Role of Cytopathology in Selecting Patients for Target Treatments.

Non Hodgkin lymphomas (NHL) are a heterogeneous group of lymphoid malignancies comprising morphologically and biologically different lymphoid neoplasms. NHL have been the subject of intensive studies and research that have produced a significant amount of new knowledge concerning the phenotypic, genetic, genomic and proteomic aspects of the different entities composing this group of diseases. This knowledge has determined a better definition of some entities and the further sub-classification of others. In the past few decades, traditional clinical trials have not led to significant improvements in patient survival, and therefore the final goal of an accurate phenotypic and molecular profiling of the single entities composing NHL is the development of medical “tailored” to individual patients treatment and not bound by the rigid application of pre-determined protocols. This treatment should utilize, in a next future, new drugs and compounds with unique mechanisms of actions against distinct molecular targets instead of traditional chemotherapy performed with the same drugs and the same doses for all patients. Fine-needle aspiration cytology (FNA) has gained a defined role in the diagnosis of NHL because, in addition to advantages concerning of the technique, it has been successfully utilized together with flow cytometry (FC) (1), fluorescence in situ hybridization (FISH) (2,3) and other molecular techniques; a recent report has also demonstrated the feasibility of gene expression profiling in NHL FNA specimens (4). The aim of this presentation is therefore to overview the potential role of FNA in selecting NHL patients for target treatment. Histone deacetylase inhibitors and BCL6 in aggressive lymphomas: BCL6 protein is normally expressed in germinal b-cells; its primary function is to repress the genes involved in lymphocytes activation differentiation and apoptosis. BCL6 is generally over-expressed in many diffuse large B-cell lymphomas (DLBCL) and chromosomal 3q27 translocation, where the BCL6 gene is located, is observed in about 35% of DLBCL. The deregulation of the gene causes the over-expression of BCL6, which inhibits the apoptosis and the differentiation of germinal centers promoting the arising of malignant clones. It has recently been claimed that, as happens to histones wrapping DNA, the activation of the BCL6 gene may be determined by the acetylation status. Acetylatation of the gene inhibits its repressor function; conversely, deacetylation restores the repressor functions of the gene. In the last few years, some drugs that inhibit histone deacetylase (iHDAC), such as depsipepside, have been shown to be effective in the treatment of NHL over-expressing BCL6. These iHDAC could deactivate the BCL6 gene promoting cell differentiation and apoptosis, and leading to tumor regression. Therefore, proof of BCL6 protein over-expression and/or the t3q27 translocation would provide useful information for therapeutic planning (5), which could be obtained on FNA samples too, enabling the target utilization of these drugs.Ubiquitin-Proteasome and cell cycle proteins: Proteins p21 and p27 are members of the family of cyclin-dependent kinase inhibitors which halt cell cycle progression at the G1-S phase by deactivating the cyclin/cdk complexes. These proteins represent the potential substrate for ubiquitin-proteasome patway, which is a lysosome independent system of intracellular protein degradation. The first anti-ubiquitin-proteasome drug was bortezomib (velcade). Whereas this drug is potentially the cause of several biological effects, bortezomib is considered a target drug for multiple myeloma and specific subtypes of NHL, such as mantle cell lymphoma (MCL). Its main effects are the inhibition of NF-kB and cyclin D1 and the decreased degradation of cell-cycle dependent kinase inibitors such as p27 and p21. From this viewpoint, FNA might be helpful in demonstrating p27 and p21 expression using conventional immunocytochemistry (ICC) (6).Signal transduction inhibitors, cyclins and mantle cell lymphoma:Farnesyltransferase inhibitors (FTIs) and rapamycin represent new classes of signal transduction inhibitors mainly targeting the Ras/MAPK and PI3K/Akt pathways. Rapamycin is a macrolide which inhibits the gene mTOR, deregulating the translation of specific mRNAs required for the progression from G¹ to S phase. mTOR mutations have not been observed in human cancers; nonetheless, mTOR is a component in the P13K/Akt pathway which has a role in the proliferation of t and b cells. Rapamycin, which inhibits mTOR, decreases cyclin D2, cyclin D3 and CDK4 and increases the expression of the CDK inhibitor p27. Rapamycin has been shown to be effective on Epstein-Barr virus positive lymphomas and mantle cell lymphomas (MCL). This is not surprising considering the dependence of MCL on Cyclin D1, which is regulated by mTOR activity. FNA may contribute to the diagnosis of MCL in various ways. In fact, despite potentially equivocal cytological features, MCL may be identified on cytological samples by immunocytochemical demonstration of cyclin D1, specific phenotype by FC (1) or t (11;14)(q13,q32) translocation by fluorescence in situ hybridization (FISH) (2). Follicular lymphoma, Bcl-2 over expression and antisense oligonucleotides: Bcl-2 is an apoptosis regulating protein, whose over-expression in follicular lymphoma (FL) is related to Bcl-2 gene translocation on the IGH locus. Bcl-2 over-expression may be associated with chemotherapy resistant disease, aggressive courses of treatment, and poor survival rates in patients with B-cell NHL. Deregulation of this oncoprotein thus represents a possible new way of targeting clinically aggressive FL. Antisense oligonucleotides are single strand, chemically modified DNA molecules that have a nucleotide sequence complementary to the target mRNA and therefore capable of inhibiting the expansion of the target gene. Preclinical studies have shown that Bcl-2 antisense oligonucleotides can effectively decrease Bcl-2 over-expression, reverse chemotherapy resistance and enhance the anti-apoptotic potential of both chemotherapeutic and biological agents. As for the role of FNA, Bcl-2 gene translocation on the IGH locus: t(14-18)(q32;q21) may be demonstrated in FNA samples by FISH (3) and chimeric protein over-expression by immunocytochemistry (ICC) or flow cytometry (FC) (7). Using this latter technique, it is possible to quantify the percentage of follicular, B-cell positive cells by the co-expression of CD10/Bcl-2.Gene expression profiling and diffuse large cell lymphomaMolecular profiles obtained by complementary DNA micro-arrays have shown that DLBCL are composed of at least two different entities with two different gene expression patterns: the germinal cell subtype (GC) and the activated b-cell subtype (ABC), each of them having a different survival rate (8). Moreover, micro-array studies in FL have investigated the gene changes involved in the FL progression into DLBCL. These studies could lead to the better subtyping of DLBCL and the identification of potential new therapeutic targets. As far as the role of FNA is concerned, recent studies have proven the feasibility of FNA-based gene expression profiling. Moreover, compared to archive samples, fresh samples obtained from FNA contain less non-tumoral cells and can be immediately processed, generating gene profiles resembling live profiles (4). Monoclonal antibodies: Monoclonal antibodies represent the first successful result of target therapy; in fact, chimeric anti-CD20 monoclonal antibody (rituximab) has been the most extensively studied and clinically used among the target drugs. The anti-CD20 antibody consists of murine variable regions grafted onto a human IgG1 constant region. The CD20 antigen is an excellent target since it is selective for mature B cells in the absence of precursor B cells. In addition to direct action, rituximab induces apoptosis of Bcl-2 positive cells and has been used as maintenance therapy following standard chemotherapy treatment. Numerous other monoclonal antibodies, unconjugates or radioconjugates have been produced, such as Epratuxumab (anti- CD22), Apolizumab (antiHLA-DR), Alentuzumab (Campat-1H), antiCD40 (antiCD52), Iodine 131 anti-CD20, 90-Yttrium anti-CD20, each of them designed to act against specific antigens. FNA may play an important role in ICC or FC evaluation and quantification of each of the potentially addressed antigens in different phases of the disease (before and after therapy).Conclusions: In conclusion, in a next future the number of therapies available for NHL will probably increase, and the task of the pathologist will be to produce specific information useful for the application of these target therapies. FNA may play a role in this process, combining the advantages of the technique (non-invasive, good tolerance levels, possible immediate evaluation, and possible repetition even in a potential series of samples pre and post-therapy) with the use of ancillary techniques.