Evaluating mismatch repair deficiency in colorectal cancer biopsy specimens.

Mismatch repair (MMR) testing on all new cases of colorectal cancer (CRC) has customarily been preferably performed on surgical specimens, as more tissue is available; however, new clinical trials for the use of immune checkpoint inhibitors in the neoadjuvant setting require MMR testing on biopsy samples. This study aims at identifying advantages, disadvantages and any potential pitfalls in MMR evaluation on biopsy tissue and how to cope with them. The study is prospective-retrospective, recruiting 141 biopsies (86 proficient (p)MMR and 55 deficient (d)MMR) and 97 paired surgical specimens (48 pMMR; 49 dMMR). In biopsy specimens, a high number of indeterminate stains was observed, in particular for MLH1 (31 cases, 56.4%). The main reasons were a punctate nuclear expression of MLH1, relatively weak MLH1 nuclear expression compared to internal controls, or both (making MLH1 loss difficult to interpret), which was solved by reducing primary incubation times for MLH1. A mean of  ≥ 5 biopsies had adequate immunostains, compared to ≤ 3 biopsies in inadequate cases. Conversely, surgical specimens rarely suffered from indeterminate reactions, while weaker staining intensity (p < 0.007) for MLH1 and PMS2 and increased patchiness grade (p < 0.0001) were seen. Central artefacts were almost exclusive to surgical specimens. MMR status classification was possible in 92/97 matched biopsy/resection specimen cases, and all of these were concordant (47 pMMR and 45 dMMR). Evaluation of MMR status on CRC biopsy samples is feasible, if pitfalls in interpretation are known, making laboratory-specific appropriate staining protocols fundamental for high-quality diagnoses.

Mutation frequencies of GNAQ, GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single case of uveal melanoma.

BACKGROUND: Uveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma. METHODS: We analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays. RESULTS: We detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse. CONCLUSION: These data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.

Somatic diversification and selection of immunoglobulin heavy and light chain variable region genes in IgG+ CD5+ chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes. Most studies have found that these leukemic CD5+ B cells, like their normal counterparts, use immunoglobulin (Ig) variable (V) region genes that exhibit minimal, if any, somatic diversity. These and other observations have suggested that CD5+ B cells may be incapable of generating Ig V gene diversity, and therefore may not be able to develop higher affinity binding sites that could be selected by antigen. However, most of the studies of CLL and normal CD5+ B cells have focused on IgM-producing cells. Since somatic mutations are most often seen in B cells that have undergone an isotype class switch, we analyzed the Ig heavy (H) and light (L) chain variable region genes of seven IgG+CD5+ CLL B cells to determine if somatic diversification and antigen selection had occurred. The data derived provide evidence for skewed use, somatic diversification, and antigenic selection of the Ig V region genes. Nonrandom use of both H and L chain V region genes was manifested by an overrepresentation of VH4 and VKI family genes and the underrepresentation of the JH4 gene segment. Furthermore, VH4 gene use was restricted to only two family members (4.21 and 4.18). In four of the seven cases, the VH and VL genes displayed > or = 5% difference from the most homologous known germline counterparts. Polymerase chain reaction and Southern blot analyses performed in two of these patients demonstrated that their unique VH CDR2 and adjacent sequences were not present in their germline DNA. In addition, a significant level of diversity was seen in the rearranged DJH segments and at the VL-JL junctions of every patient that occurred both at the time of recombination and subsequently. The localization of replacement changes to complementarity determining regions of some patients suggested that antigen selection had occurred. Furthermore, the mutations identified in the VH and VL genes of each individual patient were strikingly similar, both in number and location. Collectively, the data indicate that a subset of CD5+ CLL B cells can display Ig V region gene mutations. In addition, they are consistent with the notions that in some cases antigen selection of these mutations may have occurred, and that antigen stimulation may be a promoting factor in the evolution of certain CLL clones.

Examples of in vivo isotype class switching in IgM+ chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) usually involves the expansion of a clone of CD5+ B cells synthesizing IgM antibodies. These B cells appear to be blocked at the antigen receptor-expressing stage of B cell differentiation and are thought not to undergo an isotype class switch to IgG or IgA production. In vivo and in vitro studies suggest, however, that in some instances terminal differentiation and isotype switching can occur. To test the hypothesis that in vivo isotype class switching occurs in IgM+ B-type CLL cells, we analyzed the PBMC of 19 CLL patients for the presence of transcripts encoding the rearranged CLL V(H)DJ(H) associated with either gamma or alpha H chains. The molecular data indicate that approximately 50% of B-CLL patients have amplifications of IgM+ B cells that undergo an isotype class switch. Switching to IgA appears to occur more often than to IgG; also, switching can involve different IgG subclasses in individual patients. In many instances, these CLL-related gamma and alpha transcripts are much more plentiful than those of normal B cells that produce the same isotype. These switched transcripts do not reveal evidence for the accumulation of significant numbers of new V(H) gene mutations. The cellular data indicate that B cells with lesser amounts of surface membrane IgD and higher IgM/IgD ratios are more likely to undergo this switching process. Furthermore, B cells expressing IgG and IgA of the same idiotype or V(H) family and the same CDR3 length as those of the CLL IgM+ clone can be identified in the blood of patients studied using multiparameter immunofluorescence analyses. Collectively, these data suggest that not all members of a B-CLL clone are frozen at the surface membrane Ig-expressing stage of B cell maturation, and that some members can switch to the production of non-IgM isotypes. The occurrence of switching without the accumulation of V gene mutations indicates that the processes of differentiation and diversification are not linked.

Restricted immune responses lead to CNS demyelination and axonal damage.

Although autoreactive T-cells have a pivotal role in initiating the inflammatory process in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), recent evidence suggests a relevant role for autoantibodies specific for myelin proteins as well. To examine the role of B-cells in the cerebrospinal fluid of patients with MS, we analyzed the V(H) gene usage in ten MS patients by PCR technologies. Analysis of HCDR3 length revealed an oligoclonal accumulation of B-cells. Sequence analysis of the V(H)3 and V(H)4 gamma transcripts of two MS individuals demonstrated that this accumulation was related to the expansion and somatic diversification of a limited groups of B-cell clones. These findings are indicative of a chronic and intense antigenic stimulation occurring in the CNS. Animal models, such as EAE, are of particular importance in order to elucidate the pathogenetic effector mechanisms in autoimmune demyelination. In a non-human primate model of EAE, we describe that the immunodominant T-cell epitope is presented exclusively by a monomorphic DRB1 allele, suggesting that susceptibility to EAE may be linked to this unique restriction and, therefore, providing a possible mechanism for MHC linkage to diseases. Moreover, we report on the presence of inflammation, sharp demyelination and axonal damage in EAE induced with whole myelin as well as with recombinant myelin oligodendrocyte glycoprotein (MOG), but not with myelin basic protein alone. The presence of axonal pathology was supported by immunohistochemistry with anti-amyloid precursor protein and anti-non phosphorilated neurofilaments monoclonal antibodies within early active demyelinated plaques. These findings suggest that axonal damage may be an early event in the pathogenesis of autoimmune demyelinating diseases of the CNS and highlights the importance of animal models in which therapies targeting repair and axonal survival may be exploited.

IgG+, CD5+ human chronic lymphocytic leukemia B cells. Production of IgG antibodies that exhibit diminished autoreactivity and IgG subclass skewing.

Several questions exist regarding CD5+ B cells. These include the ability of these cells, as compared to CD5- B cells, to undergo an Ig isotype class switch, the subclasses utilized, and the effects that switching may have on antigen binding. To address these issues, ten patients with chronic lymphocytic leukemia (CLL) whose CD5+ leukemic B cell clones produced IgG were studied. Monoclonal IgG was collected from PMA-stimulated CLL cells and from heterohybridomas constructed with these cells, and then analyzed for IgG subclass utilization, autoreactivity, and DNA idiotype expression. The monoclonal B cells from 80% of the CLL patients produced IgG1 and those from 20% produced IgG3. None produced IgG2. In contrast to the known autoreactivity of IgM-producing CD5+ CLL cells (> 50% autoreactive), none of these IgG antibodies reacted significantly with the autoantigens tested. However, three did react significantly with autoantigen after artificially increasing antibody valency by crosslinking. Whereas five of the IgG molecules expressed a cross reactive idiotypic (CRI) marker characteristic of non-mutated kappa anti-DNA antibodies, three expressed a CRI displayed primarily on mutated IgG anti-DNA antibodies. Thus, some CD5+ human B cells can undergo an isotype class switch that for these CLL cells is biased against IgG2 and in favor of the IgG1 and IgG3. In their native state the IgG molecules secreted by these isotype-switched CD5+ cells have diminished autoreactivity, as compared to IgM-producing CLL cells. Since some of the IgG antibodies could be made auto- and poly-reactive by increasing antigen-binding valency, while others expressed idiotypic markers of mutated antibodies, certain of these CD5+ B cells probably utilize non-mutated Ig V genes coding for polyreactive antibodies, whereas others may use genes that have undergone somatic mutation and that code for more restricted specificities. Therefore, both valency and VH gene mutation may account for the diminished autoreactivity of these CD5+ B cell-derived IgG antibodies.

Report from the OECI Oncology Days 2014.

The 2014 OECI Oncology Days was held at the 'Prof. Dr. Ion Chiricuta' Oncology Institute in Cluj, Romania, from 12 to 13 June. The focus of this year's gathering was on developments in personalised medicine and other treatment advances which have made the cost of cancer care too high for many regions throughout Europe.

Production of hematopoietic growth factors by human B lymphocytes: mechanisms and possible implications.

In this study we have investigated the ability of human B lymphocytes to produce granulocyte-macrophage colony stimulating factor (GM-CSF) and, in preliminary experiments, granulocyte CSF (G-CSF). The sources of human B cells were surgically removed tonsils from normal individuals and peripheral blood from patients with B cell chronic lymphocytic leukemia (B-CLL). Tonsil B lymphocytes were purified by E rosetting and complement-mediated cytotoxicity with selected monoclonal antibodies and subsequently fractionated by a Percoll density gradient into in vivo activated and resting cells. The latter cell fractions were subsequently cultured with or without stimuli. GM-CSF was detected by a bioassay, G-CSF by an enzyme-linked immunoassay. In vivo and in vitro activated B cells produced GM-CSF, whereas in vivo activated, but not in vitro activated, B lymphocytes produced G-CSF. These results were confirmed by Northern blot experiments with cDNA probes specific for GM-CSF and G-CSF genes. Many B cell suspensions from B-CLL patients produced GM-CSF or G-CSF only following Staphylococcus Aureus Cowan I (SAC) stimulation; in some cases, a spontaneous production or no production at all of the two cytokines was detected. The possible implications of these results for B cell physiology and for the pathogenesis of immunologically mediated diseases will be discussed.

Production of granulocyte-macrophage colony-stimulating factor but not IL-3 by normal and neoplastic human B lymphocytes.

The ability of human B cells to produce granulocyte-macrophage (GM)-CSF and IL-3 was investigated. B cells, isolated from tonsils or from the peripheral blood of patients with B cell chronic lymphocytic leukemia using mAb and immune rosettes, were cultured with or without Staphylococcus aureus Cowan strain I. GM-CSF and IL-3 were measured in the culture supernatants using a bioassay based on the selective proliferative response of the MO7e megakaryoblastic cell line to IL-3 or GM-CSF. S. aureus Cowan I-stimulated normal B cells released measurable amounts of GM-CSF but not of IL-3 as determined in neutralization assays with specific mAb in the MO7e cell line test. Some of the unstimulated normal B suspensions also produced GM-CSF, albeit in lower quantities. When normal B cells were fractionated into small (resting) and large (activated) B cells by Percoll density gradients, spontaneous GM-CSF production was detected only in the large cell fractions, but small cells were induced to produce GM-CSF upon S. aureus Cowan I stimulation. On a per cell basis, tonsillar B cells were found capable of releasing more GM-CSF than activated peripheral blood monocytes. The amount of GM-CSF produced by B cells was always inferior to that released by stimulated peripheral blood T cells or NK cells. The purified B cell suspensions from all 14 B cell chronic lymphocytic leukemia patients studied released GM-CSF but not IL-3 in the culture supernatants, sometimes even in the absence of stimuli. Northern blot analysis on total or poly(A)+ RNA confirmed the presence of GM-CSF, but not of IL-3, mRNA transcripts in both normal and malignant B cells. The results of these studies support the notion that activated human B lymphocytes release sufficient GM-CSF to play a role in the control of both hematopoiesis and the inflammatory process.

Identification of two distinct CD5- B cell subsets from human tonsils with different responses to CD40 monoclonal antibody.

This study investigated the response of different CD5- B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)-4 or rabbit anti-human mu chain antibody (a-mu-Ab). The different CD5- B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5+ B cells and subsequently fractionated on Percoll density gradients. While resting CD5+ B cells proliferated and produced IgM molecules in response to a-mu-Ab, IL-4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL-2, resting CD5- B cells, which were co-purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5+ B cells had the typical phenotype of mantle zone B cells (IgM+ IgD+ CD39+ CD38- CD10- CDw75dim), whereas resting CD5- B cells were CD38- CD39- CD10- CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38+ CD10+ CD39- CDw75bright, IgG+) responded to CD40 mAb and IL-4 and also to SAC plus IL-2 further underlined the differences to resting CD5- B cells. However, some of the data collected suggest possible relationships between CD5- B cells and germinal center cells. The CD5- B cells isolated from the 50% Percoll fraction proliferated in response to a-mu-Ab, CD40 mAb and IL-4 as well as to SAC and IL-2. These cells had the same mantle zone B cell phenotype as the CD5+ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5+ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5- B cells from the 50% Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5- B cells did not express CD5. The CD5- B cells from the 50% Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of CD5 and CD38 by human CD5- B cells: requirement for special stimuli.

In this study the mode of expression of CD5 by human tonsillar CD5- B cells after stimulation with different agents was investigated. Resting B cells were separated into CD5+ and CD5- cells and the two cell fractions exposed to phorbol 12-myristate 13-acetate (PMA). CD5- B cells expressed CD5 and maximum CD5 expression was achieved after approximately 60 h of culture. Based upon the proportions of cells that express CD5 as well as those of the cells surviving in culture, it was calculated that 15-25% of the total CD5- B cells were induced to express CD5. Unlike CD5- B cells, CD5+ B cells proliferated vigorously in response to PMA as assessed by [3H]thymidine incorporation and cell cycle analysis in vitro. However, the expression of CD5 by CD5- B cells was not related to the selective expansion of some CD5+ B cells left over as contaminant cells since this occurred in the absence of cell proliferation. Upon exposure to PMA, CD5- B cells remained in the G0-G1 phases of the cell cycle and did not express the Ki67 antigen or incorporate [3H]thymidine. Furthermore, mitomycin C treatment of the CD5- B cells did not prevent CD5 expression. Phenotypic studies disclosed that CD5+ B cells but not CD5- B cells expressed CD39. This finding offered the opportunity to carry out an additional control experiment. Separation of the two populations according to the expression of CD39 confirmed the finding obtained by fractionating the cells into CD5+ and CD5- B cells. The cells induced to express CD5 also expressed CD38 that was not detected on resting CD5- B cells. In this respect, the CD5- B cells that converted into CD5+ cells (inducible CD5+ B cells) resembled the cells from the CD5+ B cell fractions that up-regulated CD5 and also expressed CD38 upon exposure to PMA alone. Another example of coordinate expression of these two antigens was the finding that exposure to PMA in the presence of recombinant interleukin-4 (rIL-4) resulted in inhibition of the expression of CD5 and CD38. Although virtually all of the tonsillar CD5- B cells expressed the CD69 activation marker, no cells other than those co-expressing CD5 and CD38 were induced to express CD5 by PMA alone. Resting CD5- B cells failed to express CD5 and/or CD38 when cultured with PMA in the presence of EL4 T cells and IL-4-free T cell supernatants.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for differential responsiveness of human CD5+ and CD5- B cell subsets to T cell-independent mitogens.

Tonsillar resting B cells were separated into CD5+ and CD5- cell subsets and stimulated with the thymus-independent mitogens, Staphylococcus aureus Cowan strain I (SAC) or insolubilized anti-mu monoclonal antibodies (a mu Ab). CD5+ cells incorporated [3H]thymidine more efficiently than unfractionated cells when stimulated with SAC and their response was augmented by the addition of interleukin (IL) 2 to the cultures. CD5+ cells also proliferated in response to a mu Ab provided that IL 2 was present, SAC-, but not a mu Ab-stimulated CD5+ cells produced IgM and IgG molecules when IL 2 was added to the cultures and also secreted autoantibodies with rheumatoid factor activity and sometimes also with anti-single-stranded, but not double-stranded, DNA activity. The efficient response of CD5+ cells was not explained by the fact that they contained cells already activated in vivo. Thus, they did not express the CD23, CD69, CD71 and CD39 activation markers, failed to incorporated [3H]thymidine and to secrete Ig spontaneously or in response to IL 2 and were found to be in a quiescent state by cell cycle flow cytometric analysis. In contrast to CD5+ cells, CD5- cells displayed very little or no [3H]thymidine incorporation in response to SAC or to a mu Ab and their poor responsiveness was not altered by changing either the doses of the stimulants, the timing of the cultures, by co-culturing the cells together with CD5+ cells, or by adding IL 2 or IL 4. Immunofluorescence studies showed that freshly prepared CD5- cells did not have surface activation markers but that they expressed them following SAC stimulation. Thus, unlike that observed for CD5+ cells, SAC seems to be capable of activating CD5- cells but does not appear to be a sufficient stimulus for driving the cells into the subsequent phases of the cell cycle. The above findings, that demonstrate marked differences in the response to CD5+ and CD5- cells to thymus-independent stimuli, may bear relevance for the understanding of the normal clonal expansion of CD5+ cells as well as for the pathogenesis of autoimmune diseases.

In vitro stimulation of human tonsillar subepithelial B cells: requirement for interaction with activated T cells.

Human tonsillar subepithelial B cells, which are a marginal zone-equivalent B cell subset, respond readily to T-independent type 2 antigens, but not to polyclonal B cell activators in vitro. In this study, subepithelial (SE) B cells were induced to proliferate and mature into plasma cells when co-cultured with activated T cells. The response of SE B cells was not observed when co-cultures were carried out in transwell chambers or in the presence of blocking anti-LFA-1 antibodies, demonstrating the need for a close T-B cell interaction. The presence of soluble CD40 also prevented the B cell response in vitro suggesting a pivotal role of CD40-CD40 ligand interactions. The data are discussed in terms of the T cell dependence of marginal zone (MZ) B cell response and the possible existence of various MZ B cell subsets.

CD38 signaling by agonistic monoclonal antibody prevents apoptosis of human germinal center B cells.

The present study demonstrates that an agonistic anti-CD38 monoclonal antibody (mAb) (IB4) is capable of preventing apoptosis of human tonsillar germinal center (GC) B cells as measured by either morphological methods on Giemsa-stained cytospin preparations or flow cytometry on propidium iodide-stained cells. Two other anti-CD38 mAb (Leu-17 and OKT10) consistently failed to prevent apoptosis in the same cells, even when tested over a wide range of concentrations. Furthermore, exposure of GC B cells to IB4 mAb up-regulates the bcl-2 proto-oncogene product in a manner similar to that observed with CD40 ligand (CD40L). The ability of IB4 mAb to prevent apoptosis of GC B cells was inferior to that of both anti-CD40 mAb and CD40L. No synergistic or additive effects were observed when IB4 mAb was used together with CD40L. Unlike anti-CD40 mAb or CD40L, IB4 mAb neither induced a proliferation of GC B cells nor increased their proliferative response to anti-CD40, CD40L or recombinant interleukin-4, used alone or in combination. The present results are consistent with the recent findings on either the feature of the CD38 molecules to deliver activation signals and on the mechanisms of selection of B cells that operates in the GC.