Tumour infiltrating lymphocytes correlate with improved survival in patients with oesophageal adenocarcinoma.

BACKGROUND: Oesophageal adenocarcinoma (OAC) is increasingly common in the west, and survival remains poor at 10-15 % at 5 years. Immune responses are increasingly implicated as a determining factor of tumour progression. The ability of lymphocytes to recognise tumour antigens provides a mechanism for a host immune attack against cancer providing a potential treatment strategy. MATERIALS AND METHODS: Tumour infiltrating lymphocytes (TILs: CD3+, CD4+, CD8+ and FOXp3+) were assessed by immunohistochemistry using tissue microarrays in a contemporary and homogeneous cohort of OAC patients (n = 128) undergoing curative treatment. RESULTS: Multivariate analysis identified three independent prognostic factors for improved cancer-specific survival (CSS): increased CD8+ TILs (p = 0.003), completeness of resection (p < 0.0001) and lower pathological N stage (p < 0.0001). Independent prognostic factors for favourable disease-free survival included surgery-only treatment (p = 0.015), completeness of resection (p = 0.001), increased CD8+ TILs (p < 0.0001) and reduced pathological N stage (p < 0.0001). Higher levels of TILs in the pathological specimen were associated with significant pathological response to neoadjuvant chemotherapy (NAC). On multivariate analysis increased levels of CD4+ (p = 0.017) and CD8+ TILs (p = 0.005) were associated with significant local tumour regression and lymph node downstaging, respectively. DISCUSSION: Our results establish an association of TILs and survival in OAC, as seen in other solid tumours, and identify particular TIL subsets that are present at higher levels in patients who responded to NAC compared to non-responders. These findings highlight potential therapeutic strategies in EAC based on utilising the host immunological response and highlight the immune responses biomarker potential.

Cancer testis antigens in newly diagnosed and relapse multiple myeloma: prognostic markers and potential targets for immunotherapy.

BACKGROUND: In multiple myeloma, expression of cancer testis antigens may provide prognostic markers and potential targets for immunotherapy. Expression at relapse has not yet been evaluated for a large panel of cancer testis antigens which can be classified by varying expression in normal tissue: restricted to testis, expressed in testis and brain and not restricted but selectively expressed in testis. DESIGN AND METHODS: Evaluation of cancer testis antigen expression was made in newly diagnosed multiple myeloma cases (HOVON-65/GMMG-HD4 trial; n = 320) and in relapse cases (APEX, SUMMIT, CREST trials; n = 264). Presence of expression using Affymetrix GeneChips was determined for 123 cancer testis antigens. Of these 87 had a frequency of more than 5% in the newly diagnosed and relapsed patients, and were evaluated in detail. RESULTS: Tissue restriction was known for 58 out of 87 cancer testis antigens. A significantly lower frequency of presence calls in the relapsed compared to newly diagnosed cases was found for 3 out of 13 testis restricted genes, 2 out of 7 testis/brain restricted genes, and 17 out of 38 testis selective genes. MAGEC1, MAGEB2 and SSX1 were the most frequent testis-restricted cancer testis antigens in both data sets. Multivariate analysis demonstrated that presence of MAGEA6 and CDCA1 were clearly associated with shorter progression free survival, and presence of MAGEA9 with shorter overall survival in the set of newly diagnosed cases. In the set of relapse cases, presence of CTAG2 was associated with shorter progression free survival and presence of SSX1 with shorter overall survival. CONCLUSIONS: Relapsed multiple myeloma reveals extensive cancer testis antigen expression. Cancer testis antigens are confirmed as useful prognostic markers in newly diagnosed multiple myeloma patients and in relapsed multiple myeloma patients. The HOVON-65/GMMG-HD4 trial is registered under Dutch trial register n. NTR-213. CREST, SUMMIT and APEX trials were registered under ns. M34100-024, M34100-025 and NCT00049478/NCT00048230, respectively.

Lack of allelic exclusion by secondary rearrangements of tumour B-cell receptor light chains in hairy cell leukaemia.

Analyses of the tumour immunoglobulin (Ig) gene (IG) heavy (H) and light chains show heterogeneity of mutational status, but reveal common features of ongoing IGH isotype-switching with multiple IGH isotype expression and preference of IG lambda (IGL) light chain with selective use of IGLJ3. Phenotypic and immunogenetic analyses were performed in a series of 105 HCL patients to estimate prevalence of multiple IG light chain expression by the tumour cells. By phenotype, 3/105 HCL (2.9%) expressed double tumour-related Ig kappa (K) and L light chain proteins. By immunogenetic analysis, functional mutated double IGK(I) /IGK(II) , IGK(I) /IGL(I) and IGL(I) /IGL(II) transcripts were cloned and sequenced in 3/71 (4.2%) HCL. These latter three HCL expressed multiple IGH isotypes with mutated IGHVDJ rearrangements at the time of AID transcript expression. Most interestingly, the three cases had reinduced RAG1 transcript. In the double IGL expresser, single-cell analysis documented co-expression of the tumour-related IGLs in 5/6 cells (83%). In the IGK/IGL co-expresser, evidence of surface IgK/IgL isotype proteins confirmed functionality of the tumour-derived transcripts. The evidence of double light chain expression in single HCs and the new observation of RAG re-induction suggest ongoing selective influences on the BCR that may promote or maintain the HCL clone in the periphery.

CD27 in defining memory B-cell origins in Waldenström's macroglobulinemia.

CD27 is a tumor necrosis factor receptor family glycoprotein, identified in seminal studies as an apparently robust marker for normal memory B cells. Somatic hypermutation (SHM) in immunoglobulin variable (V) region genes, however, remains the definitive memory imprint. In Waldenström's macroglobulinemia (WM), SHM defines a predominant mutated (MUT) subset and a minor unmutated subset indicative of naive B-cell origin. In MUT-WM, tumor cells can lack CD27 expression, raising suggestions of unusual memory B-cell origins. We recently identified such normal IgM+D+CD27-ve memory B-cells, with low levels of SHM in VH genes. While these could seed WM, the possibility remains that WM could derive from classical memory B cells that shed CD27. The utility of CD27 expression in defining memory in MUT-WM origins, then, is uncertain, but SHM unequivocally defines memory B-cell derivation in most WM. Patterns of SHM and additional IgH locus events furthermore reveal ongoing intra-tumoral diversification in WM.

Selective influences in the expressed immunoglobulin heavy and light chain gene repertoire in hairy cell leukemia.

BACKGROUND: We previously reported ongoing mutational and isotype switch events in the immunoglobulin (Ig) heavy chain (H) locus in hairy cell leukemia. Those analyses raised questions on the incidence and type of selective influences occurring on the tumor B-cell receptor of hairy cell leukemia. DESIGN AND METHODS: To further investigate this issue, we examined the full IGH and kappa and lambda light chains (IGkappa and IGlambda) variable and constant region transcripts expressed in a large cohort of patients with hairy cell leukemia (n=88). RESULTS: Multiple IgH isotypes were expressed in 46/56 (82%) cases of hairy cell leukemia. Comparison of tumor with normal B-cell repertoires revealed preferential usage of IGHV3-21, IGHV3-30 and IGHV3-33 in hairy cell leukemia (p=0.001, p=0.003 and p=0.001, respectively). Light chain analysis demonstrated preferential Igl use with an inverted IGk:IGl ratio (0.7:1) and universal usage of IGLJ3. Analysis of LCDR3 junctions revealed highly homologous motifs in 40% of IGL. Parallel analysis of IGH and IGL showed selective pairing of IGHV3-21/30/33 segments to specific LCDR3-J3 subsets (p=0.008). Of 40 cases of hairy cell leukemia, 38 had mutated IGHV and/or IGK/LV, with variations in 13/13 cloned cases, while two had 100% unmutated IGHV and IGK/LV. CONCLUSIONS: Overall, biased IGV usage, preference for Iglambda with universal IGLJ3 usage and a high incidence of LCDR3 homologous motifs suggest selective influences on the B-cell receptor of hairy cell leukemia. Ongoing mutations and isotype switching suggest that influences occur on the tumor B-cell receptor at ectopic sites.

Immunoglobulin heavy chain locus events and expression of activation-induced cytidine deaminase in epithelial breast cancer cell lines.

When cells transform, phenotypic and genetic profiles can be dramatically altered. Nevertheless, a recent report identifying IgG in breast cancer cells was unexpected, revealing differentiation features normally associated with B lymphocytes. To extend these findings, we focused on immunoglobulin variable (V) region gene analysis using well-defined breast cancer cell lines expressing the epithelial marker, epithelial cell adhesion molecule (EpCAM). V(H) gene transcripts were identifiable by nested reverse transcription-PCR either as single or dual V, diversity (D), and joining (J) rearrangements in four of six lines, most being potentially functional. V(D)J transcripts were observed in sequential cultures, indicating stable expression. To exclude coexisting lymphocytes, each cell line was shown to be EBV negative, with CD19/CD20 and cytoplasmic/surface immunoglobulin also absent by flow cytometry. Identified V(H) transcripts were then sought in individual tumor cells, isolated as EpCAM+ single cells by flow cytometry. Importantly, in three of three selected cell lines, V(H) genes were identifiable in a significant fraction (approximately 32%) of single cells. In five of six identified V(H) genes, somatic mutations were apparent with no intraclonal variation, indicating cessation of mutational activity. V(H) transcripts were pre- and post-isotype switch, with activation of switch events evident from expressed germ-line switch transcripts in two of six lines. Strikingly, six of six cell lines expressed activation-induced cytidine deaminase (AID) essential for mutational and switch activity. These data suggest either a de novo rearrangement and modification of V(H) genes in epithelial tumor cells or assimilation of lymphocyte-derived chromatin. Constitutive AID activation in malignant epithelial cells further raises a potential for inducing aberrant mutational activity.

A VH4-34+ myeloma protein with weak autoreactivity.

It has been suggested that VH4-34 gene segment expression is counter-selected in multiple myeloma (MM) due to a self-tolerance mechanism. We cloned and sequenced a VH4-34 gene segment from bone marrow mononuclear cells of a stage III MM patient. We show that VH4-34 was expressed by the serum IgA myeloma (M)-protein, as demonstrated by reactivity with the VH4-34 specific 9G4 mAb and mass spectrometry (MS). The M-protein had weak reactivity with nuclei. These results demonstrate that VH4-34 may be expressed in secreted IgA M-protein with weak autoreactivity. Thus, counter-selection of VH4-34 is pronounced but not absolute in MM. Mechanisms of how VH4-34 can occasionally be expressed in MM and clinical implications are discussed.

Tracking B-Cell Repertoires and Clonal Histories in Normal and Malignant Lymphocytes.

Methods for tracking B-cell repertoires and clonal history in normal and malignant B-cells based on immunoglobulin variable region (IGV) gene analysis have developed rapidly with the advent of massive parallel next-generation sequencing (mpNGS) protocols. mpNGS permits a depth of analysis of IGV genes not hitherto feasible, and presents challenges of bioinformatics analysis, which can be readily met by current pipelines. This strategy offers a potential resolution of B-cell usage at a depth that may capture fully the natural state, in a given biological setting. Conventional methods based on RT-PCR amplification and Sanger sequencing are also available where mpNGS is not accessible. Each method offers distinct advantages. Conventional methods for IGV gene sequencing are readily adaptable to most laboratories and provide an ease of analysis to capture salient features of B-cell use. This chapter describes two methods in detail for analysis of IGV genes, mpNGS and conventional RT-PCR with Sanger sequencing.

Immunoglobulin gene analysis in polyneuropathy associated with IgM monoclonal gammopathy.

Antineural antibody activity is the implicated pathogenic mechanism in polyneuropathy associated with monoclonal gammopathy. Recognition of antigen depends on immunoglobulin variable regions, encoded by V genes. We studied V(H)DJ(H) and V(L)J(L) gene use in monoclonal B cells by clonal analysis in 20 patients with polyneuropathy and IgM monoclonal gammopathy. V genes associated with bacterial responses appear over-represented and V(H)3-23 was preferentially used, without association with specific D, J(H) or V(L)J(L). V genes revealed somatic mutation and intraclonal variation was found in 9 of 20 patients. Polyneuropathy associated with monoclonal gammopathy may be caused by an immune response to bacterial antigens, which recruit somatically mutated autoreactive B cells.

Exome Sequencing in Classic Hairy Cell Leukaemia Reveals Widespread Variation in Acquired Somatic Mutations between Individual Tumours Apart from the Signature BRAF V(600)E Lesion.

In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease.

Immunoglobulin M myeloma: evaluation of molecular features and cytokine expression.

Immunoglobulin (Ig) M myeloma is a distinct entity with features of multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM). The malignant cells in IgM myeloma have a distinctive chromosomal translocation that differentiates them from WM. These cells are postgerminal-center in origin with isotype-switch transcripts. They appear to be arrested at a point of maturation between that of WM and MM. Preliminary data indicate that a pattern of osteoclast-activating factor and osteoprotegerin expression similar to that observed in classic MM is present in IgM myeloma. Additional studies on patients with this rare tumor may provide further insight into the pathogenesis of bone disease in plasma cell dyscrasias.

Identification and assembly of V genes as idiotype-specific DNA fusion vaccines in multiple myeloma.

Tumor-specific markers are important in identifying and tracking malignant cells. In this regard, functionally rearranged immunoglobulin variable (V) region genes in B-cell tumors fulfill and extend these criteria. V genes provide signature motifs in tumor cells and can delineate critical features of the clonal history of the cell of origin. They also define a tumor-specific antigen, which can be targeted for immunotherapy. Our focus has been on using novel DNA fusion vaccines to induce antitumor immunity. Here, we describe in detail the methods for identifying tumor-derived V genes at the nucleotide level in the malignant plasma cells of multiple myeloma. We further present the methodology for assembly of tumor V genes as single-chain variable region fragments (scFv), fused in frame with an immunopotentiating nontoxic bacterial sequence, Fragment C (FrC) of tetanus toxin. These scFv.FrC DNA vaccines provide protection in myeloma models and are currently in clinical trials. The vaccines are patient specific and can be rapidly assembled for clinical use.

Idiotype gene rescue in follicular lymphoma.

Beyond the morphological, immunophenotypic, and genetic information used for the diagnosis of lymphoid malignancies, molecular analyses have deepened our insights into the development of B-cell lymphomas. We have learned that B-cell tumors can be grouped according to the mutational status of their immunoglobulin variable (V) region genes, and this has become an important prognostic tool in chronic lymphocytic leukemia. The analysis of V genes also has allowed us to more precisely place B-cell lymphomas relative to their normal B-cell counterparts and to the germinal center where somatic hypermutation takes place. It has become evident that many of the common B-cell tumors arise at this site and are able to respond to stimuli, which govern normal B-cells. In this chapter, we focus on the analysis of V genes in follicular lymphomas based on the experience in our laboratory and provide a detailed guide for this analysis.

Determining mutational status of immunoglobulin v genes in chronic lymphocytic leukemia: a useful prognostic indicator.

Determining the clonal origins of malignant B-cells will have an impact on disease understanding and management. In this regard, immunoglobulin variable (V) region gene analysis already is having a significant impact in delineating the tumor cell of origin. It can identify, among other features whether such a cell has undergone somatic mutation, which usually occurs within germinal centres. Remarkably, in chronic lymphocytic leukemia (CLL), the mutational status of V genes has allowed researchers to identify two subsets of disease, one originating from an unmutated B-cell with a markedly poorer disease outcome and the other from a mutated B-cell, which associates with long-term survival. The V gene status in CLL thus provides a robust indicator of disease outcome, which is beginning to shape clinical treatment. This chapter describes in detail the methodology for determining V gene usage in CLL, from acquisition of patient sample to generating the V-gene readout.

Massive parallel IGHV gene sequencing reveals a germinal center pathway in origins of human multiple myeloma.

Human multiple myeloma (MM) is characterized by accumulation of malignant terminally differentiated plasma cells (PCs) in the bone marrow (BM), raising the question when during maturation neoplastic transformation begins. Immunoglobulin IGHV genes carry imprints of clonal tumor history, delineating somatic hypermutation (SHM) events that generally occur in the germinal center (GC). Here, we examine MM-derived IGHV genes using massive parallel deep sequencing, comparing them with profiles in normal BM PCs. In 4/4 presentation IgG MM, monoclonal tumor-derived IGHV sequences revealed significant evidence for intraclonal variation (ICV) in mutation patterns. IGHV sequences of 2/2 normal PC IgG populations revealed dominant oligoclonal expansions, each expansion also displaying mutational ICV. Clonal expansions in MM and in normal BM PCs reveal common IGHV features. In such MM, the data fit a model of tumor origins in which neoplastic transformation is initiated in a GC B-cell committed to terminal differentiation but still targeted by on-going SHM. Strikingly, the data parallel IGHV clonal sequences in some monoclonal gammopathy of undetermined significance (MGUS) known to display on-going SHM imprints. Since MGUS generally precedes MM, these data suggest origins of MGUS and MM with IGHV gene mutational ICV from the same GC B-cell, arising via a distinctive pathway.

Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response.

Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the immunoglobulin (Ig) M, IgG, and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n = 16) in comparison with healthy controls (HCs; n = 16). The Complementary Determining Region 3 region of the immunoglobulin heavy chain variable region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis, and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show interindividual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B-cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p = 0.01). Postvaccination, significant differences emerged in MGUS in relation to M-protein levels. High M-protein concentration is associated with a more oligoclonal IgG and IgA response at day 7 postvaccination, and, in contrast to HCs, vaccination also induced significant perturbations in the MGUS IgM repertoire at day 7 (p = 0.005). Monoclonal expansion in MGUS thus has an effect on the baseline B-cell repertoire and influences the recruited repertoire upon vaccination.

Origins of the malignant clone in typical Waldenstrom's macroglobulinemia.

Histopathology and phenotypic considerations in IgM-secreting B-cell tumors allow distinction between different diseases, but insight into pathogenesis is restricted. Here, variable region (V) gene analysis can complement classification but, more importantly, can also reveal disease origins and clonal history. We used V(H) gene analysis to probe origins in Waldenstrom's macroglobulinemia (WM), and contrasted these with the less malignant counterpart, IgM-secreting monoclonal gammopathy of undetermined significance (MGUS). Limited data on WM sequences had previously shown evidence for somatic mutation, but with conflicting analysis of intraclonal variation in tumor sequences. To further the investigation, we analyzed seven cases of WM and compared these with three cases of IgM MGUS. In both diseases, V(H) genes were somatically mutated with no evidence of intraclonal variation, even at the MGUS stage. A sensitive V(H) gene-probe assay revealed no evidence for isotype switch transcripts in any of the WM and IgM MGUS cases. These findings reveal an origin of WM and IgM MGUS from a IgM cell, which transforms after cessation of somatic mutation but without initiating switch events. In contrast, IgM-secreting multiple myeloma arises at a later stage in differentiation, when isotype switch mechanisms have been engaged.

Immune defects in the risk of infection and response to vaccination in monoclonal gammopathy of undetermined significance and multiple myeloma.

The plasma cell proliferative disorders monoclonal gammopathy of undetermined significance (MGUS) and malignant multiple myeloma (MM) are characterized by an accumulation of transformed clonal plasma cells in the bone marrow and production of monoclonal immunoglobulin. They typically affect an older population, with median age of diagnosis of approximately 70 years. In both disorders, there is an increased risk of infection due to the immunosuppressive effects of disease and conjointly of therapy in MM, and response to vaccination to counter infection is compromised. The underlying factors in a weakened immune response in MGUS and MM are as yet not fully understood. A confounding factor is the onset of normal aging, which quantitatively and qualitatively hampers humoral immunity to affect response to infection and vaccination. In this review, we examine the status of immune alterations in MGUS and MM and set these against normal aging immune responses. We focus primarily on quantitative and functional aspects of B-cell immunity. Furthermore, we review the current knowledge relating to susceptibility to infectious disease in MGUS and MM, and how efficacy of conventional vaccination is affected by proliferative disease-related and therapy-related factors.

Variant B cell receptor isotype functions differ in hairy cell leukemia with mutated BRAF and IGHV genes.

A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD(+ve) mult-HCL, IgD mediated persistent Ca(2+) flux, also evident via >1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD(-ve) mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with sIgD. This suggests the possibility that mutant BRAF by-passes BCR constraints in mult-HCL.