Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry

Since its introduction over 3 decades ago, flow cytometry (FC) immunophenotyping has provided relevant information for the diagnosis, classification and monitoring of hematological malignancies on blood, bone marrow, cerebrospinal fluid, lymph nodes (LNs) and extranodal fine-needle cytology (FNC) [1]. Regarding LN FNC, the FC data provided consist mainly of the clonality assessment of lymphoproliferative processes, the classification of corresponding entities, and prognostic and predictive information. The advantages and achievements of LN FNC and FC have been extensively reported in the literature and are summarized in the present issue; nonetheless, looking at the same data, specific limits of the procedure also emerge. In fact, despite a general high sensitivity in clonality assessment by light chain and specific phenotypes, a small but definite percentage of undefined cases occurs in all the series reported [2]. The accuracy rate in subtyping classifications of specific non-Hodgkin lymphoma (NHL) entities by LN FNC/FC also ranges between 51 and 95%, with the unclassified cases only defined as B-cell or T-cell NHL [2,3,4,5,6]. In the meantime, spectacular progress in genetic and molecular knowledge and procedures has progressed the diagnostics of tumors, and specifically of NHL, toward a more individual definition of the single entities and personalized corresponding therapies. Therefore, an accurate phenotypical definition of NHL, in addition to not being always feasible by FNC/FC, may be insufficient for diagnostic and predictive needs. Despite these limitations, FNC is still the most accessible, feasible, inexpensive, well-tolerated and effective procedure with which to obtain vital cells from LNs, without invasive procedures. An additional advantage of LN FNC is that its adequacy can be immediately assessed by rapid on-site evaluation (ROSE). Moreover, since chronic NHL diseases present with a high rate of relapses or concomitant reactive processes, FNC offers the opportunity to control the corresponding processes over time and in different sites, with a high homogeneity of the samples and minimal discomfort for patients. Numerous studies have shown that FNC samples are suitable for different molecular procedures (fig. 1) [7,8]. Even the drawback of the small amount of material obtained by FNC, which is the main limitation of this procedure, may be overcome by new technologies that are able to maximize the use of a few nanograms of good-quality genetic material (fig. 2) [7,8]. Therefore, once again, FNC may be conveniently used in NHL management, and the new challenge for cytopathologists, starting with FC/FNC and ROSE, will be to acquire updated knowledge and apply new technologies to FNC, exploiting its own basic qualities. In this review, some of the molecular procedures mostly utilized for NHL are briefly described and evaluated in this perspective.

Most NHLs are characterized by specific cytogenetic abnormalities and are routinely investigated using genetic tests, such as classical cytogenetics, fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) (fig. 1, 2). These tests provide useful information regarding clonality, or distinctive chromosomal/molecular abnormalities associated with specific entities and lymphoid cell lineages, confirming NHL diagnosis - albeit with some exceptions and limitations [5,9,10,11,12,13,14,15,16,17,18]. Classical cytogenetics include the routine analysis of G-banded chromosomes and other cytogenetic banding techniques for the study of cell structure and chromosomes.

FISH is considered the gold standard for the detection of NHL primary karyotypic abnormalities. FISH, by using fluorescent probes that bind parts of the chromosomes with a high degree of sequence complementarity, can identify the most frequent translocations and gene region amplifications of specific NHL subtypes [11]. In the absence of specific translocations, FISH may also identify genes involved in rearrangements that are secondarily translocated to other chromosomes [11,19]. Commercially available probes cover almost all the variable breakpoints of chromosomal alterations; as for their clinical applications, they are selected on the basis of initial clinical, (cyto)morphological and immunophenotypical data. FISH can be performed on tissues and any cytological sample, including LN FNC, where it is generally used after FNC/FC [3,5,9,10,11,12,13,14,15,16,17,18,20,21,22,23,24]. FISH on interphase nuclei is generally performed using fusion-signal FISH probes, which consist of two fluoresceinated probes that hybridize two regions proximal to the breakpoint of the chromosomes involved in a supposedly reciprocal translocation [3,5,10,12,13,14,16,17,18,20,21,22,23,24]. In the absence of any chromosomal aberration, two pairs of distinct color signals are generally detected. Conversely, in the case of specific translocations involving the labeled loci, two differently labeled probes will juxtapose, giving fusion signals [3,5,10,12,13,14,16,17,18,20,21,22,23,24]. FISH has been successfully applied on LN FNC to detect specific chromosomal translocations in diffuse large B-cell lymphoma (DLBCL) [9], follicular lymphoma (FL) [9,11,16,17,23], mantle cell lymphoma (MCL) [9,11,22,] small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) [9], marginal zone lymphoma [9], Burkitt lymphoma (BL) [9] and anaplastic large-cell lymphoma (ALCL) [11,24]. These studies have generally used one or two probes to identify a specific NHL subtype, namely IGH/BCL2 to detect the t(14;18)(q32;q21.3) in FL; IGH/CCND1 to detect the t(11;14)(q13;q32) in MCL; IGH/MYC:CEP8 to detect t(8;14)(q24;q32) (Dual Color Dual Fusion Probes) for BL; immunoglobulin (Ig)H, MALT1, MYC (Break Apart Probes), as well as the enumeration/copy number probes CEP 3 (D3Z1), CEP 12 (D12Z3), 13q14 (D13S319), 13q34 (D13S25/LAMP1), ATM/D11Z1 and 17p13 (TP53), and CEP17 (D17Z1). In DLBCL, FISH is used to identify BCL6 mutations (10% of cases) [9] and translocations with numerous partners (20-30% of cases) [9,25], which may lead to overexpression of the BCL6 protein. In FL, FISH has detected twice as many t(14;18)(BCL3-IGH@)-positive patients as polymerase chain reaction (PCR) assays [10] by disclosing the cryptic translocations producing overexpression of the BCL2 protein [9,26]. In MCL, FISH has disclosed the degree of karyotypic complexity [11] determining not only the presence of the t(11;14) translocation, but also of the SOX11 alterations in CCND1 negativity, the gains of 3q and the losses of 17p, which are associated with poor survival [6,9,22,27,28,29]. Conversely, FISH is not helpful in MCL cases where the t(11;14) involves cyclin D2 or D3 [9]. In SLL/CLL, FISH is performed to detect 13q14 deletion, 11q22-23 deletion, trisomy 12q, 17p13 and 6q21 deletions, and ZAP70 expression. The latter is mainly useful in patients in advanced stages of the disease or those carrying the TP53 mutations, which are correlated to an unfavorable prognosis [9,30]. FISH also distinguishes SLL/CLL from MCL, which share the CD5/CD19 cluster coexpression [10,11,12,13,14,16,17]. In ocular adnexa, salivary gland, and lung MALT, FISH may be useful to detect the IGH@-MALT1 [9], the t(3;14)(p14.1;q32) FOXP1-IGH@, the t(1;14)(p22;q32) and the IGH@-BCL10, with the latter rarely being found in the lung, intestine, or salivary glands. BL generally shows a quite typical immunophenotype: CD10+ BCL6+ BCL2-, Ki67+ (100%) [9]. FISH can confirm FC/FNC diagnosis by the detection of the t(8;14)(q24;q32) involving MYC and IGH@, and the variant translocations involving MYC and the j or k light chain loci (2p12 and 22q11, respectively) in a minority of cases. Moreover, since the breakpoint sites of the translocations above described are widely dispersed, the use of standard PCR-based assays is precluded. Conversely, the FISH probes used for the identification of MYC translocations are more sensitive. As far as DLBCL is concerned, FISH can identify all of the more frequent translocations [9]. ALCL is characterized by t(2;5)(p23;q35) involving NPM1 and ALK, resulting in the upregulation of ALK (a receptor tyrosine kinase). ALK overexpression may be detected by immunohistochemistry but, since different variants of ALK translocation have been identified, FISH uses an ALK break-apart probe suited to capture all of these abnormalities, as compared with RT-PCR [9]. On the other hand, FISH is not indicated in cases of ALK-negative ALCL for the lack of translocations involving ALK or any other recurrent genetic abnormality. In these cases, PCR may show clonal rearrangement of the T-cell receptor (TCR) genes, as in ALK+ ALCL [9].

CISH combines the chromogenic signal detection method of immunohistochemistry techniques with in situ hybridization [31]. This procedure was developed as an alternative to FISH for the detection of HER-2/neu oncogene amplification [31]. CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complex fluorescence microscopes used in FISH [31]. CISH performed after FISH double checks the data provided by the latter; in addition, the persistence of a signal beyond the decay of the fluorescence allows a protracted reaction and a reevaluation of the sample [10]. CISH analysis on interphase nuclei is generally performed using split-signal FISH probes, which hybridize two regions of the same chromosome proximal to a supposed breakpoint. In normal cells, the two hybridized regions are proximal to each other, generating two fusion signals [10]. Split-signal FISH has some advantages over fusion-signal FISH; in fact, the detection of a translocation is independent of the partner genes involved and is useful for detecting translocations involving multiple partner genes, as is the case of the IGH locus at chromosome 14q32. What is generally considered another advantage of split-signal FISH is the absence of the false-positive cases reported using fusion-signal FISH probes [10]. The IGH locus is most frequently involved in different NHL translocations [10,28,32,33], therefore the detection of any breakage involving the IGH locus at chromosome 14q32 should identify a B-cell NHL independently of the specific subtype on cytological samples. In this case, the split-signal IGH FISH/CISH DNA probe is a mixture of two fluorochrome-labeled DNAs: a green fluorescein-labeled DNA probe (IGH-Flu) that binds to a 612-kilobase (kb) segment telomeric, and a red-labeled DNA probe (IGH-TR) that binds to a 460-kb segment centromeric, both to the IGH breakpoint. In the case of a translocation involving the IGH locus, whatever the fate of the sequence detached, two distinct split signals are observed. Therefore, the split-signal IGH FISH/CISH DNA probe should be highly sensitive, detecting any translocation involving the IGH locus at chromosome 14q32 and assessing the clonality of the corresponding processes, although it is not specific for any of the corresponding NHL entities [10]. In fact, the IGH split probe, other than in FL and MCL, identifies two distinct split signals in 14% of malignant mesothelioma cases, 13% of CLL cases, and 50% of DLBCL, 17% of SLL/CLL and 7% of marginal zone lymphoma cases [10,34]. Since the IGH split-signal DNA probe spans much of the constant and variable regions of the IGH, split signals should be generated even in cases of aberrant breakpoints. Another application of FISH/CISH assays is the identification of the so-called dual-translocation or double-hit lymphomas, which involve MYC and BLC2 rearrangements, with MYC thought to represent the second hit in a t(14;18)-positive DLBCL. These cases may have morphologic and immunohistochemical properties of a typical DLBCL, although no reliable morphologic features are seen on cytology [9,10,22,35]. Finally, double-hit lymphomas are usually characterized by complex karyotypes, highly aggressive behavior, and a poor prognosis [35]. MYC translocations are best documented using appropriate FISH probes rather than by PCR. In summary, on the basis of FNC/FC data, FISH may be conveniently used on LN FNC samples to achieve a definitive classification of NHL. The use of cytospin could optimize the preparations of multiple slides useful for multiple tests, including FISH assays. However, FISH chromosomal abnormalities are usually investigated when a specific pathological entity is highly suspected or has already been diagnosed since very few translocations are shared among different entities; therefore, specific chromosomal abnormalities are not routinely investigated to determine clonality in different B-cell lymphoproliferative processes [10].

PCR-based molecular tests are routinely used in lymphoproliferative processes for the identification, characterization and quantification of lymphomatous cells. The most common applications of PCR are the identification and quantification of translocations by qRT-PCR strategies, Ig and TCR rearrangements by clonality testing, and the identification of IGVH (IG variable segment of heavy chain) somatic hypermutations either by Sanger sequencing, single-strand conformational polymorphisms or RT-PCR strategies (fig. 1, 2). All these procedures are generally chosen based on the different requests, the availability of the corresponding technologies and the experience of each laboratory. Corresponding technologies have been used on any type of sample, such as paraffin-embedded tissues, fresh cells or tissues, and even cells obtained by FNC and cryopreserved or stored on FTA cards [8,36,37,38,39,40,41,42,43].

The most frequent NHL translocations identified by PCR assays are the t(14,18)(q32;q21), involving the BCL2-IGH loci, and the t(11;14)(q13;q32), involving the BCL1-IGH loci, which occur in 60-70% of FL and in 30-40% of MCL [43], respectively. The t(14;18) occurs in 90% of FL and 20% of DLBCL [43]. As a consequence of this translocation, the BCL2 gene from the 18q21 locus is placed under the control of the IGH-Em enhancer, resulting in the deregulation of the BCL2 apoptotic pathway. In particular, in most MCLs, the BCL1 breakpoints are clustered on an 85-bp region of chromosome 11, named as the major translocation cluster region, and JH breakpoints lie on IGH genes juxtaposing the IGH-Em enhancer to chromosome 11q13 sequences. As a result of the translocation, the cyclin D1 gene is constitutively activated [43]. PCR for the detection of both of these translocations is routinely used on embedded or frozen tissues and has also been successfully applied to FNC samples [43,44]. Because of consistent differences in primers, procedures and results utilized in different laboratories, BIOMED-2 was established. BIOMED-2 is a consortium of multiple institutions that has standardized the BCL1/JH and BLC2/JH tests by validating primers and PCR systems among the participants and many groups outside the consortium as well [43]. BIOMED-2 Multiplex PCR strategies utilize a consensus JH primer in combination with a single BCL1 primer and nine BCL2 primers, respectively achieving a sensitivity of 10^-3 and 10^-4 on agarose gel. In addition to having diagnostic purposes, these translocations are investigated to evaluate the minimal residual disease by qRT-PCR assay in NHL follow-up [45,46,47].

Ig and TCR antigen receptor gene rearrangements are the mostly applied targets for clonality testing in NHL [42,43,48,49,50]. Ig and TCR rearrangements occur in the earliest stages of B and T cell development by the random joining of many V, (D) and J genes in a unique V(D)J exon that encodes the final antigen-binding moiety of the Ig or TCR chain [51]. Therefore, each lymphocyte has a unique antigen receptor molecule on its cell membrane. Conversely, identical rearrangements cannot occur in independently generated cells but reflect the clonality of a lymphoid cell population. These differences largely dependent on the number of N sequences added by the terminal deoxynucleotidyl transferase, at the time of V-(N)-(D)-(N)-J rearrangement [43]. Monoclonal Ig and TCR gene rearrangements are distinguished from the polyclonal ones by the homogeneity of PCR size compared to the heterogeneity of the different-sized fragments in polyclonal cell populations, and distinguish benign from lymphomatous cell proliferations. Clonality analysis has been defined in the EuroClonality/BIOMED-2 consortium by the standardization of multiplex PCR assays for nearly all Ig/TCR targets (Igκ and TCRβ, incomplete IGH D-J and TCRB D-J rearrangements). These tests collectively show a high rate of detection in the most common B- and T-cell lymphomas [41,42,43,48,49]. EuroClonality/BIOMED-2 have also produced guidelines for the interpretation and for reporting results in suspected lymphoma [37]. In clinical practice, these procedures are generally used when histology and immunohistochemistry are equivocal or difficult to perform [37,43], but have also been used on cytological samples, including nodal and extranodal FNC samples [36,52,53]. In particular, IGH/TCR PCR can confirm FNC/FC diagnoses of lymphoma or assess polyclonality, adding clinical value to the FNC diagnoses of nodal and extranodal processes [40,54]. Mayall et al. [55] maintain that, because of its high efficiency and short turnaround time, IGH/TCR PCR may replace FC on FNC samples, whereas Davidson et al. [56] suggested that their combined use should be advisable because of a relatively high rate of monoclonality detection by PCR only. Maroto et al. [4] stressed that IGH/TCR PCR enhances the LN FNC and FC diagnosis and cautioned on the risk of false positives and negatives by using a single primer pair PCR amplification over seminested methods. False positives and negatives of IGH PCR also occurred on FNC samples in another early investigation [44]. Therefore, to avoid this inconvenience, the combined use of IGH and BCL2 PCR has been suggested [44]. Moreover, new procedures and EuroClonality/BIOMED-2 guidelines have improved PCR specificity, reducing dramatically the risk of false negatives and positives on both tissues and FNC samples [36]. TCR-PCR has been less utilized on FNC samples of T-cell NHL whereas corresponding diagnoses are generally more complex than those of the B-cell counterpart [52,53]. In particular, TCR-PCR may be extremely useful in cutaneous lymphoma staging by the corresponding NHL FNC [57] or in cerebrospinal fluid evaluation [52].

Another application of the PCR clonality tests is the identification of somatic hypermutations of Ig gene variable regions. These mutations are physiologically important in the affinity maturation of antibodies, and their frequencies in vivo are generally high enough to provide sufficient point mutations to generate a large number of different antibodies. IGVH mutations are characterized by single base-pair substitutions in the RGYW hotspot motifs, often resulting in amino acid changes. IGVH hypermutations have different clinical applications; in fact, they are considered a reliable prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL) because hypermutated patients have a longer survival than nonmutated B-CLL patients. The hypermutation rate of human BL is approximately 10-fold less than the normal [43]. Different methods for a rapid detection of mutated VH region genes have been developed, including single-strand conformational polymorphism, direct sequencing of PCR products and denaturing high-performance liquid chromatography [58,59]; however, to the best of our knowledge, there is no experience of IGVH testing on the FNC samples reported.

High-throughput technologies (HTT) encompass numerous and different new technologies that share the capacity of multiple molecular target investigations and sample processing in a fast and reproducible manner. The development of fast and simultaneous multiple molecular investigations has allowed the search for global genomic alterations responsible for the development and progression of different neoplasms, including lymphoma, with important clinical implications. The number and applications of these technologies are large and variable, as are their potential clinical applications. The most utilized HTT in biomedical research are gene expression profiling (GEP), comparative genomic hybridization (CGH), single-nucleotide polymorphism (SNP) arrays and next-generation sequencing (NGS) technologies (fig. 1, 2). An exhaustive description of these technologies is impossible in the present article, where only the general principles and applications are summarized.

GEP has been conceived for the study of RNA and DNA changes, including chromosomal copy number alterations, genotyping and epigenetic modifications [60]. GEP platforms consist of numerous oligonucleotide probes immobilized on a solid surface, hybridized with DNA/RNA samples [60,61]. The signal obtained by the fluorochrome-labeled DNA/RNA reflects the concentration of the corresponding transcript, can be quantified [60,62] and is able to measure a high number of genes simultaneously [60,61]. The resulting data can be analyzed and validated by bioinformatic tools and other technologies, such as PCR or immunohistochemistry, and in other additional independent series of samples. GEP analysis of NHL provides information on the corresponding different subtypes, revealing how each displays a unique gene expression program where the genes involved in different pathways are potential therapeutic targets [60]. The incorporation of GEP array information requires the extraction of good-quality DNA and RNA from tissues or blood, and often the need for fresh samples is a logistic challenge that is difficult to overcome in routine practice. Therefore, not surprisingly, FNC samples have been conveniently used for the GEP of different tumors [63,64], including lymphomas [65], exploiting the specific advantages of FNC - namely to harvest vital tumor cells avoiding stroma, fat and other nontumor components. GEP improves the current prognostic indexes based on clinical criteria, such as the International Prognostic Index (IPI) [60,66], because the corresponding GEP-based prognostic models are different in each pathological entity. Information generated by GEP may be translated into clinical practice when specific genetic or phenotypic features are associated with different GEP profiling. One of the major contributions of GEP has been the identification of two major subgroups of DLBCL, namely the germinal center type (GCB) and the activated B cell (ABC), since the gene expressions of germinal center cells are detected in GCB-DLBCL and the expression pattern of mitogenically activated B cells with a secretory function are detected in ABC-DLBCL [60,67,68]. The latter also shows the constitutive activation of the NFκB pathway through BCR signaling, with acquired activating mutations in CD79a, CARD11, and MYD88 and with inactivating mutations of the NFκB inhibitor A20, which are absent in GCB-DLBCL [60,69,70]. The different gene profiling tools determine prognostic and predictive differences, with relevant clinical impacts that may be reproduced by specific immunophenotypic profiles utilized to classify single cases in clinical practice [71,72]. GEP data need to be confirmed by other analyses, including NGS or in vitro studies in which the target genes are cloned into cell lines to evaluate their effective role in the start and/or progression of NHL. Bodor et al. [73] compared and confirmed the FL expression data obtained by the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) using NGS. The high-throughput sequencing showed that the mutations in the SET domain of the EHZ2 gene act in a dominant fashion by increasing H3K27 trimethylation, which confers a gain of function and involves EHZ2 in tumorigenesis. These SET mutations are stable during disease progression, and thus represent a good therapy monitoring target [73]. Again dealing with GEP data confirmation, Iqbal et al. [74] confirmed the MYC target microRNAs in GCB- and ABC-DLBCL, identified by GEP signature, direct sequencing and in vitro studies. The in vitro analysis confirmed that the high expression of miR-155 is associated with R-CHOP treatment failure and suggests different treatment options for resistant DLBCL. Finally, the information generated by GEP may be confirmed and translated into clinical practice by FISH, qPCR or other mRNA detection techniques [60,75].

The CGH technique is carried out with high-density long (50-75 mer) oligonucleotide arrays, labeled with different fluorochromes, covering the whole genome with probes spaced about 1-5 kb apart. Oligonucleotide arrays competitively hybridize the normal chromosome metaphase of tumoral DNA and reference normal DNA of the same gender (possibly from the same individual). The different intensity of the hybridized tumoral and normal DNA signals indicates the gains or losses in specific chromosomal regions. In this manner, CGH detects unbalanced DNA copy number changes and small alterations, such as copy number variants (CNV) - considered gains or losses in tumoral DNA. CNV are DNA segments of 1 kb or larger, in single-copy tandem duplication or in complex gains/losses/inversions of homologous sequences. CNV are present in a variable copy number between tested DNA and a reference genome. CNVs are responsible for most individual genetic variations [60]. The systematic analysis of chromosomal imbalances by GCH has shown that most NHL carry a high number of secondary chromosomal alterations other than those targeting specific oncogenes, such as BCL2, MYC and CCND1 [60]. These secondary chromosomal alterations seem to be specific for different NHL entities and even for different patients suffering from the same NHL. Therefore, CGH might have a role in defining the biological behavior of different patients suffering from the same NHL subtype [60,76]. CGH has shown frequent deletions in 13q and 11q, gains of the 12 and 3q and losses of 1p chromosomes in MCL [60,77]. In CLL, CGH has shown the same deletions of MCL, but not the gains of 3q and the losses of 1p [60,77]. In ABC-DLBCL, CGH has shown gains or trisomy of 3 and gains of 18q chromosomes [60,78,79]. In GCB-DLBCL, CGH showed the gains of 2p chromosome that are uncommon in ABC-DLBCL. Differences in genomic profiles have also been observed in different types of peripheral T-cell and NK cell NHL [79]. Like other types of HTT, CGH needs high-quality DNA that can be easily obtained by FNC. Consequently, FNC has been used to harvest cells from different tumors to perform CGH [80,81,82], whereas no experiences on LN FNC CGH are so far available.

SNP arrays use short oligonucleotides (25 mer) as probes to distinguish different genotypes on the basis of the different number and distribution of SNPs. Signals of the same areas from different genomes are unique because they are differently covered by corresponding probes, which are able to identify CNV, uniparental disomy (UPD) and DNA copy-neutral loss of heterozygosity. UPDs are different chromosomal alterations that may occur through different mechanisms. The most frequent UPD is generated by the deletion of one allele and the correction of the defect by the duplication of the remaining allele. Consequently, the mutated allele is reduced to homozygosity after the deletion of the normal allele and the duplication of the mutated one [60]. The DNA copy-neutral loss of the heterozygosity is represented by stretches of DNA where both strands are identical and, therefore, all the SNPs are homozygous. The reduced intensity of the hybridization signal identifies the presence of two identical strands of DNA in the regions covered, revealing the homozygosity generated by the deletion of the corresponding chromosomal region. SNP arrays on many NHL types have revealed the following: (1) the deletion of several genes of the Hippo signaling pathway in MCL, with loss of proliferation and apoptosis control [60,83]; (2) the deletions at known chromosomal fragile sites that confirm the NHL genomic instability [60,84]; (3) the significantly high CNV and segmental duplications in the regions flanking somatic UPD in MCL, suggesting that these regions may facilitate DNA recombination; (4) the inactivation of TP53 or CDKN2A in 17p or 9p21, respectively, with direct implications for the patient's outcome [60,77,79], and (5) large gains of chromosome 3 in ABC-DLBCL and MCL [60,77,79]. As a consequence, SNP arrays in NHL show that genomic complexity is an important and independent prognostic parameter [60,77]. To avoid missing the small clones that are important in the evolution of NHL, high-quality DNA is needed, which may be obtained by blood spots on FTA Whatman cards [85] or by FNC [86]. Consequently, FNC has been used to harvest cells from different tumors, such as choroidal melanoma, where high-density SNP arrays were more effective than FISH in detecting chromosome 3 aberrations and monosomy [86], although no experience with NHL is so far available.

NGS technologies are a fast, relatively inexpensive and versatile tool with which to analyze the mutational spectrum in NHL [60]. All the methodologies developed require DNA fragmentation, subsequent amplification of generated multiple fragments and their simultaneous sequencing in parallel, producing millions of sequenced reads for each given position of the genome, which are then aligned against the reference genome. The number of reads per stretch of DNA is called the coverage [60]. A high coverage is useful to detect tumor mutations in samples where there is some contamination of the normal cells, as in NHL, since it is able to filter out errors and noise. A high coverage may generate bias that can be avoided using reliable bioinformatic algorithms to interpret the sequences. A number of reads above or below the mean coverage per DNA region indicates the presence of gains, amplifications and hemizygous or homozygous deletions [60]. In addition to these large structural alterations, NGSs detect translocations, single nucleotide changes, somatic mutations, individual polymorphisms, small insertions or deletions (indels) using whole genome, whole transcriptome (WT), exome (specific regions of the genome including all coding exons) and specific-targeted genomic region sequencing strategies [60,87]. WT or RNA sequencing starts with mRNA, total RNA, microRNAs, or other RNA cDNA, and quantify transcripts, disclose new fusion transcripts or alternative splice form transcripts. Exome and specific-targeted genomic regions are based on a selective capture of the genomic fragments of interest using tagged complementary oligonucleotides and can be used to minimize costs and increase speed by reducing the volume of data to analyze. All NGS methodologies can be performed on different platforms, such as Ion Torrent (Applied Biotechnologies), the 454 GS-Junior (Roche), semiconductor-based Ion Personal Genome Machine Sequencer, HiSeq 2000 Illumina, GAIIx Illumina, MiSeq Illumina, and others. The data obtained with NGS need to be confirmed by additional functional and clinical investigations. NGS WG, WT, exome and specific-targeted genomic region sequencing have been carried out in CLL [60,87,88], HCL [60], FL [60,89], DLBCL [60,89] and plasma cell myeloma [60,90]. These studies have shown that in most NHL cases the mutations lie in a few genes, tend to cluster in common pathways depending on the NHL type and cell of origin, are not specific, and that the same mutations may occur in different NHL subtypes. In CLL, NGS has demonstrated that mutations in the genes of NOTCH1 signaling, RNA splicing, and processing machinery, inflammatory response, DNA damage and cell cycle control, and WNT pathways are differently distributed in IGVH-mutated and unmutated CLL. Conversely, IGVH hypermutations are equally distributed [60]. In DLBCL, NGS has confirmed the mutational data in ABC concerning CD79b, MYD88, A20 inhibitor, BCR signaling and NFκB pathway mutations, and in GCB concerning BCL2 and EZH2, obtained with the HTT described above [60,89]. NGS has also highlighted new mutations in the histone methyltransferase MLL2 (in 32% of DLBCL and 89% of FL cases) [60]. Other mutations detected lie on EZH2, MLL2, CREBBP, BRAF, B2M and EP300 (involved in the chromatin remodeling pathway). Other mutated genes identified by NGS are involved in the postgerminal center differentiation program, in protein translation machinery (including genes of the unfolded protein responses) and in mechanisms of the normal plasma cell secretor function [60,90]. In T-cell NHL, NGS has shown a high mutation rate in STAT3, causing additional neutropenia and rheumatoid arthritis [60,91]. Many of the mutated genes have become therapeutic targets, as is the case of the BRAF inhibitors used in NHL where this gene is mutated [60]. Like in all other HTT, NGS requires good-quality DNA and RNA from tissues, blood or fresh samples, which are difficult to obtain in routine practice. Therefore, different NGS platforms use FNC to harvest cells from different tumors [92,93,94,95,96,97,98,99,100]. These studies demonstrated that NGS-based mutational profiling can be performed with a few nanograms of DNA (∼40 ng/μl), which can be easily obtained from FNC in different tumors [93,94,95,96,97,98]. Therefore, NGS may enhance the molecular FNC potential, providing mutational information on genes with high diagnostic and predictive relevance, such as EGFR, BRAF, K/N/HRAS, KIT, PTEN, CDKN2A, just to mention some, thus contributing to personalized therapies.

The application of all the molecular procedures described above requires a sufficient amount of cells and corresponding good-quality genetic material. As previously reported, 10-40 ng of DNA/RNA generally represent a sufficient amount to perform any molecular study. In clinical practice the main problem is represented by the number of cells obtained by FNC and the corresponding genetic content. Corresponding amounts are not constant and depend on different factors, such as the size of the needles, the FNC technical procedures and the nature of the lesions. It has been calculated that, using 23-gauge needles, a mean of 4 × 10⁶ cells is obtained by LN FNC, 2.5 × 10⁶ from breast carcinoma and 1.65 × 10⁶ from thyroid carcinoma [101]. Considering that 40 ng of DNA/RNA may be obtained from 4 × 10⁶ cells, a couple of additional passes from LN FNC may be sufficient to obtain genetic material sufficient for any procedure. Considering the technical support, genetic material has been successfully obtained from different cytological supports, such as smears, cell blocks or cryopreseved cells [7,36,102,103]. This variability in technical supports is due to the usage of archival material or FNC performed by clinicians for routine diagnostic purposes that is only subsequently utilized for retrospective molecular studies. In the case of LN FNC, cytopathologists should personally perform FNC, ROSE, additional passes and the disposal of diagnostic material. In this way the same material may be capitalized for different basic ancillary techniques (ICC, FC, FISH) and stored on different supports depending on the needs and the technical availability of the single laboratories [36,104].

Despite their high number, not all NGS-identified alterations can play a role in lymphoma genesis or can be used as biomarkers for therapy monitoring, prognostic and predictive evaluation, or as therapeutic targets. Moreover, to reduce costs and analysis time to make the detection of the new molecular alterations compatible with the routine diagnostic purposes, many other strategies have been set to rapidly analyze any molecular alterations in a high number of samples. These strategies are often conceived as detection of the targets by qRT-PCR amplification followed by Sanger sequencing. Other procedures discriminate samples harboring molecular alterations by DNA/RNA multiple quantitative amplifications, allele-specific PCR-based evaluations or fragment analysis discriminations. Park et al. [105] used a multiplex real-time PCR method that incorporates melting curve analysis (Real-Q assay) to detect the BRAF V600E mutation in FNC samples of papillary thyroid carcinoma. The results obtained were comparable to those produced by an allele-specific PCR-based kit using dual-priming oligonucleotides (AS-PCR). da Cunha Santos et al. [8] used DNA retrieved from lung carcinoma FNC stored on Whatman FTA cards to detect small in-frame deletions in EGFR exon 19, using fragment analysis of fluorescently labeled PCR products subjected to ABI genetic analyzer capillary electrophoresis and sequencing. The same samples were then used to detect the EGFR exon 21 L858R mutation and exon 2 deletion of KRAS by direct sequencing. This study demonstrated that FTA cards could maximize and simplify sample storing for multiple mutational analyses providing high molecular weight DNA in sufficient amounts and quality. Saieg et al. [7,38] and da Cunha Santos et al. [106] investigated the mutational status of EZH2, CD79B and MYD88 in B-cell NHL using DNA from FNC stored on FTA cards [7,38] and DNA extracted from archival cytospin preparations previously tested at the time of diagnosis for the presence of MYC rearrangement and/or IGH/BCL2 translocation [106]. Samples harboring mutations were identified by MassARRAY spectrometry followed by Sanger sequencing. MassARRAY is a high-throughput multiplex spectrometry that permits mutation profiling and SNP genotyping by the simultaneous detection of multiple mutations. In conclusion, the application of high-throughput multiplex platforms, other than NGS, on minimal samples, such as FNC samples, reinforces the idea that these tests exponentially increase sample availability for molecular analysis and may facilitate future studies on NHL-related molecular events, in addition to assisting the design of individualized therapies.