. Leukemic cells of patients with chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and chronic myelomonocytic leukemia (CMML) will all undergo substantial differentiation toward dendritic cells (DC) and may be used to drive autologous T cells to acquire anti-leukemic cytotoxicity. This article describes the use of these human leukemia-derived dendritic cells for stimulation of allogeneic donor lymphocytes and presents a clinical trial of autologous CML DC-stimulated lymphocytes.

HER2 is a promising target for immunotherapeutic interventions with T cell-based approaches since it is amplified and overexpressed in 20-30% of breast cancers. However, several previous studies including ours showed that HER2-overexpressing tumors may escape cytotoxic T lymphocyte-mediated lysis by downregulating MHC Class I and components of the antigen-processing machinery. The aims of the present study were to analyze the relationship between HER2 and MHC Class I expression and to elucidate the mechanisms underlying MHC Class I downregulation in breast cancer. We explored expression of HER2, MHC Class I, PTEN, Ki67, estrogen and progesterone expression in 70 breast cancer patients by immunohistochemistry (IHC) and analyzed their correlation. We also explored the components of the signal transduction pathway that are involved in the regulation of MHC Class I expression using small-interfering RNAs targeting HER2 as well as an inhibitor of HER2 signaling. HER2 expression in breast...

At the present time, regulatory T cells (Tregs) are an integral part of immunology but the route from discovery of "suppressive" lymphocytes in the 1980s to the current established concept of Tregs almost 20 years later has been a rollercoaster ride. Tregs are essential for maintaining self-tolerance as defects in their compartment lead to severe autoimmune diseases. This vitally important function exists alongside the detrimental effects on tumor immunosurveillance and antitumor immunity. Beginning with the identification of CD4(+)CD25(+) Tregs in 1995, the list of Treg subsets, suppressive mechanisms, and knowledge about their various origins is steadily growing. Increase in Tregs within tumors and circulation of cancer patients, observed in early studies, implied their involvement in pathogenesis and disease progression. Several mechanisms, ranging from proliferation to specific trafficking networks, have been identified to account for their systemic and/or local accumu...

Lung cancer represents one of the malignancies in which the 3 elements of conventional therapy (ie, surgery, radiotherapy, and chemotherapy) have limited effectiveness in curbing progressive disease. In this context, there is burgeoning interest in the use of vaccine therapy as a nontoxic adjunct to increase the treatment success rates over those obtained with traditional regimens alone. Several clinical trials using a variety of vaccination strategies have been reported or are ongoing. In this review, we have provided an overview of these trials, with a special focus on the clinical efficacy of the vaccines. The future prospects and challenges of vaccine therapy in lung cancer have also been discussed.

B-cell chronic lymphocytic leukaemia (B-CLL) is characterised by the progressive accumulation of monoclonal CD5(+) B cells. In a previous study, we have analysed the expression profile of apoptosis-regulating genes using a cDNA-based microarray and found overexpression of the antiapoptotic bcl-2 family member, bfl-1, in B-CLL cells with an apoptosis-resistant phenotype. In this study, bfl-1 mRNA levels have been determined by competitive PCR in an extended population of B-CLL patients to characterise its role in disease progression and development of chemoresistance. bfl-1 levels were significantly higher in patients with no response (NR) to last chemotherapy than in patients responding (partial response (PR)) to last chemotherapy (P<0.05) and in patients who had not required treatment (P<0.05). We found no correlation between bfl-1 mRNA levels and disease progression, IGHV mutational status or other clinical parameters. In addition, bfl-1 mRNA levels were inversely correlated...

Breast cancer is globally the most common malignancy in women. Her2-targeted monoclonal antibodies are established treatment modalities, and vaccines are in late-stage clinical testing in patients with breast cancer and known to promote tumour-killing through mechanisms like antibody-dependent cellular cytotoxicity. It is therefore increasingly important to study immunological consequences of conventional treatment strategies. In this study, functional tests and four-colour flow cytometry were used to detect natural killer (NK)-cell functions and receptors as well as T-cell signal transduction molecules and intracellular cytokines in preoperative breast cancer patients, and patients who had received adjuvant radiotherapy or adjuvant combined chemo-radiotherapy as well as in age-matched healthy controls. The absolute number of NK cells, the density of NK receptors as well as in vitro quantitation of functional NK cytotoxicity were significantly higher in preoperative patients than th...

Active, specific immunotherapy for cancer holds the potential of providing an approach for treating cancers, which have not been controlled by conventional therapy, with very little or no associated toxicity. Despite advances in the understanding of the immunological basis of cancer vaccine therapy as well as technological progress, clinical effectiveness of this therapy has often been frustratingly unpredictable. Hundreds of preclinical and clinical studies have been performed addressing issues related to the generation of a therapeutic immune response against tumors and exploring a diverse array of antigens, immunological adjuvants, and delivery systems for vaccinating patients against cancer. In this chapter, we have summarized a number of clinical trials performed in various cancers with focus on the clinical outcome of vaccination therapy. We have also attempted to draw objective inferences from the published data that may influence the clinical effectiveness of vaccination app...

Her-2/neu (HER-2) is a 185-kDa receptor-like glycoprotein that is overexpressed by a variety of tumors such as breast, ovarian, gastric, and colorectal carcinomas. Overexpression of this oncogene is directly associated with malignant transformation of epithelial cells. The frequency of HER-2 overexpression varies among the different types of cancers, but universally represents a marker of poor prognosis. The critical role of HER-2 in epithelial oncogenesis as well as its selective overexpression on malignant tissues makes it an ideal target for immunotherapy. Antibodies and T cells reactive to HER-2 are known to naturally occur in patients with HER-2 positive tumors, confirming the immunogenicity of the molecule. Both antibodies as well as T cells reactive to HER-2 have been utilized for immunotherapy of HER-2 positive tumors. The "humanized" monoclonal antibody Herceptin has been tested in several clinical trials and found to be an effective adjuvant therapy for HER-2 pos...

Fludarabine and alemtuzumab are routinely used for treatment of B-cell chronic lymphocytic leukemia (B-CLL). The present study aimed to compare the expression of signaling molecules and cytokine production by T cells of B-CLL patients in long-term unmaintained remission/plateau phase following fludarabine or alemtuzumab treatment with that of indolent/untreated B-CLL patients and healthy donors. The frequency and intensity of TCR-CD3zeta chain, p56lck, p59fyn, ZAP-70, PI3-kinase and interferon (IFN)-gamma/interleukin (IL)-4 production in CD4 and CD8 T cells was examined by flow cytometry. T-cell function was assessed by stimulation with purified protein derivative (PPD) and phytohemagglutinin (PHA). Despite a reduction in number, the expression of IFN-gamma/IL-4 in T-cells in patients was significantly higher than in healthy donors. The intensity of most signaling molecules in treated patients was relatively unaffected vs. healthy donors but lower than untreated-indolent patients. However, the total number of T cells which expressed each of the signaling molecules was decreased in patients, with no difference between fludarabine- and alemtuzumab-treated patients. The T-cell response to PHA but not PPD was reduced in treated patients. The results suggest that, despite some alterations in signaling molecules and a reduction in T-cell number, overall T-cell functions may be relatively well preserved long-term after treatment with fludarabine and alemtuzumab.

Patients (n = 34) with previously untreated, slowly progressive asymptomatic stage I/II multiple myeloma or with stage II/III multiple myeloma in stable response/plateau phase following conventional anti-tumor therapy were immunized repeatedly with the antigen-specific cancer immunotherapeutic agent tecemotide (L-BLP25). Additionally, patients were randomly allocated to either single or multiple low doses of cyclophosphamide to inhibit regulatory T cells (Treg). Immunization with tecemotide resulted in the induction/augmentation of a mucin 1-specific immune response in 47% of patients. The immune responses appeared to involve a Th1-like cellular immune response involving CD4 and CD8 T cells. The rate of immune responses was similar with single versus multiple dosing of cyclophosphamide and in patients with vs. without pre-existing mucin 1 immunity. On-treatment reductions in the slope of M-protein concentration over time (but not fulfilling clinical criteria for responses with conventional anti-tumor agents) were observed in 45% of evaluable patients, predominantly in those without versus with pre-existing mucin 1 immunity and in patients with early stage disease. No differences were seen in patients receiving single or multiple cyclophosphamide dosing. Treatment with tecemotide was generally well tolerated. Repeated vs. single dosing of cyclophosphamide had no impact on Treg numbers and was stopped after a case of fatal encephalitis that was assessed as possibly study-related. Tecemotide immunotherapy induces mucin 1-specific cellular immune responses in a substantial proportion of patients, with preliminary evidence of changes in the M-protein concentration time curve in a subset of patients.

The human myeloid leukemias are a diverse group of disorders characterized by massive clonal expansion of myeloid cells showing variable degrees of differentiation block. Leukemic dendritic cells were generated in culture from chronic myelogenous leukemia (CML). These were used to stimulate autologous T cells to develop leukemia-specific cytotoxicity. Available data suggest that the cells responsible for the cytolytic activity are at least in part CD8+ and HLA restricted in their function. Additional data suggest that some anti-CML cellular activity may be Fas mediated. T-cell receptor studies provide evidence for an oligoclonal response implying a recognition of a limited number of antigens. We have used culture techniques similar to those used for CML to study the ability of AML cells to differentiate toward dendritic cells. Four of five patients have shown acute leukemia-derived dendritic cells. This work offers an avenue for the development of novel strategies for the control of human myeloid leukemias.

Lapatinib is a dual tyrosine kinase inhibitor of the EGFR and HER2 tyrosine kinase domains. EGFR is expressed in 33.3% and HER2 in 30.3% of esophageal squamous cell carcinomas (ESCCs). To explore the potential utility of Lapatinib for therapy of ESCC patients, we evaluated the effect of Lapatinib on a panel of ESCC cell lines. EGFR and HER2 expression by the cell lines was established, and the effects of Lapatinib on inhibition of the phosphorylation of HER2, antiproliferative effect, apoptosis-inducing activity and accumulation of HER2 and EGFR on cell surface were evaluated. Additionally, the combined effect of Lapatinib together with Herceptin or Cetuximab on cell-mediated cytotoxicity was evaluated. Lapatinib inhibited HER2 phosphorylation in HER2-overexpressing, HER2 gene amplification positive ESCC cell line. Lapatinib also inhibited cell proliferation, induced apoptosis and caused the surface accumulation of HER2 and EGFR in ESCC cell lines. Addition of Lapatinib increased Herceptin-mediated antibody-dependent cell-mediated cytotoxicity by 15-25% with three ESCC target cell lines. Similarly, Cetuximab-mediated antibody-dependent cell-mediated cytotoxicity also increased by 15-30% in two ESCC cell lines on addition of Lapatinib. Cumulatively, the data indicate that Lapatinib has activity in EGFR- and/or HER2-expressing ESCC cells, and the combination therapy of Lapatinib and Cetuximab/Herceptin is a promising strategy in ESCC.

We have previously demonstrated that IT9302, a nonameric peptide homologous to the C-terminal domain of human IL-10, mimics several effects of the cytokine including down-regulation of the antigen presentation machinery and increased sensitivity of tumor cells to NK-mediated lysis. In the present report, we have explored a potential therapeutic utility for IT9302 related to the ex vivo production of tolerogenic dendritic cells (DCs). Our results indicate that IT9302 impedes human monocyte response to differentiation factors and reduces antigen presentation and co-stimulatory capacity by DCs. Additionally, peptide-treated DCs show impaired capacity to stimulate T-cell proliferation and IFN-γ production. IT9302 exerts its effect through mechanisms, in part, distinct from IL-10, involving STAT3 inactivation and NF-κB intracellular pathway. IT9302-treated DCs display increased expression of membrane-associated TGF-β, linked to a more effective induction of foxp3+ regulatory T cells. These results illustrate for the first time that a short synthetic peptide can promote monocytes differentiation to tolerogenic DCs with therapeutic potential for the treatment of autoimmune and transplantation-related immunopathologic disease.

The aim of this study was to examine the effect of two of the most commonly used viral vectors, that is, retrovirus and adenovirus, on the antigen presentation of dendritic cells (DCs). DCs were generated from CD34(+) hematopoietic precursors and CD14(+) monocytes of the same prostate cancer patients. Adenoviral transduction of monocyte-derived DCs (MO-DCs) resulted in upregulation of CD80, CD86, and CD83 expression. Adenovirus-transduced MO-DCs were also more potent stimulators of allogeneic lymphocytes, produced increased amounts of the cytokines tumor necrosis factor alpha and interleukin 12 p70, and exhibited increased expression of NF-kappaB and antiapoptotic molecules Bcl-X(L) and Bcl-2. Enhanced expression of the antiapoptotic molecules correlated with increased resistance of adenovirus-transduced MO-DCs to spontaneous as well as Fas-mediated cell death. In contrast to the adenoviral construct, no significant transduction of MO-DCs with the retrovirus could be obtained. Transduction of CD34(+) cell-derived DCs with the retrovirus or the adenovirus did not significantly alter expression of the costimulatory molecules or cytokines studied. At lower stimulation ratios, CD34(+) cell-derived DCs transduced with retrovirus were less potent in their ability to stimulate allogeneic lymphocytes in comparison with nontransduced DCs. Our results indicate that adenoviral vectors may be more suitable for gene delivery to DCs for immunotherapy.

T cells may have a role in sustaining the leukemic clone in chronic lymphocytic leukemia (CLL). In this study, we have examined the ability of T cells from CLL patients to support the survival of the leukemic B cells in vitro. Additionally, we compared global gene expression of T cells from indolent CLL patients with healthy individuals and multiple myeloma (MM) patients. Apoptosis of purified leukemic B cells was inhibited in vitro when co-cultured with increasing numbers of autologous T cells (p < 0.01) but not autologous B and T cells of normal donors. The anti-apoptotic effect exceeded that of the anti-apoptotic cytokine IL-4 (p = 0.002) and was greater with CD8+ cells (p = 0.02) than with CD4+ cells (p = 0.05). The effect was depended mainly on cell-cell contact although a significant effect was also observed in transwell experiments (p = 0.05). About 356 genes involved in different cellular pathways were deregulated in T cells of CLL patients compared to healthy individuals and MM patients. The results of gene expression profiling were verified for 6 genes (CCL4, CCL5 (RANTES), XCL1, XCL2, KLF6, and TRAF1) using qRT-PCR and immunoblotting. Our results demonstrate that CLL-derived T cells can prevent apoptosis of leukemic B cells and have altered expression of genes that may facilitate the survival of the leukemic clone.

Olfactory neuroblastoma is an uncommon neoplasm. Typically, these tumors are indolent with long-standing symptomatology, but the fact that the lesions are indeed malignant has been proven by the repeated demonstration that they can metastasize to distant organs. Suitable prognostic factors are lacking and therapeutic strategy still remains controversial. Expression of human telomerase reverse transcriptase (hTERT) is associated with most human malignancies and high levels have been correlated with poor prognosis in many cancers. In comparison, overexpression of cyclin-D1 occurs in several malignancies and has been associated with aggressive tumor behavior and poorer prognosis. In this study, we collected 16 olfactory neuroblastomas from the Kaohsiung Medical University Hospital. The aim was to investigate the value of immunoexpression of hTERT and cyclin-D1 in correlation with clinicopathologic features of olfactory neuroblastoma. Low and high cyclin-D1 expression was found in 6 and 10 cases, respectively. For hTERT, low and high protein expression was detected in 5 and 11 tumors, respectively. Cyclin-D1 expression was not correlated with selected parameters. However, high hTERT expression was significantly correlated with high Kadish stage. In conclusion, high hTERT expression can be considered a potential indicator of aggressive olfactory neuroblastoma.