The results of the cytologic examination on the FNAB material provide the basis for the further clinical assessment. However, the assessment of the cells can be challenging in some instances and assessed into the Bethesda Class III (indeterminate), or class IV (suspicious of follicular neoplasm), thereby resulting in a resection where more than 70% are benign nodules (**Table 3**)[19].

Hence, the use of molecular markers in routine assessment of such nodules would be helpful to assess the risk of malignancy, as recommended in the second edition of Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) and in the American Thyroid Association(ATA) guidelines. Additionally, the introduction of a new diagnosis by the WHO in 2017 is an important aspect in the diagnosis of thyroid cancer and included a new section termed "other encapsulated follicular patterned thyroid tumors" (**Figure 1**) [19, 20].

In lesions like the (NIFTP), the features of invasion are required to exclude the malignancy, and the cytomorphologic features alone are not helpful. Hence, the use

**Table 3.**

*Bethesda system for reporting thyroid cytopathology (TBSRTC) [18].*

*Recent Advances and Researches in the Field of Fine Needle Aspiration Cytopathology DOI: http://dx.doi.org/10.5772/intechopen.110854*

**Figure 1.**

*Preoperative management of thyroid nodules [19, 20]. (AUS-Atypia of unknown significance, FLUS-follicular lesion of unknown significance).*

of molecular markers is indicated to assess the malignancy in these lesions to avoid an unnecessary surgical treatment.

The use of immunohistochemical (IHC) markers that assess the malignancy in the lesions would increase the sensitivity of FNAC. The postulated markers included galectin 3, HBME, fibronectin 1 and cytokeratin 19, which proved insufficient for assessment of the biopsy material. Hence, the molecular markers become the next option for assessment of these lesions.

One of the first molecular markers to be used was the commonly observed molecular markers in papillary thyroid carcinoma (PTC) such as BRAF gene mutations, RET and NTRK rearrangements or PAX8/PPARγ fusion characteristic of follicular tumors.

Multi gene panels with mutations characteristic of follicular and papillary thyroid cancers like point mutations within BRAF, KRAS, HRAS and NRAS for follicular and RET/PTC1,RET/PTC3 and PAX8/PPARγ rearrangements for papillary carcinoma, respectively. The sensitivity of the techniques is further improved with the introduction of the oligonucleotide arrays and next-generation sequencing (NGS), which allows the assessment of many gene alterations in one analysis. The classifiers are grouped into two broad groups—rule out a malignant lesion "Rule out test" or to confirm a malignant lesion "Rule in test" determined by the negative predictive value (NPV) or the positive predictive value (PPV). The higher negative the NPV, the higher probability of benign lesion. Similarly, the higher positive the PPV, the higher probability of malignant lesion (**Table 2**, **Figure 1**) [21].

The use of ultrasound guided biopsies has become a common practice in all nodules measuring >10 mm [7].

Rapid on site evaluation (ROSE) is a useful adjunct to the thyroid FNAC for the specimen adequacy and diagnosis. A comparison of the ROSE and non-ROSE groups improves the specimen adequacy with a decrease in the number of needle passes [8].

Hurthle cell predominant nodules present with a confounding picture on FNAC in terms of their malignant potential and hence assigned a Bethesda Category III or IV. Thyroseq V3 molecular profiling can be used for tailoring the surgical management of the Hurthle cell neoplasms. A molecular profiling with ThyroSeq V3 helps in predicting the malignant potential of these nodules [22].

Mutation of BRAF gene occurs almost exclusively in papillary thyroid carcinoma. (PTC), which can be detected by the IHC marker BRAF(V600E) [23].

Low-cost qualifiers based on the PCR method which support FNAC are being developed which need to be validated.

Telomerase activity is shown to distinguish benign from malignant lesions in various studies. Telomerase activity with hTERT gene expression is seen in malignancy along with in few inflammatory conditions like lymphocytic thyroiditis and follicular adenomas in some cases.

Molecular testing is a useful adjunct to surgical decision making. Mutational profiling of thyroid nodule, and mainly the BRAF and RAS can be done using droplet digital PCR. Gene expression profiling by RT-PCR helps in the distinction of benign from malignant thyroid nodules. Upregulation of genes like (extracellular matrix protein (ECM1) and transmembrane protease serine 4 (TMPRS4) mRNA was determined to be an independent predictor of malignant thyroid neoplasm [24]. Classification of the thyroid neoplasms can be done on cell blocks using FISH [25].

Other molecular tests are being increasingly used which can help in differentiating benign from the malignant nodules more accurately. The Afirma gene sequencing classifier (Thryoseq v2) and other molecular tests are based on micro-RNA alterations [26, 27]. The tests can help in improving the diagnosis for appropriate therapy. ThyGEnX/ThyraMIR is another combination test waiting for validation.

TERT promoter mutations have a diagnostic and prognostic significance in the thyroid FNAC. TERT mutation positive nodules might be indeterminate on FNAC and present with aggressive clinicopathological behaviors like extra thyroid invasion, lymph node metastases, distant metastases, disease recurrence or patient death, underscoring the importance of the TERT analysis.

Telomerase activity is shown to distinguish benign from malignant lesions in various studies. Telomerase activity with hTERT gene expression is seen in malignancy along with in few inflammatory conditions like lymphocytic thyroiditis and follicular adenomas in some cases [28].

Use of analytical chemistry procedures allows for the potential recognition of cancer-based metabolites for the purposes of advancing the era of personalized medicine. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) coupled with separation techniques, e.g., gas chromatography (GC) and liquid chromatography (LC), are the main approaches for metabolic studies in cancers [29].

The immense metabolite profiling has provided a chance to discover novel biomarkers for early detection of thyroid cancer and reduce unnecessary aggressive surgery.

An assessment of the cyclooxygenase-1 and 2 gene expression levels in chronic autoimmune thyroiditis, papillary thyroid carcinoma and non-toxic nodular goiter *Recent Advances and Researches in the Field of Fine Needle Aspiration Cytopathology DOI: http://dx.doi.org/10.5772/intechopen.110854*

showed that an up-regulation of cyclooxygenase-2(COX-2) activity is associated with the papillary thyroid cancer (PTC) [30].

The metabolic alterations in thyroid cancer can serve as potential therapeutic targets. Altering the balance between cancer and stromal cells might serve as a promising therapeutic strategy [31].

#### **4.6 Lymph nodes**

PCR for selected markers has been studied for their utility in increasing the sensitivity of cytomorphology for detecting the metastasis and their histogenesis, particularly pertaining to the endoscopic ultrasound (EUS)-guided FNAB, and detection of micro-metastasis.

Various studies involving the aberrations in genes like the Cyclin D1 by FISH for squamous cell carcinoma and FNA-PCR for tyrosinase to detect metastasis in melanoma [1, 3, 32].

The results could serve as an independent marker for prognosis, tumor aggressiveness and recurrence.

#### **4.7 Pancreatic neoplasms**

The EUS-FNA for the diagnosis of pancreatic neoplasms has the potential drawbacks of a paucity of diagnostic material along with the overlapping features of a low-grade tumor and reactive processes like chronic pancreatitis [33].

Franseen Needle used for EUS-guided FNAB has a role in tissue acquisition for study of microsatellite instability in unresectable pancreatic lesions, required for a treatment with the immune checkpoint inhibitors for the unresectable pancreatic lesions [34].

Mutations of the K-ras oncogene have been most frequently studied as an adjunct to conventional cytology to aid in the diagnosis of pancreatic adenocarcinoma. Other markers being studied include PLAT by RT-PCR and Lipocalin 2.

An expression of S100P, IMP3 and Maspin with a non-expression of von Hippel-Lindau gene product (pVHL) were significantly correlated with pancreatic ductal adenocarcinoma (PDAC) [35].

#### **4.8 Renal neoplasms**

Molecular tests have been studied as an ancillary adjunct for the diagnosis of malignancy in renal FNAB. However, a very few reports are available on the same [1, 3].

#### **4.9 Infectious diseases**

A PCR is one of the most popular diagnostic adjuncts in addition to the cytomorphology with microbiologic staining and cultures for the diagnosis of tuberculous lymphadenitis. Molecular tests can be used to increase the rate of detection and identify the resistant strains [1].

Various studies have advocated the use of RT-PCR to detect M. Tuberculosis with a higher sensitivity. Primers and Probes for the real-time PCR were designed on the basis of the internal transcribed spacer sequence, enabling the recognition of Mycobacterium Avium and Mycobacterium Tuberculosis. The tests showed no false

positive with other mycobacterial species and other pathogens causing lymphadenitis. The technique needs no hybridization or further processing time for analysis [36].

Even in the absence of epithelioid granulomas/necrosis, Avidor et al. developed a PCR method for amplification of Bartonella Henselae DNA for an accurate diagnosis of Cat Scratch disease, obviating the need for excisional biopsy [1].

Starac et al. reported the presence of human papilloma virus (HPV) DNA by PCR which can be successfully performed on FNA material. This can guide the clinicians in assigning the site of origin of metastatic squamous cell carcinoma, since the anogenital SCC has a high prevalence of high-risk HPV DNA particularly in the cervix [37].

#### **4.10 Salivary glands**

FNAB has a well-established role in the evaluation of salivary gland tumors. However, they have a significant morphologic diversity with a confounding presentation on FNAC which may not allow a specific diagnosis in many cases, making it a challenging area in cytopathology.

There has been an emergence of newer entities in the salivary gland tumors over the last decade, along with the characterization of specific translocations in a subset of these tumors.

Mammary analogue secretory carcinoma is a recently described entity characterized by a t(12;15)(q21;p13) translocation resulting in ETV6-NTRK3 fusion. Hyalinizing clear cell carcinoma is a low-grade tumor with infrequent nodal and distant metastasis, shown to harbor EWSR1-ATF1 gene fusion. The CRTC1-MAML2 fusion gene resulting from a t(11,19)(q21:13) translocation is now known to be a feature of mucoepidermoid carcinoma associated with an improved survival. A t(6,9) (q22–23:p23–24) translocation resulting in MYB-NFIB gene fusion has been identified in majority of adenoid cystic carcinomas.

Polymorphous low-grade adenocarcinoma is characterized by hot spot E710D mutations in PRKD1 gene, whereas the cribriform carcinomas are characterized by translocations in PRKD1–3 genes.

Notably, salivary duct carcinoma is a high-grade adenocarcinoma with the molecular and morphologic features similar to the invasive ductal carcinoma of breast, including the HER2 gene amplification, mutations of TP53, PIK3CA and HRAS and loss or mutation of PTEN. A subset of SDC with apocrine morphology is associated with overexpression of the androgen receptors [38].

As these mutations are recurrent, they serve as powerful diagnostic tools in the salivary gland tumor diagnosis and refinement of salivary gland cancer classification, along with serving as promising prognostic biomarkers and targets of therapy.

Although the molecular consequences of these translocations and their potential prognostic and therapeutic values are not well characterized, the resulting fusion oncogenes and oncoproteins can be used as diagnostic clues in salivary gland FNAB material in order to overcome the limitations of the cytomorphological evaluation alone [33].

#### **4.11 Upper gastrointestinal lesions**

Automated multiband imaging system can be used for EUS-FNA biopsy specimen for the evaluation of upper gastrointestinal subepithelial lesions.

Sample isolation processing by spectromicroscopy (SIPS) has been introduced as an alternative to rapid on-site cytologic evaluation, which is a useful but complicated procedure.

*Recent Advances and Researches in the Field of Fine Needle Aspiration Cytopathology DOI: http://dx.doi.org/10.5772/intechopen.110854*

Automated multiband imaging system (AMUS) helps in calculating the whitish core amounts in EUS-FNA samples in patients with subepithelial lesions, which is found to be more useful than SIPS for an on-site evaluation for the gastrointestinal SEL [39].

#### **5. Conclusions**

The effectiveness of FNAB for rendering a specific diagnosis can be improved tremendously by the application of several ancillary modalities. Although in many cases cytomorphologic features alone might be sufficient for making a diagnosis, the use of ancillary tests is often necessary not only for rendering a specific diagnosis but, where malignant lesions are involved, for determining prognostic and predictive factors from the procured aspirate. Most currently available ancillary techniques can be used on FNAB specimens. Immunocytochemistry performed on direct smears, monolayered preparation and cell block sections of FNAB is the most commonly utilized ancillary technique. This can be widely routinely used on FNAB specimens for determining the organ of origin of a metastatic tumor, for classification and typing of tumors and for determining prognostic and predictive markers.

There are, however, no immunostains that can help in the distinction of benign from malignant lesions on FNAB. In comparison with immunocytochemistry, molecular tests can aid in the distinction of benign from malignant lesions, in determining the genetic abnormalities and genetic makeup of tumors that can be useful not only for making a more specific diagnosis but also for determining prognosis, response to therapy, and determining the presence or absence of specific molecular targets for selection of patients for targeted therapy.

The emerging concepts and technologies have a potential to affect the field of cytopathology and clinical practice. Although many of the advances are in a trial phase, further researches in the fields like FAST-FNA, Kisher image analysis, along with a biomarker cocktail would be useful to predict treatment options, targeted drug therapy and outcomes.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Abbreviations**

*Advances in Fine Needle Aspiration Cytopathology*

### **Author details**

Anjali Goyal Department of Pathology, Smt NHL MMC Ahmedabad, Gujarat, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Recent Advances and Researches in the Field of Fine Needle Aspiration Cytopathology DOI: http://dx.doi.org/10.5772/intechopen.110854*

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#### **Chapter 4**

## Lymph Node Cytology: Morphology and Beyond

*Meeta Singh, Kirti Balhara, Deepika Rana, Rabish Kumar, Nimisha Dhankar, Shabnam Singh, Priyanka Bellichukki, Sreoshi Paul and Sathiyanesan Mariana Chartian*

#### **Abstract**

Fine needle aspiration cytology (FNAC), being minimally invasive, rapid, cost-effective provides a valuable first-line diagnostic tool in the evaluation of lymphadenopathies both benign and malignant. Various ancillary techniques namely immunocytochemistry, flow cytometry, cell blocks, and molecular studies further improve the diagnostic accuracy of FNACs. Targeted FNAC under ultrasound guidance optimizes cellular yield in palpable and non-palpable lymphadenopathies. FNAC proves to be indispensable at establishing tissue diagnosis in cases when surgical excision is unfeasible, as in elderly patients with comorbidities or in metastatic settings. Nevertheless, lymph node FNAC represents a daunting task owing to the multitude of benign and malignant causes of lymphadenopathy. To aid categorization and better communication to the clinician, an emphasis on classification and reporting of lymph node cytopathology using Sydney system is laid upon.

**Keywords:** fine needle aspiration cytology, lymphadenopathy, non-neoplastic, neoplastic, ancillary techniques, Sydney system

#### **1. Introduction**

Lymph node enlargement can occur due to a variety of causes including benign, primary neoplastic, and metastatic. Fine-needle aspiration cytology (FNAC) provides a valuable tool in the diagnostic workup of lymphadenopathy due to a variety of causes namely reactive, infective, metastatic, and neoplastic. It is rapid, accurate, reliable, economic, safe, and more or less painless. FNAC can provide material for various ancillary investigations such as flow cytometry (FCM), immunocytochemistry (ICC), microbial culture, cell block, and molecular studies.

#### **1.1 Normal anatomy and histology of lymph node**

The lymph node (LN) is a bean-shaped, capsulated spongy sieve, which filters the lymph entering via the afferent lymphatics on the convex side and flowing out via the efferent lymphatics through the hilus [1]. The lymph node is a secondary lymphoid

organ along with spleen, mucosal, and cutaneous lymphoid tissues. Thymus and bone marrow constitute the primary lymphoid organs [2].

LNs are aggregates present along the lymphatic channels. The LNs are situated at strategic sites in our body which helps in a quick and effective immune response [3]. The lymphocytes enter the LNs through blood or afferent lymphatics and get organized in T- and B-cell areas in the LN similarly, antigens enter the LN via afferent lymphatic channels, and are presented to the lymphocytes by dendritic cells (DC) which are the most efficient antigen-presenting cells (APC) [4].

The LN is divided into compartments by the connective tissue trabeculae which provide the framework for circulatory sinuses which are lined by the endothelial cells and divided into three regions: cortex, medulla, and paracortex [1]. Cortex is the area beneath the capsule and medulla is toward the hilus; cortex and medulla represent Bcell region. The paracortex is the area between cortex and medulla; represents T-cell region having a CD 4:CD 8 ratio of 3:1 along with presence of DC [1]. The cortex, depending on the activity of the LN, contains primary as well as secondary follicles. Primary follicles (unstimulated node) have small resting B cell aggregates and secondary follicles (stimulated LN) have an outer narrow mantle zone (containing B lymphocytes) and an inner germinal center (containing centrocytes and centroblasts) [5]. **Table 1** highlights the antigen expression of B lymphoid cells according to the stage of maturation. **Table 2** highlights the antigen expression according to their location.

#### **1.2 Approach to lymph node FNAC**

#### *1.2.1 Clinical history and examination*

A prior detailed clinical history is required in each case to correlate the cytological findings. Patient's age, duration of LN enlargement, history of fever, malaise, significant weight loss are all pertinent to be included. Any previous history of malignancy, surgery, or prolonged illness along with relevant blood and radiological investigations

**Table 1.**

*Immunohistochemistry (IHC) of B lymphocytes according to their maturation.*

**Table 2.**

*IHC of cells according to location in LN.*

*Lymph Node Cytology: Morphology and Beyond DOI: http://dx.doi.org/10.5772/intechopen.110607*

if available should be duly noted. The site, size, consistency, mobility, and number of LN involved should be carefully palpated and recorded. Any LN >2 cm is significant. Supraclavicular lymph nodes mostly harbor metastatic disease as compared to posterior cervical which is often reactive [6]. Soft, tender lymph nodes are seen in the inflammatory process while hard, fixed tend to be often malignant whereas rubbery consistency is suspicious of lymphoma.

#### *1.2.2 Sample collection and cytologic preparation*

Peripheral LNs can be easily palpable, thus FNAC of such LN is a simple procedure, which can be done blindly, preferably by a cytopathologist and does not require any local anesthesia. Site of FNAC is properly palpated, node fixed between two fingers of one hand and needling followed by aspiration done using the other hand. The needle used should be 22-24G and hand movement be brisk to avoid dilution of sample with blood since LNs are highly vascular structures (**Figure 1**). Once aspirated, the material should be checked for color and consistency as this provides a valuable information about the disease process. For example, infective lesions such as tuberculosis yield a thick purulent aspirate while that involved by lymphoma usually yield blood mixed aspirate. A necrotic aspirate is often seen in large metastatic LNs with areas of tumor necrosis. Aspirating only the cystic necrotic focus in such cases leads to a misdiagnosis if the firm areas containing viable tumor cells are missed. It is therefore important to completely aspirate the purulent material followed by needling of the firm areas. The sample obtained is gently spread over a glass slide with the help of another slide in a smearing fashion. Both air-dried and wet fixed smears are prepared and stained with routine stains such as May-Grunwald Giemsa (MGG), Papanicolaou (PAP) stain, and Hematoxylin& Eosin (H&E) stains. Extra smears can be air-dried

**Figure 1.** *Needle aspiration of lymph node by using 24-guage needle with plunger.*

and fixed with methanol for further usage. Additionally, cell blocks must be prepared for ancillary studies.

#### *1.2.3 Ancillary techniques*

Ancillary studies in LN aspirates include cell block preparation, special stains, immunocytochemistry (ICC), flowcytometry (FCM), molecular studies, and electron microscopy (EM). Adding to the advantages of FNAC, the ancillary studies help to arrive at a diagnosis, almost always, thus avoiding the need for an open biopsy. Cytology report when combined with the reports of ancillary techniques is well accepted by the clinicians and other cytopathologists [7].

It is, thus, advisable to always aspirate extra material which could be further helpful in performing the ancillary techniques. The cases are triaged based on examination of the MGG, PAP, and H&E-stained slides. In cases which are suspected to be infective, mycobacterial, or fungal in origin, special stains such as Ziehl-Neelson (ZN) stain and Periodic Acid Schiff (PAS) stain along with culture and PCR can be performed [5].

In cases of clinically suspected metastasis to the lymph nodes, cell blocks are prepared using various methods such as plasma thrombin method, fixed sediment method, scraping of cytology smears, histogel method, gelatin embedding method, colliding bag method, albumin method, bacterial agar method, and many more [8]. Cell blocks are micro biopsies embedded in paraffin section using the FNA technique. The residual clot or tissue in the hub of the needle is carefully removed and rinsed in 10% buffered formalin, centrifuged at 4000 rpm for 6 mins to form pellet. Supernatant is discarded and the sediment is placed on filter paper and processed as a routine histopathology specimen. Cell blocks are useful in better preservation of the morphology, architecture, and further help in performing IHC to find the primary [8, 9].

When evaluating a case of lymphoproliferative disorder, FCM and ICC are both helpful. FCM helps in immunologically characterizing the aspirated cells which are suspended in borate buffered saline (BBS) solution at pH 7.4. For complete characterization, cell concentration of approximately 1 million cells per ml buffer is required. FCM utilizes the principle of light scatter which measures the physical properties of a cell. Forward scatter (FS) depicts cell size and side scatter (SS) depicts cytoplasmic complexities. The fluorescence data is collected by a photodetector. Various antibody panels are used depending upon the cell morphology on giemsa stained smears.

In suspected cases of B-cell lymphoma antibodies which may be used are CD5, CD10, CD19, CD20, kappa, and lambda. In cases of suspected T-cell lymphoma CD 3, CD4, CD8, CD5, CD2, and CD7 are utilized. For immature blast identification TdT, CD34, HLA-DR, and CD38 are helpful markers. FCM provides the advantage of performing discrete measurements on millions of cells in a single sample.

FCM has a rapid turnaround time as compared to ICC, however, the morphology and staining characteristics are more preserved in ICC on cell blocks. Also, large cells, such as Reed-Sternberg (RS) cells, are better appreciated by ICC/IHC on cell blocks [9].

Transmission electron microscope (TEM) uses a fine electron beam, created by a tungsten filament heated by a high-voltage, electric current-heated and focused by magnetic lenses. The electron beam passes through an ultrathin plastic section in which tissue and cellular components have been post-fixed in osmium and impregnated with heavy metals such as uranyl and lead. A differential transmission of electrons occurs due to difference in densities of these heavy metals. An image is then *Lymph Node Cytology: Morphology and Beyond DOI: http://dx.doi.org/10.5772/intechopen.110607*

captured on a fluorescent screen and viewed through a binocular microscope. It enables the visualization of details not apparent via light microscopy. TEM has an important role in cytopathology. Cells in effusions and FNAC samples can be readily prepared for TEM. It allows easy distinction of epithelial cells from mesothelial cells. They can be optimally fixed by directly injecting the aspirate into glutaraldehyde and treated as a cell suspension. As compared to IHC stains, TEM often is at least as specific and is also more economical in solving a diagnostic problem in specific conditions [10].

Molecular studies such as fluorescence in situ hybridization (FISH), Polymerase chain reaction (PCR), mutation analysis, laser-assisted microdissection (LMD), and DNA sequencing (classical and next-generation sequencing [NGS]) are valuable tools for the detection of genetic changes and hence provide improved early diagnosis of malignancy, prognostication, and prediction of response to therapy [11]. Detailed use of these studies will be discussed in subsequent sections.

FNAC from LN, benign or malignant, usually have a high cell count. The aspirates show lymphoid cells having fragile cytoplasm along with the presence of cytoplasmic fragments in the background called "lymphoglandular bodies," measuring around 8 microns [1, 12]. They are round, small, pale and basophilic and their presence is important to identify the lymphoid origin of any lesion [1]. The cells seen in a normal LN lesion include mature lymphocytes (B or T phenotype), plasma cells, centrocytes (B cells, with cleaved nucleus), centroblasts (larger than centrocytes, marginal nucleoli), immunoblasts (largest of all lymphoid cells, with an eccentric round nucleoli) and macrophages [5].

#### **2. Non-neoplastic lymphadenopathy**

#### **2.1 Reactive lymphoid hyperplasia**

Reactive or follicular lymphoid hyperplasia is a common cause of lymphadenopathy, particularly in the younger age group and children. FNAC smears are highly cellular comprising a polymorphous population of small lymphocytes, follicular center cells (centrocytes and centroblasts), immunoblasts, plasma cells, dendritic cells, tingible body macrophages, occasional eosinophils, and polymorphs in a background of lymphoglandular bodies. It is divided into germinal center type, paracortical type, mixed type, and histiocytic reactions.

#### **2.2 Granulomatous lymphadenitis**

Granulomatous lymphadenitis is a chronic inflammatory response to foreign bodies or infectious agents. It is characterized by syncytial aggregates of epithelioid cells forming granulomas, varying number of multinucleated giant cells surrounded by small lymphocytes and plasma cells. Granulomas can be associated with necrosis (necrotizing granulomas) or be present without it (non-necrotizing/naked granulomas).

#### *2.2.1 Tuberculosis*

Tuberculosis is the commonest cause of granulomatous lymphadenitis in India. It is caused by infection by *Mycobacterium tuberculosis*. Tuberculous lymphadenitis is the

most common manifestation of extrapulmonary tuberculosis and can be seen in any age group [7]. Patient usually presents with painless enlargement of LN accompanied with or without fever, cough, and weight loss.

Cytology smears show multiple epithelioid cell granulomas, Langhans type of multinucleated giant cells, and reactive lymphoid cells in a necrotic background. Epithelioid cells are histiocytes with characteristic elongated nuclei resembling the sole of a shoe [12]. The nuclear chromatin is fine, granular and the cytoplasm is abundant, pale, without distinct cell outlines. Multinucleated giant cells have abundant cytoplasm with multiple nuclei arranged peripherally in a horse-shoe shape pattern (Langhans giant cells). Necrosis seen in tuberculous lesion is characteristic caseous necrosis, getting its name from its cheesy appearance which on microscopy appears extracellular, eosinophilic, and granular in appearance. In cases where only finding is necrosis, demonstration of acid-fast bacilli (AFB) by Z-N staining or AFB culture helps clinch the diagnosis.

#### *2.2.2 Sarcoidosis*

Sarcoidosis is a disease of unknown etiology, seen more commonly in women and African Americans in the 3rd–4th decade [12]. It is associated with non-necrotizing granulomatous lymphadenitis and is often indistinguishable from other causes of granulomatous lymphadenitis, hence is a diagnosis of exclusion. Cytology smears show multiple non-caseating epithelioid cell granulomas, multinucleated giant cells, reactive lymphoid cells, and plasma cells. Calcium oxalate crystals and asteroid bodies may sometimes be seen in giant cells of sarcoidosis. However, no necrosis or any organism is demonstrable.

#### *2.2.3 Leprosy*

Leprosy, a systemic chronic infection caused by *Mycobacterium leprae* was completely eradicated in 2000 from 98 countries of the world, although it is found in underdeveloped countries with variable prevalence [13]. Cytology smears show clusters of foamy histiocytes formed by cells having multivacuolated bubbly cytoplasm, central nuclei, fine nuclear chromatin, and prominent nucleoli. Epithelioid cell granulomas and multinucleated giant cells may also be seen depending on the immune status of the patient. The air-dried Romanowsky stained smears may show presence of negative shadows representing the bacilli. When the bacillary load is high as in case of lepromatous leprosy, the cytoplasm of the histiocytes is filled with parallel arrangement of bacilli in the form of globi (Virchow's/globus cell). Modified Z-N staining helps stain these bacilli.

#### *2.2.4 Fungal lymphadenitis*

Fungal lymphadenitis can be caused by a variety of fungal organisms such as *Histoplasma capsulatum, Cryptococcus neoformans, Coccidioides immitis,* etc. Cytology aspirates from fungal lymphadenitis are variable, having: (1) purely neutrophilic infiltrate, (2) only granuloma, (3) an admixture of the two, or (4) only demonstrate fungal organisms, with or without necrosis [7]. *Histoplasma* measures around 2–3 μm in diameter, has a rigid cell wall and resides in the cytoplasm of macrophages. *C. neoformans* measures around 5–10 μm and has a thick mucopolysaccharide capsule. Both demonstrate a narrow-based budding [7]. Histochemical stains such as

methenamine silver, mucicarmine, and PAS along with fungal culture help confirm the diagnosis.

#### *2.2.5 Foreign body granulomas*

Granulomatous lymphadenitis can be elicited against foreign substances which are large enough to be removed by the body's immune system. Some examples of the same include talc, silicone, suture material, or beryllium [7]. Cytology smears are mainly composed of giant cells containing foreign body particles, mature lymphocytes, and histiocytes.

#### *2.2.6 Leishmania*

*Leishmania donovani* is the causative agent of visceral leishmaniasis and is carried by sandfly which injects the promastigote form of leishmania into the skin. The promastigote form converts into amastigote form inside the histiocytes, which are released into the reticuloendothelial system (RES) upon rupture of the histiocyte. Cytology smears reveal amastigotes as oval structures of 1–3 μm size with a fine membrane, large nucleus, and a rod-shaped kinetoplast, present both intra and extracellularly admixed in a population of lymphocytes, histiocytes, and plasma cells [7]. Epithelioid cell granulomas and giant cells may also be seen occasionally.

#### *2.2.7 Filaria*

*Wuchereria bancrofti, Brugia malayi,* and *Brugia timori* are the commonest nematodes causing filariasis. Cytology smears can sometimes show microfilarial parasite in the LN aspirates. The microfilariae of both Wuchereria and Brugia are sheathed ranging in size from 200 to 300 μm in length, 2–8 μm in diameter, the former has a pointed tail that is free of nuclei and the latter has two distinct nuclei in the tip of the tail [14]. Smear may also show many eosinophils and epithelioid cell granulomas in the background.

#### *2.2.8 Toxoplasma*

Toxoplasmosisis a zoonotic disease caused by *Toxoplasma gondii.* Cytology smears show necrotic, polymorphous lymphoid background admixed with loose collections of epithelioid cell histiocytes and tingible body macrophages. Numerous small, elongated, crescent-shaped organisms may be seen [15].

#### **2.3 Rosai Dorfman disease**

Destombes first described Rosai Dorfman disease (RDD) in 1965. Later Rosai and Dorfman observed as separate entity in 1969 as Sinus histiocytosis with massive lymphadenopathy (SHML). It is a rare self-limiting benign disease. Etiology is unknown, is more prevalent among African Negros and males have more predilection (male: female = 2:1). Although any age group can be affected, most of the patients are younger than 20 years [16]. It presents with gradual painless bilateral cervical lymphadenopathy, fever, raised ESR, hypergammaglobulinemia, and occasionally anemia [17].

FNAC aspirate shows the proliferation of histiocytes with abundant eosinophilic to vacuolated cytoplasm, vesicular nuclei, and lymphophagocytosis or emperipolesis

[16]. The diagnosis can be confirmed by using IHC markers. Characteristically, S100 is always positive along with other markers, like CD68, CD163, α1 anti-chymotrypsin, and α1 anti-trypsin, negative for CD1a and Langerin (CD207) [17]. The differential diagnoses include reactive lymph node hyperplasia (RLH), infectious lymphadenitis, Langerhans cell histiocytosis (LCH), non-Hodgkin's lymphoma, and metastatic carcinoma [18].

#### **2.4 Kimura's disease**

It is a rare chronic inflammatory disorder of unknown etiology. Patients often have enlarged lymph nodes predominantly in the head and neck region [19]. Caused by trauma, allergic reactions to arthropod bites or parasites [20]. It is seen commonly in young adults with the age range of 27–40 years and male to female ratio of 3:1. It is endemic in Asia, especially in China and Japan [19]. Peripheral blood eosinophilia and raised serum IgE levels are usually noted.

FNAC smears shows features of RLH with numerous eosinophils, Warthin-Finkeldey polykaryocytes and epitheloid cell granuloma. IHC cell block for IgE and CD3 may help in cytodiagnosis [21].

#### **2.5 Kikuchi-Fujimoto disease**

Kikuchi-Fujimoto disease (KFD) also known as histiocytic necrotizing lymphadenitis is self-limited benign disease. Kikuchi and Fujimoto et al. first described this disease in Asia. It often manifests in young adults (younger than 40 years) [22], but it can occur in any age group with male to female ratio of 1:1 [23]. Most commonly seen in posterior cervical LN, with concomitant involvement of axillary and/or supraclavicular LNs. Infrequently lymphadenopathy is generalized. Most frequent extranodal involvement is skin, bone marrow, and occasionally in the liver. Affected LNs are tender and painful.

Smears examined in such patients show large accumulations of histiocytes and abundant karyorrhectic nuclear debris in a background of necrosis. Neutrophils and eosinophils are rare or absent, an important clue in the diagnosis of this entity. Lysozyme, myeloperoxidase, CD68, CD163, and CD4 are expressed in the histiocytes. Lymphocytes are mostly CD3 positive T-cells. The differential diagnosis includes infectious lymphadenitis, autoimmune lymphadenopathy (primarily SLE lymphadenopathy), and non-Hodgkin lymphoma [22].

#### **2.6 Dermatopathic lymphadenitis**

Dermatopathic lymphadenitis (DLN) was first coined by Hurwitt et al. It is also known as lipomelanotic reticulosis [24]. It is a benign form of LN hyperplasia. It is often seen in patients with skin diseases exfoliative or eczematoid inflammatory erythrodermas, neoplastic conditions like mycosis fungoides, Sezary syndrome and uncommonly in the absence of skin disease. Involvement of axillary and inguinal LNs are most commonly seen. Head and neck region LNs involvement is also seen in few patients. DLN is rare entity described in association with human immunodeficiency virus (HIV) infection. FNAC from such patients show numerous noncohesive, pale histiocyte-like cells. Macrophages contain pigment—either hemosiderin or melanin. These have small oval nuclei and a better-defined cytoplasm. Eosinophils may also be seen. The background is predominantly of small lymphocytes which may appear

slightly atypical but blast forms are less common [24]. IHC should be considered for confirmation. Histiocytes are S-100, langerin and CD1a positive. DLN is self-limited and often does not require any therapy [25]. Differential diagnosis include Hodgkin's lymphoma and Mycosis fungoides.

#### **2.7 Langerhans cell histiocytosis**

Langerhans cell histiocytosis (LCH) is a rare disease, and the estimated annual incidence ranges from 0.5 to 5.4 cases per million persons [26]. LCH may occur at any age, but majority of the cases are seen in children younger than 15 years [26]. It has a varied clinical spectrum from a solitary lesion, to multifocal unisystem to multisystem lesions. The unifocal form usually involves the bone while the multifocal unisystem form always involves the bone. The multifocal multisystem form can involve multiple organs such as bone, skin, liver, spleen, hematopoietic system, and lymph node.

The characteristic cytological features are high cellularity composed of sheets and isolated Langerhans cells (LC) admixed with polymorphous population of cells composed of eosinophils, neutrophils, lymphocytes, plasma cells, and histiocytes. The key to the diagnosis is to identify the LC with its characteristic nuclear features; indentation and nuclear grooves. LCs show positivity for S-100, CD1a, and langerin (CD207). Ultrastructural hallmark is the presence of Birbeck granule [27].

#### **2.8 HIV lymphadenitis**

Human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) is a disease caused by the human immunodeficiency virus. LNs are the major sites of emergence and spread of infection. Hence, frequently these patients present with lymphadenopathy. Cytologically it is associated with spectrum of changes from follicular hyperplasia to follicular depletion [28]. Cytological findings are florid follicular hyperplasia in the background of mature lymphocytes, plasma cells, and macrophages. These findings are however not pathognomonic [29]. Presence of many immature cells may be suggestive of lymphoma. Immunophenotyping is helpful in confirming a polyclonal nature of these cells and are mostly B-cells but mature T-cells can also be seen [15, 29].

#### **2.9 Rheumatoid arthritis, systemic lupus erythematosus (SLE)**

Smears from patients show numerous small lymphocytes, transformed lymphocytes, and tingible body, some containing Russel bodies [29].

#### **2.10 Castleman's disease**

Castleman's disease, also known as giant lymph node hyperplasia, is a disease of unknown etiology. Microscopically, based on histological pattern, two types are described: hyaline vascular (HV) and plasma cell type.

Hyaline vascular type is more common. It presents as localized enlarged LN whereas plasma cell type presents with systemic manifestations. FNAC findings in HV type include follicular hyperplasia admixed with lymphocytes, eosinophils, immunoblast, and hyalinised capillaries [30]. Cytological findings in plasma cell type are lymphoid follicular hyperplasia with numerous plasma cells [31].

#### **2.11 Infectious mononucleosis**

Infectious mononucleosis (IM), also known as the kissing disease, is a benign, selflimited lymphoproliferative disorder due to exposure to Epstein–Barr virus (EBV) or cytomegalovirus (CMV). The commonest clinical manifestations are pharyngitis, tonsillitis, LN enlargement (typically affecting cervical LNs) [32]. FNAC show lymphohistiocytic aggregates, loose granuloma formations of epithelioid histiocytes and a spectrum of immunoblast maturation. Some of these immunoblast are atypical with large irregular nuclei [33].

#### **2.12 Progressive transformation of germinal center (PTGC)**

Benign peripheral lymphadenopathy is often under diagnosed. Etiology and pathogenesis of PTGC is unknown. It can precede or coexist Hodgkin's lymphoma (HL) or nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL). FNAC is characterized by reactive follicular hyperplasia along with mantle zone expansion into the adjacent sinusoids and germinal centers (GC), the follicles with GC become enlarged and are replaced by small lymphocytes [34]. Differential diagnosis are nodal marginal zone B-cell lymphoma, small lymphocytic lymphoma (SLL) and mantle cell lymphoma. Immunostain help in ruling out differential diagnosis. CD3 and CD20 are positive in PTGC in B and T cell regions and are negative for CD15, CD30, and EMA. It is also negative for bcl-2 which is positive in follicular lymphoma [35].

#### **3. Lymphomas**

Lymphomas are neoplastic proliferations of lymphoid origin. Broadly classified as Hodgkin's and Non-Hodgkin's lymphomas, they can be both picked up on FNAC which is also the first line of investigation in their workup. Role of FNAC here is to diagnose, subclassify and stage the lymphomas. World Health Organization's (WHO) classification [36] is relatively easy to apply to cytology smears and requires detailed cytomorphological, immunophenotypic and molecular features which are easily obtained by using ancillary techniques on cytology material.

#### **3.1 Hodgkin's Lymphoma**

The global incidence of Hodgkin's lymphoma is 0.98 per 100,000 population [37]. It shows bimodal peak with first peak in young adults of 15–30 years and second peak in old age [7, 12]. A third peak is believed to occur in childhood in developing countries. About 70% patients present with lymphadenopathy affecting the cervical group of lymph nodes, followed by mediastinal, axillary, and paraaortic nodes [5, 12]. The systemic manifestations including fever, night sweats, and significant weight loss may be seen in advanced disease. The WHO has classified Hodgkin lymphoma in following categories [36].

	- Nodular sclerosis
	- Lymphocyte rich

*Lymph Node Cytology: Morphology and Beyond DOI: http://dx.doi.org/10.5772/intechopen.110607*

#### *3.1.1 Classical Hodgkin lymphoma*

The smears show the presence of Reed-Sternberg (R-S) cells lying in a polymorphic cell population comprising of small mature lymphocytes with eosinophils, plasma cells, histiocytes, and immunoblasts in varying proportions. The classical R-S cells are large in size ranging from 20 to 50 μ in diameter, have moderate to abundant amphophilic cytoplasm with usually bilobed nuclei however true binucleation or multinucleation may also be seen. The nuclei have coarsely clumped chromatin and two centrally placed prominent nucleoli which appear pale gray with MGG stain [6, 7, 12]. A perinuclear clear halo is also frequently seen. The classical R-S cells are easily demonstratable in mixed cellularity HL however this typical morphology is often lacking in other subtypes of CHL. The variants include mononuclear cells which contain single nucleus with all the above-described features (Hodgkin cells). The lacunar type of R-S cell is characteristic of nodular sclerosis Hodgkin lymphoma in histological sections however is occasionally found in cytology smears. These are large cells having abundant pale cytoplasm with indented or overlapping segmented nuclei. The nodular sclerosis HL is the most common subtype, yield scanty aspirate with an increased number of fibroblasts and collagen material. The hypocellular smears often result in false negative interpretations. The R-S and Hodgkin cells in mixed cellularity HL are present in a background comprising of small- to medium-sized lymphocytes, eosinophils, plasma cells, centroblasts, and histiocytes with an epithelioid morphology. This subtype may mimic suppurative lymphadenitis in cases where neutrophils dominate. The lymphocyte-rich HL shows rare R-S cells and Hodgkin cells of the abovedescribed features but a background rich in lymphoid cells exhibiting full spectrum from mature lymphocytes to germinal center formation. Eosinophils and histiocytes are usually not seen in this subtype.

#### *3.1.2 Nodular lymphocyte predominant Hodgkin lymphoma*

The smears of NLPHL show the presence of L and H cells in a background chiefly composed of small mature lymphocytes. These are giant cells with scant cytoplasm and multilobated extremely folded popcorn-like nucleus with vesicular nuclear chromatin and many small nucleoli. The diagnosis of NLPHL on FNA smears is challenging as L and H cells are usually difficult to find and are outnumbered by a sea of reactive small B and T lymphocytes.

Cytologically, NLPHL may be confused with T-cell-rich large B-cell lymphoma which shows abundant reactive small lymphocytes with only a few neoplastic cells [10]. These cells on IHC show uniform positivity for CD20 and are negative for CD15 and CD30, thus ruling out the possibility of CHL.

Anaplastic large cell lymphoma (ALCL) also shows large multinucleated cells which may be indistinguishable from R-S cells though neoplastic cells in ALCL are much more numerous in contrast to HL, wherein the R-S cells comprise only about 0.1–10% of the total cell population [10]. In addition, ALK1 positivity and gene rearrangement studies are helpful in the diagnosis of former [15].

Nasopharyngeal carcinoma commonly metastasizes to cervical LNs and shows large neoplastic cells simulating R-S cells in an abundant lymphoid background on cytology. Immunostaining with CK and EMA may be used to exclude HL in such cases.

A variety of other benign conditions like infectious mononucleosis, toxoplasmosis, drug-induced lymphadenopathy, post vaccinal lymphadenopathy may show large atypical immunoblasts with similar morphology as R-S cells, however these cells usually have coarse nuclear chromatin and smaller nucleoli.

The Hodgkin and R-S cells of CHL show positive immunostaining with both CD15 and CD30 and are negative for CD45, EMA and usually negative for both B and T-cell markers. Those associated with EBV are positive for EBV-LMP [5, 7, 12].

The L and H cells of NLPHL are positive for CD45 and CD20 and are positive for EMA in 50% cases. They are however negative for CD15 and may show occasional positivity for CD30 [5, 7, 15].

There is limited role of FCM in diagnosis of HL. An increase in the CD4+ T cells with a CD4+/CD8+ of more than 4:1 may serve as a clue, however, is not diagnostic. NLPHL may show an increased proportion of CD4 + CD8+ (double positive) or CD57 + T-cells [6, 10].

#### **3.2 Non-Hodgkin lymphoma**

#### *3.2.1 Small cell lymphoma*

Small cell lymphomas are a group of lymphomas, with predominate monotonous cell type on first inspection of cells (**Table 3**). They are encountered on regular basis by cytopathologist and needs a good understanding of various differentials to make correct diagnosis highlighted in **Table 4**.

#### **Table 3.**

*Simplified WHO classification for Non-Hodgkin's lymphoma.*

*Lymph Node Cytology: Morphology and Beyond DOI: http://dx.doi.org/10.5772/intechopen.110607*

#### **Table 4.**

*Types of small cell lymphomas and their distinguishing features [7].*

Next, individual small B cell NHLs are discussed in detail, to provide all the available information to the cytopathologists and help them in differentiating these similar pathologies.

#### *<IP_ADDRESS> Mantle cell lymphoma*

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma, which mainly affects elderly men. MCL represents 3–10% of all NHLs. Most common presentation is generalized lymphadenopathy and hepatosplenomegaly, with lymphoma cells seen in blood of 25% of cases. Sometimes extranodal sites like gastrointestinal tract are involved. The median survival is 3–5 years.

Cytology smear shows a monomorphic population of small to intermediate cells with nuclear membrane irregularities and indentations, fine nuclear chromatin, inconspicuous nucleoli, and scant cytoplasm. An aggressive blastoid variant can have intermediate to large cells with large nuclei having irregular margin, finely dispersed chromatin, and multiple small nucleoli. Blastoid variant has high mitotic activity [7, 38, 39].

Neoplastic cells express CD19, CD20, CD5, CD79b, Cyclin D1, and FMC7 while negative for CD10 and CD23. Light chain restriction is seen mostly in the form of kappa restriction.

Mantle cell lymphoma shows diploid cells while blastoid variant mostly shows a tetraploid population with high proliferative activity, suggestive of a high-grade lymphoma [39].

Characteristic genetic abnormality seen is t(11;14) (q13q32) translocation, leading to overexpression of cyclin D1, a cell-cycle protein [40].

#### *<IP_ADDRESS> Follicular lymphoma*

Follicular lymphoma is a tumor of follicular center B-cells. It is a common NHL, constituting 20–35% and is seen mostly in elderly patients. Most common presentation is generalized lymphadenopathy with spleen and bone marrow involvement [7, 36]. This is a mildly aggressive lymphoma with high 5 years survival rate.

Follicular lymphomas show a mixture of small, irregular lymphocytes, and larger cells. The small lymphocytes, have irregular nuclear contours and inconspicuous nucleoli while larger cells (centroblasts) are characterized by sharply demarcated basophilic cytoplasm, round, noncleaved nuclei with finely granular chromatin and prominent 2–3 peripheral nucleoli.

FL is positive for CD19, CD20, CD10, and bcl-2 while it is negative for CD5.

Neoplastic cells show t(14;18)(q32; 21) translocation that results in the

rearrangement of the bcl-2 gene and in the expression of the "antiapoptosis" gene [41].

#### *<IP_ADDRESS> Lymphoplasmacytic lymphoma*

Lymphoplasmacytic lymphoma (LPL) accounts for only 1.5% of all nodal lymphoma and is seen mostly in elderly. Most common involvement is seen in lymph node, spleen, and bone marrow along with peripheral blood. Elevated levels of monoclonal serum paraprotein of the immunoglobulin M type are seen in most of the patients that sometimes result in symptoms of hyperviscosity (Waldenstrom macroglobulinemia). LPL shows an indolent course and may very rarely transform into DLBCL.

Cytology smears show abundant small lymphocytes, plasmacytoid, and plasma cells. Occasionally, PAS-positive intranuclear inclusions known as Dutcher bodies may be observed.

Neoplastic cells are positive for CD19, CD20, CD22, CD79a, CD38 and shows light chain restriction while being negative for CD5, CD10, CD23.

Neoplastic cells may show a variety of immunoglobulin heavy chain and light chain genes rearrangement, but no single characteristic chromosomal abnormality is known.

#### *<IP_ADDRESS> Marginal zone lymphoma*

Marginal zone lymphoma (MZL) is a relatively uncommon low-grade B-cell lymphoma. MZL may be nodal or extranodal. Extranodal MZL is commonly known as MALT lymphoma and constitute 7–8% of all lymphomas. Most common sites of extranodal MZL are stomach, eye, thyroid, salivary gland, lung, and skin [42]. MALT lymphoma shows a strong association with autoimmune diseases, such as Hashimoto's thyroiditis, Sjogren's syndrome, and Helicobacter pylori infection-induced gastritis.

Cytology smear shows a heterogeneous population of monocytoid cells, small, cleaved cells, large cells, and plasma cells. The monocytoid cells have moderate to abundant amounts of pale cytoplasm. The nuclei can vary from oval to reniform shape, with vesicular or coarse chromatin and inconspicuous nucleoli [43].

Neoplastic cells are positive for CD19, CD20, CD22 and surface Ig while are negative for CD5, CD10, CD23, and CD103.

Trisomy 3 and t(11;18), (q21;q21) translocations have been reported in extranodal MZL [44].

#### *<IP_ADDRESS> Small lymphocytic lymphoma*

Small lymphocytic lymphomas (SLL) are seen mostly in older adults, with bone marrow and peripheral blood involvement in many cases. It is an indolent disease. It *Lymph Node Cytology: Morphology and Beyond DOI: http://dx.doi.org/10.5772/intechopen.110607*

may undergo transformation into high grade lymphoma like prolymphocytic lymphoma (PLL) or large-cell lymphoma (Richter syndrome).

SLL shows a monomorphic population of small, round lymphocytes with nuclei that have a checkerboard pattern of clumped chromatin. Prolymphocytes and paraimmunoblasts are scattered in the background. Prolymphocytes are slightly larger than paraimmunoblasts, but both have gray-blue cytoplasm, round nuclei and a prominent nucleolus (**Figure 2**). Unfavorable cytological features are, increase in the number of paraimmunoblasts, presence of plasmacytoid cells, mitotic figures, apoptotic bodies, necrosis, and a myxoid, dirty background [45].

Neoplastic cells are positive for CD19, CD20, CD5,CD43, CD23 and shows light chain restriction while they are negative for CD10 and FMC7.

Deletion of chromosome 13q14 is the most common genetic abnormality, followed by trisomy 12 [46].

#### *<IP_ADDRESS> Burkitt lymphoma*

Burkitt lymphoma (BL) is a highly aggressive and potentially curable lymphoma commonly occurring in children. It can occur in three clinical settings. (1) The endemic BL is seen almost exclusively in children aged 4–7 years old in Africa and to a lesser extent in Middle east and south America. Jaw and facial bones are the most common sites, other extranodal sites being intestine, breast, and ovary. (2) The sporadic variant commonly presents as an abdominal mass and occurs in children and young adults. (3) The immune deficiency-associated variant is commonly seen to be associated with HIV. BL has a very high tumor burden with a doubling time of 24 hours in some cases [47]. EBV is seen to be commonly associated with BL: almost all cases of endemic, 30% cases of sporadic and 25–40% cases of immune deficiency associated BL [48].

The aspirates are hypercellular with a uniform monotonous population of medium-sized cells with round nuclei, coarse chromatin, and multiple (2–5) small but

#### **Figure 2.**

*Small lymphocytic lymphoma showing mature lymphocytes with round nucleus, coarsely clumped chromatin and indistinct nucleoli (MGG-600).*

prominent nucleoli. The cytoplasm is abundant, blue, and vacuolated (especially in air dried Romanowsky smears) (**Figure 3**). The formalin-fixed cell blocks as well as tissue sections have a starry sky appearance due to the abundance of tingible body macrophages and numerous apoptotic cells (**Figure 4**).

The diagnosis of BL is imperative because it warrants a more aggressive chemotherapy regimen than DLBCL. The cells of BL are positive for CD19, CD20, CD10, sIg, IgG heavy chain or kappa/lambda light chains and BCL6. Ki67 index is usually 100%. The diagnosis is done based on morphology, characteristic immunophenotype, and demonstration of MYC translocation via karyotyping or FISH. Demonstration of MYC translocation can also be done by ICC/IHC, with nuclear immunoreactivity in >40% of cells termed as positive [49]. The most common translocation (80%) is t(8,14) where MYC translocate from chromosome 8 to Ig heavy chain on 14. Other translocations seen are t(2,8) and t(8,22) where the translocation occurs with the light chain. MYC translocation is highly characteristic but not specific.

#### *3.2.2 Large cell lymphomas*

#### *<IP_ADDRESS> Diffuse large B cell lymphoma*

Diffuse large B cell lymphoma is a very commonly encountered NHL and comprises 30–40% of adult NHLs. Extranodal disease is seen in about 40% of patients, with stomach being the most common site [50]. Previously, three morphologic subtypes were recognized which later proved to be clinically irrelevant. Two molecular subtypes are now recognized based on gene expression profiling: germinal center B cell (GCB) like and activated B cell (ABC) like with the latter having poor prognosis and poor survival after chemotherapy.

FNA smears show variable cellularity with mediastinal and extranodal sites frequently having low cellularity. This might be because of extensive sclerosis or sparse

#### **Figure 3.**

*Large cell lymphoma (Burkitt lymphoma) dirty proteinaceous background with cells showing marked pleomorphism, scanty to moderate cytoplasm, indented nuclei and frequent mitosis (MGG; 400).*

#### **Figure 4.**

*Large cell lymphoma (Burkitt lymphoma) dirty proteinaceous background with cells showing marked pleomorphism, scanty to moderate cytoplasm, indented nuclei and frequent mitosis (Hematoxylin and eosin 200 cell block preparation).*

distribution of cells commonly seen in these sites. However, most smears are moderately cellular with sheets of large, atypical cells. The cells are categorized as large when the nucleus size is more than histiocytes nucleus or twice more than a lymphocyte's nucleus.

The centroblastic variant comprises of a predominant population of centroblasts with characteristic round nuclei and multiple small nucleoli. The immunoblastic variant has a pleomorphic cell population with large blast cells having abundant blue cytoplasm, perinuclear pale zone and round-irregular nucleus with single prominent nucleoli. Anaplastic variant has cells with bizarre pleomorphic nucleus with multinucleation. These cells may resemble RS cells or ALCL cells (**Figure 5**).

Cell blocks are useful to differentiate these large cell lymphomas from indolent low-grade ones by providing architectural insight. DLBCL is positive for CD19, CD20 and CD10 (**Figure 6**). Approximately 40–60% of these lymphomas are positive for MUM1 and BCL6 is positive in majority (60–90%) [51]. Aberrant co-expression of CD5 and aberrant loss of immunoglobulin expression can also be seen. The GCB type DLBCL is positive for BCL6, negative for MUM1, and positive/negative for CD10. The non-GCB subtype (including ABC) are either double negative for CD10 and BCL6 or positive for both BCL6 and MUM1. Positivity for both BCL2 and MYC on IHC classifies it as double expressor DLBCL, seen in about 30% of DLBCL, NOS cases [52]. Positivity is defined as >30% cells expressing the marker. These cases portend a poor prognosis for the patient with relapse and CNS involvement being more commonly seen. If MYC and BCL2 rearrangements are confirmed by FISH, it used to be termed double hit DLBCL, but it has been categorized as a separate entity now: high grade lymphoma with MYC and BCL2 rearrangement. Virtually all cases of DLBCL have heavy or light chain gene rearrangements. Some cases might have t(14,18), if there is a history of follicular lymphoma. On FCM, the large cells may appear outside of the usual lymphoid gate with higher SS and FS.(Sometimes, the cell count in FCM is low

#### **Figure 5.**

*Diffuse large B-cell lymphoma-highly cellular smear with large atypical lymphoid cells, marked nuclear pleomorphism and high N:C ratio (MGG; 1000).*

#### **Figure 6.**

*Diffuse large B-cell lymphoma-CD20 positive large atypical lymphoid cells (ICC; 600).*

as the large cells are fragile and may not survive processing. However in most of the cases on gating CD45 dim population with CD10 (mayor may not be present) CD19 and CD79 expression with light chain expression (**Figure 7**).

#### *<IP_ADDRESS> T cell/histiocyte rich B-cell lymphoma*

It is subtype of DLBCL where histiocytes and reactive T cells are present in such abundance as to obscure the tumor cells. It commonly affects men in middle age [53]. The smears are cellular with a predominant population of small mature lymphocytes. Variable number of scattered histiocytes are also seen along with scattered large cells such as anaplastic, centroblasts, popcorn like or occasionally RS cells. It is diagnosed on cytological smears with difficulty and usually immunophenotyping on cell/tissue blocks is needed for definitive diagnosis. The smaller cells are positive for CD3, CD5, and CD8 and the large cells express DLBCL phenotype.

*Lymph Node Cytology: Morphology and Beyond DOI: http://dx.doi.org/10.5772/intechopen.110607*

**Figure 7.** *Expression of CD19 and kappa chain restriction (FCM).*

#### *<IP_ADDRESS> Precursor lymphoblastic lymphoma/leukemia*

Precursor Lymphoblastic Lymphoma/Leukemia (LBL) is commonly seen in older children or young adults and is a neoplasm of immature lymphoid ells. T-LBL is more common and constitutes 90% of lymphoblastic lymphomas [54]. The patients usually present with a mediastinal mass and can rapidly progress to involve the peripheral blood, bone marrow, CNS, and gonads. B-LBL is less common and involves lymph nodes, skin, and even bones.

The FNA smears are hypercellular and show a homogenous population of small to medium-sized cells with high N:C ratio, lobulated, convoluted, or round nuclei and speckled chromatin with 2–3 prominent nucleoli. There will be frequent mitosis and B and T LBLs are morphologically similar. Certain clues like exaggerated convolution, cerebriform nuclei point toward T-LBL.

Demonstration of immunoexpression of TdT by IHC or FCM that occurs exclusively in LBL help in differentiating them from rest of the lymphomas. T-LBL would demonstrate positivity with surface/cytoplasmic CD3 and other T cell markers like CD5, CD7 depending on the stage of development. Other markers include CD2, CD34, CD4, and CD8. 20% of T-LBLs show positivity for NK cell markers like CD16 and CD56 [55].

B-LBL expresses B lineage markers like CD19, CD79a, and PAX5. CD20 is positive in 50% of the cases. CD10 is positive in the CALLA variant. While surface IG are usually negative, cytoplasmic Ig can be expressed in pre-B LBLs.

#### *<IP_ADDRESS> Anaplastic large cell lymphoma (ALCL)*

Anaplastic large cell lymphoma (ALCL) is a separate entity for T-cell and null cell types in WHO classification of haematolymphoid neoplasms. It accounts for about 3% of adult non-Hodgkin lymphomas and 10–20% of childhood lymphomas [56]. ALCL affects children and young adults but can occur in any ages. Apart from lymph nodes, the extranodal sites involved are skin, bone, lung and soft tissues.

The aspirate smears are moderately to highly cellular. The most common morphologic variant of ALCL is pleomorphic type in which large pleomorphic cells with eccentric, horseshoe-shaped or kidney-shaped nuclei are present: hallmark cells. These cells have fine or clumped chromatin with prominent basophilic nucleoli and usually abundant amphophilic cytoplasm. Another characteristic cell type are Doughnut cells which are multinucleated giant cells with abundant clear or basophilic cytoplasm and multilobate nuclei in wreath like pattern. There is a paucity of lymphoglandular bodies. Necrosis and inflammation are also seen commonly. Small cell variant of ALCL has small to medium sized cell with irregular nuclei and moderate amount of cytoplasm. The other two common morphologic types are small cell variant (that might resemble PTCL) and lymphohistiocytic variant which has numerous histiocytes that might mask the tumor cells. Both small cell and lymphohistiocytic variant have a worse prognosis with more tendency for leukemic spread [57].

IHC on cell block with CD30 markers shows perinuclear dot-like or strong membranous positivity while being negative for CD15. ALCL also expresses T-cell markers such as CD2 and CD4 but 15% cases have null-type phenotype [58]. The chromosomal translocation t(2,5) is seen where ALK gene on chromosome 2 fuses with NPM (nucleophosmin) gene on chromosome 5. This gene product (ALK) can also be demonstrated by ICC. EMA immunoexpression also helps to differentiate ALCL from other lymphomas. FCM is rarely useful with necrosis and surrounding inflammation confounding the results. Demonstration of TCR-gamma/delta is usually helpful to establish clonality. FISH can also be used for demonstration of the translocation using the break apart probe.

#### *<IP_ADDRESS> Peripheral T-cell lymphoma*

Peripheral T-cell lymphoma (PTCL), has a mature or post thymic phenotype, unspecified accounts approximately half of the case of PTCL. PTCL affects adult population more commonly but can also affect children. Along with generalized lymphadenopathy, there might be involvement of bone marrow, liver, spleen, and extranodal tissues too.

The aspirate smears of PTCL are highly cellular and pleomorphic. They show a spectrum of atypical cells from large to small cell type [59]. The larger cells have large single to multiple nuclei that have fine to clumped chromatin, smooth to irregular border and huge nucleoli (**Figure 8**). The presence of multiple nuclei and prominent nucleoli may give the appearance of RS cells/variants. However, cytoplasm is usually abundant and pale gray and may point toward it being a T-cell lymphoma rather than B-cell. The small morphologic type has tumor cells which are only slightly bigger than small mature lymphocytes, however have irregular nuclear contour with nuclear indentations, cleaving and folding. The nuclei have clumped chromatin and inconspicuous nucleoli. Intermediate sized cells of PTCL show features which are a mix of both large and small cell type.

**Figure 8.** *T-cell lymphoma; large pleomorphic cells with indented, folded nuclei and 1–2 distinct nucleoli (MGG; 1000).*

ICC can be used to demonstrate the T cell phenotype. There is variable expression of pan T-cell markers like CD3, CD5, and CD2. CD7 expression is seen to be frequently lost. There is variable expression of CD4 and CD8 and CD4 positivity is more commonly seen than CD8 (**Figure 9**). The cells can also be dual negative or dual positive for CD4 and CD8. CD30 and CD52 are other markers that can be positive in 50% of the tumor cells [60, 61]. FCM can be helpful to isolate the clonal subset with aberrant T-cell phenotype but usually falls short of making a definitive diagnosis. PCR can be used to demonstrate T-cell receptor rearrangement.

**Figure 9.** *Expression of CD7 and CD34 (on FCM).*

#### **4. Metastatic lymph node disease**

Lymph nodes contain blood, lymphatic vessels and stromal tissue.

Metastatic disease of LN often shows diffuse involvement and can be picked up on cytology. FNAC is of considerable value in disease staging and documentation of metastasis in both known and occult tumors. In more than 90% cases of lymph node metastasis, primary site of origin is already known [62].

Carcinomas show lymphatic pattern of dissemination thus lymph nodes are commonly involved. In well-differentiated squamous cell carcinoma (SCC), the tumor cells have hyperchromatic nuclei with individual cell keratinization. The tumor cells often show necrotic background. Thus, in case of low cellularity smears with abundant necrosis, a careful search for the tumor cells is required (**Figure 10**) [63]. Branchial cleft cyst and epidermal inclusion cyst are close differentials due to presence of amorphous debris with necrotic parakeratotic cells. Poorly differentiated squamous cell carcinoma which might demonstrate cell clusters showing thick nuclear membrane and prominent nucleoli pose a diagnostic challenge and often becomes difficult to distinguish from adenocarcinoma [64].

In Metastatic adenocarcinoma, the cells are usually arranged in acinar or glandular pattern and are large, cuboidal to columnar with moderate amount of cytoplasm and pleomorphic nuclei with prominent nucleoli. Vacuolated cytoplasm usually indicates intracellular mucin accumulation (**Figure 11**). Presence of intracellular mucin and necrosis, usually suggests gastro intestinal tract as primary site of origin. Moderately pleomorphic glandular cells with gland-in gland formation suggests metastasis from prostatic carcinoma. Metastasis from renal cell carcinoma shows large cells with abundant pale, granular vacuolated cytoplasm and prominent nucleoli. Similarly, large cells with prominent nucleoli supports the diagnosis of anaplastic carcinoma of lung and nasopharynx. Metastatic deposit of Papillary thyroid carcinoma displays a

#### **Figure 10.**

*Squamous cell carcinoma cellular smears with necrotic background showing polygonal cells with intercellular bridges, blue cytoplasm, Ink dot pyknotic nuclei and bizzare nuclear forms. Also seen are tumor giant cells (MGG; 400).*

**Figure 11.**

*Cuboidal to columnar cells in acini with palisaded glands. Cells show cytoplasmic extension, vacuolation, moderate nuclear pleomorphism, and compact chromatin (MGG; 40).*

papillary pattern with central fibrovascular core along with nuclear grooving and intranuclear inclusion. Metastatic ductal carcinoma of breast shows high cellularity with several loose clusters of tumor cells having moderate to abundant cytoplasm with pleomorphic nuclei and prominent single to multiple nucleoli [63].

The role of FNA for searching unknown primary tumor can be facilitated by ICC on aspirated smears/cell block. In metastasis from epithelial tumors, the IHC/ICC expression of CK7 and CK20 profile helps to triage according to the origin from primary tumor. **Table 5** summarizes CK7 and CK 20 immunohistochemical markers in various epithelial malignancies.

Though the risk of LN metastasis is low in malignant round cell tumors (SRCT), they may be seen on LN aspirates, for example, Ewing sarcoma, rhabdomyosarcoma, retinoblastoma, neuroblastoma, hepatoblastoma, Wilms tumor and osteogenic sarcoma (small cell variant). These tumors can be differentiated with the help of IHC markers. Cytologically, these tumors are composed of uniform population of round to oval cells with scanty, basophilic cytoplasm; mixed populations of small and large cells present in some tumors like neuroblastomas. These tumors consist of cells size of approximately three times the small mature lymphocyte and hyperchromatic nucleus with evenly distributed chromatin with cytoplasm usually scanty with lack of nucleoli and very high nucleo-cytoplasmic ratio [63]. Metastasis from small cell carcinoma undifferentiated type arising from lung show cohesive clusters of tumor cells with scant cytoplasm, coarse chromatin, frequent mitosis and necrosis thus, it is important to differentiate from small and medium sized lymphoma and large cell lymphoma [65].

Metastasis from malignant melanoma have dissociated cells, well defined cytoplasm, eccentric nuclei, dense chromatin, and rarely cells have intracytoplasmic pigment. IHC analysis for S-100, Melan A and HMB45 are of great value in diagnosing the melanoma [66].

#### **Table 5.**

*Immunohistochemistry for carcinoma of unknown primary.*

Metastatic sarcomas are very uncommon in lymph nodes [67]. Cytological features of sarcomas are varied in different types. More common sarcomas metastatic to lymph node include synovial sarcoma, Kaposi sarcoma, Follicular dendritic cell sarcoma, epitheloid sarcoma rhabdomyosarcoma. and epithelioid sarcoma.

#### **5. Lymph node FNA cytology reporting using Sydney system**

The current WHO classification of lymphoproliferative disorders incorporates clinical, morphological, and ancillary data that are required for specific diagnoses. Despite the tremendous progress made in performing and interpreting LN-FNAC and its correlation with ancillary tests, the technique is not uniformly accepted by clinicians and pathologists. This is mainly due to the lack of widely shared and accepted guidelines and a cytopathological classification that directly relates to management. The proposal of the Sydney system for performing classification and reporting of lymph node cytopathology was published by an expert panel. **Table 6** highlights the proposed Sydney classification for reporting the lymph node cytopathology [68].

Fine needle aspiration cytology is an invaluable tool in the workup of lymphadenopathy, neoplastic and non-neoplastic. Lymph nodes offers the most fulfilling diagnostic experience to a cytopathologist. This is considered first line investigation for superficial or deep seated and inoperable lymphoid masses.

The introduction of a standardized reporting system *viz* The Sydney system shall help in streamlining the quality of procedure, optimal utilization of material for ancillary testing and better understanding of the report communicated to the clinician, overall aiding patient care. With proper utilization of FNAC, unnecessary surgical interventions in cases of infective, lymphoproliferative, and metastatic disorders might be avoided. The ever increasing use of FCM and molecular testing in the diagnostic workup of lymphoproliferative disorders and the ease and suitability of FNAC in obtaining tissue sample for these testing, an increasing reliance on FNAC as preferred modality can be foreseen.

*Lymph Node Cytology: Morphology and Beyond DOI: http://dx.doi.org/10.5772/intechopen.110607*

**Table 6.**

*Sydney system classification.*

### **Author details**

Meeta Singh\*, Kirti Balhara, Deepika Rana, Rabish Kumar, Nimisha Dhankar, Shabnam Singh, Priyanka Bellichukki, Sreoshi Paul and Sathiyanesan Mariana Chartian Maulana Azad Medical College, New Delhi, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 5**

## Histopathologic Diagnosis of Neuroendocrine Neoplasms of Head and Neck, Lung and Gastrointestinal Tract

*Liberty Bonestroo and Emilian Racila*

#### **Abstract**

Neuroendocrine neoplasms are classified as epithelial and non-epithelial based on their origin being from epithelial neuroendocrine progenitor cells or derived from the neural crest. The latter are negative for cytokeratin (hence non-epithelial) and mostly result from neoplastic transformation of paraganglia. Here, we are reviewing the most important histologic and immunophenotypic characteristics of neuroendocrine carcinomas as well as the current WHO classification guidelines. The terminology of neuroendocrine neoplasms is confusing due to various classification systems employed for each internal organ. In the lung and GI tract, for example, "neuroendocrine tumors" comprise carcinomas of different degree of differentiation and histologic grade. While in the lung the term refers strictly to low-grade neuroendocrine carcinomas, in the GI tract it comprises both low- and high-grade neuroendocrine carcinomas. Despite concerted efforts to unify the overall classification of neuroendocrine carcinomas across organs, major differences continue to persist.

**Keywords:** neuroendocrine neoplasms, histopathologic diagnosis, immunohistochemistry, Ki-67, small cell carcinoma

#### **1. Introduction**

The diagnosis of neuroendocrine neoplasms of the head and neck, lung, ad GI tract share many common features, however, as mentioned, terminology can be confusing among the entities. We aim to provide clarity and simplify the schema for making a histopathologic diagnosis of neuroendocrine neoplasms while maintaining the current WHO guidelines for neuroendocrine tumor classification based on each organ. Misdiagnosing neuroendocrine carcinomas is not unusual and lead to improper therapeutic management. We occasionally come across such cases in our practice. The most common cause for misdiagnosis of a low-grade neuroendocrine tumor of the lung as small cell lung carcinoma (SCLC), for example, is limited tissue obtained as part of a biopsy combined with lack of experience and inadequate or insufficient use of ancillary studies that can aid in proper classification. We would strongly recommend the

use of Ki-67 immunohistochemistry study whenever typical morphologic features of SCLC are not seen in a biopsy specimen (e.g., rather nested growth pattern than large sheets of tumor cells, no or only focal necrosis, mitoses are not readily observed etc.). In general, the pathologist should refrain from classifying a neuroendocrine neoplasm as SCLC if Ki-67 proliferation index is less than 50% overall. Rather, obtaining more tissue for diagnosis may be suggested. The management of typical and atypical carcinoids is entirely different than SCLC, as in the later surgical resection is not an option in most patients. Neuroendocrine neoplasms variably express markers of neuroendocrine differentiation (synaptophysin, chromogranin, neural specific enolase (NSE), CD56 and INSM1), are comprised of tumor cells of epithelial or neuronal/neuroectodermal origin and occur at various anatomic sites throughout the body. Since in poorly differentiated tumors the expression of commonly used neuroendocrine markers like chromogranin and synaptophysin may be lost, the use of second-generation neuroendocrine differentiation biomarkers, especially the INSM1 (nuclear transcription factor that regulates neuroendocrine differentiation) has been shown to be helpful. Within the category of "neuroendocrine neoplasms" these tumors share similar morphologic and cytologic features, though features can vary depending on anatomic site and degree of differentiation. A word of caution regarding use and interpretation of neuroendocrine markers: there are malignancies that are not neuroendocrine but may be misinterpreted as either small cell or large cell neuroendocrine carcinomas due to aberrant expression of above-mentioned markers. In alveolar rhabdomyosarcomas of nasal cavity, for example, in addition to neuroendocrine markers, even unusual expression of cytokeratin may be seen. In such cases, the reviewing pathologist's experience in pattern recognition and keeping an open mind to alternative possibilities, is of most importance. Equally important is good communication among all medical staff involved in patient's care. In larger centers, it is common practice to discuss complex cases in interdisciplinary tumor boards, where all those involved, surgeons, oncologists, radiologists and pathologists, are present, ensuring that the therapy is appropriately tailored to the needs of every patient.

The histopathologic diagnosis of neuroendocrine neoplasms begins with the identification of neuroendocrine morphology. Classification and grading of the neoplasm varies based on the anatomic site. Generally, poorly differentiated neuroendocrine neoplasms are high-grade, with well-differentiated neoplasms falling into the low-grade category. The immunohistochemical profile and expression of neuroendocrine differentiation markers (synaptophysin, chromogranin, INSM1 and CD56) are essential for diagnosis. One should also evaluate for the presence and degree of necrosis. Mitotic count and Ki-67 proliferation index are important, with mitotic activity expressed as mitoses per 2 mm2 area or 10 high-power fields (hpfs). When evaluating the Ki-67 proliferation index, the count should be performed on "hotspot" regions within the neoplasm, per WHO classification guidelines. When finalizing the pathologic report, the number of mitoses counted within the total area should be included in the final report, either as number of mitoses per 10hpfs or per mm2 . Additionally, in some cases, neuroendocrine tumors may have both neuroendocrine and non-neuroendocrine elements such as squamous cell or adenocarcinoma, encompassing the so-called "mixed" or "combined" malignancies.

#### **2. Head and neck**

Head and neck neuroendocrine carcinomas are rare neoplasms, accounting for less than 5% of all head and neck malignancies. With the 5th edition of the WHO

#### *Histopathologic Diagnosis of Neuroendocrine Neoplasms of Head and Neck, Lung… DOI: http://dx.doi.org/10.5772/intechopen.109096*

head and neck neuroendocrine neoplasms (NEN), classification has been revised to include both the WHO and IARC unified terminology framework. With this classification, the upper aerodigestive tract and salivary gland NENs are included as a group. The classification of neuroendocrine neoplasm (NEN) can be further subclassified into well-differentiated neuroendocrine tumors (NETs) and assigned to proliferative grades 1, 2, and 3, while the poorly differentiated neuroendocrine carcinomas (NECs), including small cell and large cell neuroendocrine carcinoma, are grouped separately, similarly to general classification used for GI tract malignancies. To date, the G3 NET category remains provisional (**Figure 1**, **Table 1**).

#### **Figure 1.**

*Low-grade neuroendocrine tumors of lung. Carcinoids are characterized by organoid histologic pattern, frequently nested or trabecular, with no or only focal areas of necrosis (a, 100X; B, 200X). There is mild to moderate cytologic/nuclear atypia and mitoses are either absent or rarely observed (C, 400X). Cytokeratin is diffusely expressed (D, cytokeratin AE1/AE3 immunostain, 200X). These tumors are also diffusely positive for neuroendocrine markers (E, synaptophysin, 100X; F, chromogranin, 100X).*

*\*Provisional criteria applied at this time.*

*\*\*Small cell NEC cytomorphology: minimal cytoplasm, hyperchromatic molded nuclei, finely granular chromatin, inconspicuous nucleoli, and cell size smaller than combined diameter of three lymphocytes, prominent apoptotic bodies, and necrosis.*

*\*\*\*Large cell NEC cytomorphology: nested, organoid or trabecular growth with abundant cytoplasm, round nuclei with prominent nucleoli, peripheral palisading, rosette formation, comedo-pattern necrosis.*

#### **Table 1.**

*Epithelial neoplasms of the upper aerodigestive tract and salivary glands.*

#### **2.1 Neuroendocrine tumor (NET)**

Neuroendocrine tumors of the head and neck are well-differentiated neoplasms of neuroendocrine differentiation and can arise in the nasal cavity, paranasal sinuses, nasopharynx, oropharynx, larynx, and rarely in salivary glands and the oral cavity. Sinonasal NETs occur within the sinuses and the nasal cavity. The tumors macroscopically appear as polypoid, nodular or exophytic masses that may ulcerate and bleed. Histologically, these tumors are well-differentiated epithelial neoplasms. Architecturally, they are comprised of nests, trabecula, or cords of neuroendocrine cells with monotonous nuclei containing salt-and-pepper finely granular chromatin and moderately abundant cytoplasm, in a background of highly vascular stoma. It is important to assess for the degree of necrosis and mitoses per 2 mm2. The overall Ki-67 proliferation index is generally <20% and can be useful in determining Grade 1 versus Grade 2 NETs. All NETs will show diffuse positivity for neuroendocrine markers (INSM1, synaptophysin, chromogranin-A), keratins (CK7/8), and are negative for TTF-1 in most cases. NETs do not exhibit anormal p53 staining and show no loss of Rb. Rare cases of mixed neuroendocrine and non-neuroendocrine tumors have been described in the nasal cavity and larynx.

#### **2.2 Small cell neuroendocrine carcinoma (SCNEC)**

SCNEC of the head and neck is a poorly differentiated neuroendocrine carcinoma. By the time of clinical detection, the submucosal SCNECs are generally large, with areas of necrotic tumor or ulceration commonly seen. SNECs are comprised of sheets

*Histopathologic Diagnosis of Neuroendocrine Neoplasms of Head and Neck, Lung… DOI: http://dx.doi.org/10.5772/intechopen.109096*

or nests of epithelial tumor cells with hyperchromatic nuclei, finely granular chromatin, inconspicuous nucleoli, and scant cytoplasm. The tumor cells are small (less than the combined diameter of three lymphocytes), with peripheral palisading, rosettes, or trabeculae often seen and often show nuclear molding. SCNECs have high mitotic counts (required: >10 per 2mm2 ) and necrosis. The SCNEC immunohistochemical profile includes reactivity for at least one cytokeratin (usually low-molecular weight), TTF1 (subset), and variable routine neuroendocrine markers (synaptophysin, chromogranin). Ki-67 proliferation index is >20% (often >70%), though the use of Ki-67 in grading has not yet been standardized and thus it is not established. p16 can be expressed independent of HPV infection. Particular anatomic sites have a higher frequency to occur as a combined lesion, with 5% of laryngeal SCNEC and up to 80% of oropharyngeal SCNEC occurring in combination with another histology, usually squamous cell carcinoma [1, 2].

#### **2.3 Large cell neuroendocrine carcinoma (LCNEC)**

LCNEC of the head and neck, like SCNEC, is a poorly differentiated neuroendocrine carcinoma most frequently occurring in the larynx, oropharynx, and sinonasal tract [3]. Of carcinomas arising in the larynx, 80% arise in the supraglottis [4]. Anatomic site LCNECs have independent risk factors, with laryngeal LNCECs associated with tobacco use in greater than 90% of cases, oropharyngeal and sinonasal LCNECs associated with high-risk HPV and smoking history, and the rare nasopharyngeal LCNECs associated with EBV positivity [5, 6]. LCNECs are comprised of nests or sheets of tumor cells with moderately hyperchromatic nuclei, vesicular or speckled chromatin, prominent nucleoli, and abundant cytoplasm. The tumor cells are large (greater than the combine diameter of three lymphocytes), with trabecular or organoid growth and peripheral palisading, rosettes, or comedo necrosis often seen. LCNECs have high mitotic counts (required: >10 per 2mm2 ) and necrosis. The immunohistochemical profile of LCENC is similar to that of other well differentiated neuroendocrine carcinomas and includes reactivity for at least one cytokeratin (usually low-molecular weight), TTF1 (minority of cases), p63 (variable), p40 (variable), and variable routine neuroendocrine markers (synaptophysin, chromogranin). Ki-67 proliferation index is always >20% (>40% in most cases), and while increased, is typically less than that of SCNECs. p16 can be overexpressed independent of HPV status.

#### **3. Lung**

Within the lung, neuroendocrine neoplasms are classified as follows: low grade typical carcinoid (TC), intermediate grade atypical carcinoid (AC), and high grade large cell neuroendocrine carcinoma (LCNEC) and small cell lung carcinoma (SCLC) (**Table 2**). These classifications can be grouped collectively as lung neuroendocrine neoplasms (NENs), with further subclassification of well differentiated typical carcinoid and moderately differentiated atypical carcinoid as neuroendocrine tumors (NETs), and the poorly differentiated neuroendocrine carcinomas (NECs) including SCLC and LCNEC. Among primary lung neuroendocrine carcinomas, SCLC is most frequent, with a prevalence of approximately 14% among lung cancers in the United States. In contrast, typical and atypical carcinoids account for less than 2% of all lung malignancies.

#### **Table 2.**

*Histologic and immunophenotypic characteristics of low and high-grade neuroendocrine tumors of the lung.*

LCNEC and SCLC are poorly differentiated and can contain different histologic components of other non-small cell carcinomas (usually adenocarcinoma or squamous cell carcinoma) and can be further classified as combined LCNEC or combined SCLC. For typical and atypical carcinoids, it is exceedingly rare to contain these other histologic components. These tumors are classified according to the degree of necrosis and the mitotic count, and for high grade LCNEC and SCLC, the cytologic features. Ki-67 is also commonly used by pathologists to differentiate the carcinoid tumors from the high-grade tumors, although it is not among the WHO diagnostic criteria. Further research is required to determine the optimal approach for using Ki-67 to differentiate typical versus atypical carcinoid, and carcinoids from LCNEC and SCLC. Classification is based primarily on mitotic counts per mm2, presence or absence of necrosis, and small cell versus large cell cytological features. Mitoses should be counted in areas of highest mitotic activity. Ki-67 is used to aid in distinguishing carcinoid tumors (NETs) from LCNEC and SCLC (NECs). Regarding Ki-67 proliferation index, based on most experts opinion, tumors with a Ki-67 index <5% are likely TC, >5% to <20% are AC, and > 40% are most likely high grade NEC (LCNEC or SCLC).

#### **3.1 Diffuse idiopathic pulmonary neuroendocrine hyperplasia (DIPNECH)**

Diffuse idiopathic pulmonary neuroendocrine hyperplasia (DIPNECH) represents the precursor lesion in a subset of carcinoid tumors and is comprised of multifocal areas of neuroendocrine cell hyperplasia associated with tumorlets in small airways. While the pathogenesis remains unclear, the underlying etiology is thought to be secondary to chronic pulmonary (airway) injury. The proliferating neuroendocrine cells have round to oval nuclei with salt-and-pepper chromatin, with moderate amounts of amphophilic cytoplasm. Proliferations of >5 (single cells or in clusters) measuring <5 mm that invade beyond the bronchoalveolar wall are classified as tumorlets.

*Histopathologic Diagnosis of Neuroendocrine Neoplasms of Head and Neck, Lung… DOI: http://dx.doi.org/10.5772/intechopen.109096*

DIPNECH expresses neuroendocrine markers (chromogranin, synaptophysin, CD56), TTF-1, and pancytokeratins, and is negative for p40, p63, and high-molecular weight cytokeratins. All neuroendocrine hyperplastic nodules larger than 5 mm are classified as carcinoids.

#### **3.2 Typical carcinoid (G1 NET), atypical carcinoid (G2 NET), and carcinoid tumor NOS**

Well differentiated carcinoid tumors are rare and commonly seen in younger patients with no smoking history. They occur both centrally and peripherally (TC = more central; AC = more peripheral). Current terminology proposed by the International Agency for Research on Cancer (IARC) and WHO Classification of Tumors Group includes well differentiated grade 1 (G1 NET) corresponding to TC, and well differentiated grade 2 (G2 NET) corresponding to AC. Carcinoids are associated with the precursor lesion diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) in 60–75% of cases [7]. The tumors have well differentiated architecture and neuroendocrine morphology. Tumor cells are small to intermediate in size, cuboidal to polygonal and uniform, with finely granular salt-and-pepper chromatin and abundant eosinophilic cytoplasm and inconspicuous nucleoli. Trabeculae, rosettes, palisading, and organoid nesting may be seen. Carcinoid tumors are typically positive for low molecular weight cytokeratins, chromogranin A, synaptophysin, CD56, INSM1, and can express TTF-1, the latter being more common in peripheral lesions (**Figure 1**). Ki-67 is not required for diagnosis, with still no consistent threshold for Ki-67 proliferation index in distinguishing TC (G1 NET) from AC (G2 NET), with its main function in excluding NEC (LCNEC and SCLC). Typical carcinoids have a Ki-67 index of <5% with <2 mitoses/2 mm2 without necrosis. Atypical carcinoids exhibit a Ki-67 index of 5–20% with 2–10 mitoses/2 mm2 and foci of necrosis (**Table 2**). The distinction of TC versus AC on biopsy can be challenging and should be reserved for the resection specimen, with the terminology of "carcinoid tumor NOS" being recommended. If features suggestive of AC are present (higher Ki-67 index, punctate necrosis) this can be mentioned in a comment favoring classification as AC even on limited tissue. For patients with metastatic pulmonary carcinoids, the term "metastatic carcinoid tumor NOS" is preferred. 3.3 Large Cell Neuroendocrine Carcinoma (LCNEC).

Large cell neuroendocrine carcinomas (LCNEC) are primarily located in the lung periphery. Metastases are present in 40–50% of patients at presentation, with common sites including liver and bone, with brain metastases occurring in approximately 50% of patients [8]. LCNEC is highly associated with smoking and represents 3% of resected lung carcinomas [9]. On gross appearance, the cut surface appears tan-red, well circumscribed, and necrotic. The tumor is high grade, poorly differentiated, and exhibits a neuroendocrine growth pattern with organoid nesting, trabecular, peripheral palisading, and rosettes. The tumor cells are large (usually greater than 3 lymphocytes), with coarsely granular chromatin, moderate to abundant cytoplasm, and prominent nucleoli. There is abundant necrosis and high mitotic counts (>10 mitoses/2 mm2, generally very high) (**Figure 2**). A majority of tumors express at least two neuroendocrine markers (synaptophysin, chromogranin A, CD56), pankeratins, and 50% also express TTF1 [10]. Tumor cells usually lack napsin A, with weak staining in a minority of cases, therefore the combination of strong TTF1 staining with negative/weak napsin A is likely to represent a NEC

#### **Figure 2.**

*Large cell neuroendocrine carcinoma (LCNEC). In LCNEC, neoplastic cells form large nests with extensive areas of central necrosis (a, 40X; B, 100X). At higher magnification the contrast to small cell carcinoma is obvious: The cytoplasm is conspicuous, there is no nuclear molding, the chromatin is condensed or clear with prominent nucleoli, crush artifact is absent or, at most, focal (C, 200X). Pan-cytokeratin is diffusely positive (D, AE1/AE3, 100X). Neuroendocrine markers may be weak or patchy, but usually are more consistently positive than in SCLC (E, synaptophysin, 200X). CD56 is very useful as a surrogate neuroendocrine marker when diffusely and strongly positive (F, CD56, 100X).*

#### **Figure 3.**

*Small cell lung carcinoma (SCLC). SCLC is comprised of sheets of atypical cells with extensive necrosis and crush artifact due to nuclear fragility (a, 200X). Tumor cells display high nuclear to cytoplasm ratio and there is nuclear molding due to close nuclear vicinity. Mitotic activity is brisk, nuclei are hyperchromatic or have granular (salt and pepper) appearance with inconspicuous or very small nucleoli (B, 400X). Cytokeratin is present in majority of cases, but frequently is discontinuous or punctate (C, AE1/AE3, 200X). Neuroendocrine markers, when expressed, are seen in most neoplastic cells, but occasionally may be patchy, focal or entirely absent (D, chromogranin, 200X). In cases with absent neuroendocrine differentiation, the diagnosis relies solely on morphology. Proliferation index is markedly increased, typically more than 70% (E, Ki-67, 200X). Expression of nuclear TTF-1 is not organ specific, but when present, indicates that the neoplasm is more likely to originate from lung than other organs (F, TTF-1, 200X).*

*Histopathologic Diagnosis of Neuroendocrine Neoplasms of Head and Neck, Lung… DOI: http://dx.doi.org/10.5772/intechopen.109096*

(LCNEC or SCLC) versus adenocarcinoma. The Ki-67 index is >40%. LCNEC can exist as a combined tumor, combined LCNEC, with components of adenocarcinoma, squamous cell carcinoma, spindle cell carcinoma, or giant cell carcinoma. 3.4 Small Cell Lung Carcinoma (SCLC).

Small cell lung carcinomas are typically centrally located in major airways, and can frequently involve mediastinal lymph nodes, with metastatic spread to other organs common at the time of presentation. Patients may present with acute symptoms due to intrathoracic tumor growth, extrapulmonary spread, or paraneoplastic syndromes, the latter being most common in SCLC and can be the patient's initial presentation. SCLC is a malignant epithelial tumor comprised of small cells with finely granular chromatin, scant cytoplasm, and inconspicuous or absent nucleoli. The tumor cells are small to mid-size (usually less than the size of 3 resting lymphocytes) and are oval to spindle shape. They have a high mitotic count (>10 mitoses/2 mm2) with frequent and vast necrosis (**Figure 3**). Most SCLCs express neuroendocrine differentiation markers (synaptophysin, chromogranin, CD56). SCLC may be negative for neuroendocrine markers in 5–10% of cases. INSM1 is consistently positive in SLCL with positive staining for pancytokeratins and TTF1, and is generally negative for CK20, Napsin A, p63, and p40 [11]. On gross examination, the tumor appears as a large perihilar mass with a tan, necrotic cut surface. SCLC can exist as a combined lesion of non-small cell carcinoma which can include LCNEC, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, spindle cell carcinoma, or giant cell carcinoma. In combined SCLC, the second component should make up > = 10% of the tumor. While not part of the diagnostic criteria, Ki-67 can be useful in crushed biopsies to avoid the pitfall of misdiagnosing carcinoid tumors, which may also show significant crush artifact, with a very high Ki-67 index (>70%) excluding TC or AC.

#### **4. Gastrointestinal tract**

Overall, based on the NCI-SEER database, the gastrointestinal tract has the highest incidence of neuroendocrine tumors (NETs) in the body, comprising about 55% of all NETs. By contrast the second most common site is the bronchopulmonary system, accounting for 25% of NETs. In the gastrointestinal tract, the small intestine is by far the site with highest incidence, accounting for approximately 45% of all neuroendocrine tumors, followed by rectum (20%), appendix (17%), colon (11%) and stomach (7%).

#### **4.1 Esophagus**

Esophageal neuroendocrine neoplasms (NENs), like other organ systems, are comprised of well differentiated neuroendocrine tumors (NETs), poorly differentiated neuroendocrine carcinomas (NECs), and mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs), which includes mixed adenoneuroendocrine carcinoma (MANEC). NEN are typically small lesions most commonly occurring in the lower esophagus, often associated with Barrett esophagus, and infiltrative at the time of diagnosis [12, 13]. Esophageal NEN share similar features of NEN of other anatomic sites. While NET of the esophagus has been described in the literature, the majority of cases are in fact NECs, and are further subclassified as small cell NEC (SNEC) and large cell NEC (LCNEC) [14]. The NEC is comprised of medium-sized to large cells

(SNEC and LCENC, respectively) arranged in solid, rosette-like, or palisading architecture with elongated nuclei with fine chromatin, inconspicuous nucleoli in SNEC and large nucleoli in LCNEC, and scant basophilic cytoplasm. Mitotic activity and necrosis are substantial. The immunohistochemical profile of NEC is similar to that of other neuroendocrine carcinomas and includes variable reactivity for neuroendocrine markers (synaptophysin > chromogranin), TTF1 (majority of cases), and absence of p40 and CK5/6 expression. NET share similar histopathologic features with NEC, however NET will have less necrosis, with stronger reactivity for chromogranin (versus NEC). Additionally, many NETs express hormone immunoreactivity (glucagon, PP, gastrin, and calcitonin) [15, 16]. MiNENs of the esophagus usually consist of NEC and either squamous cell carcinoma or adenocarcinoma (if underlying Barrett mucosa or ectopic gastric mucosa is identified) [17]. Grading of esophageal NENs is performed using the same grading system as used for gastroenteropancreatic NENs.

#### **4.2 Stomach**

The classification for neuroendocrine neoplasms of the stomach is identical to that of the esophagus, as follows: neuroendocrine neoplasms (NENs) comprised of well differentiated neuroendocrine tumors (NETs), poorly differentiated neuroendocrine carcinomas (NECs), and mixed neuroendocrine–non-neuroendocrine neoplasms (MiNENs) including MANEC. NEN of the stomach occur with a site-specific anatomic distribution based on tumor subtype. Enterochromaffin-like–cell (ECLcell) NETs arise in the corpus/fundus, D-cell and G-cell NETs in the antrum, and enterochromaffin-cell (EC-cell) NETs in both the antrum and corpus/fundus [18]. NECs and MiNENs usually occur in the antral or cardiac regions but can occur at any site. NETs are comprised of a population of monotonous cells with round nuclei and finely stippled chromatin, with NECs composed of sheets of small or large poorly differentiated cells. The immunohistochemical profile of gastric NENs is like that of other gastrointestinal neuroendocrine neoplasms at other anatomic sites. Gastric NECs are comprised of sheets of poorly differentiated small or large cells, and are further subtyped as small cell NEC (SCNEC) and large cell NEC (LCNEC) based on cell cytomorphologic size. LCNECs are comprised of large cells with vesicular nuclei with prominent nucleoli and abundant eosinophilic cytoplasm. SCNECs are comprised of small to mid-sized cells with scant cytoplasm and hyperchromatic nuclei and inconspicuous or absent nucleoli. NECs have a high mitotic count (> 20 mitoses/mm2 ) and high Ki-67 proliferation index (>40–50%). NECs express positivity for neuroendocrine markers (synaptophysin > chromogranin), may be focally positive for TTF-1, and have high mitotic counts and an increased Ki-67 proliferation index. Mixed adenocarcinoma neuroendocrine carcinomas (MANECs) are MiNENs composed of adenocarcinoma associated with NEC component. The neuroendocrine component of MANEC has a high Ki-67 proliferation index (> 55%) [19]. Mixed adenocarcinoma-NETs are comprised of areas of tubular, papillary, or mucinous adenocarcinoma mixed with areas of G1 or G2 NET.

Gastric ECL-cell NETs are histamine-producing, and typically show smaller microlobular or trabecular architecture and lack necrosis. They are comprised of monotonous well differentiated cells with abundant cytoplasm and round nuclei and have inconspicuous or absent nucleoli. Mitotic figures are rare to absent. Type 1 ECL-cell NETs usually infiltrate beyond the muscularis mucosae, with a majority of cases classified as G1 or G2. Type 2 ECL-cell NETs are confined to the mucosa and submucosa when they are G1, with Type 3 NETs (G1 to G3) invading into the

#### *Histopathologic Diagnosis of Neuroendocrine Neoplasms of Head and Neck, Lung… DOI: http://dx.doi.org/10.5772/intechopen.109096*

gastric wall with distant and lymph node metastases common. ECL-cell NETs are immunoreactive for VMAT2, HDC, and SSTR2A, and can show scattered positivity for serotonin, ghrelin, somatostatin, and α-hCG [20, 21]. Gastric EC-cell NETs produce serotonin and have histomorphological features similar to ileal EC-cell NETs, with nests of uniform tumor cells with peripheral palisading. EC-cell NET cells are immunoreactive for serotonin, SSTR2A, and CDX2. Gastric G-cell NETs produce gastrin and usually show monotonous cells with scant cytoplasm in a trabecular or gyriform pattern, and are immunoreactive for gastrin and SSTR2A. Gastric D-cell NETs produce somatostatin and are comprised of well differentiated monomorphic cells that are immunoreactive for somatostatin, chromogranin, synaptophysin, and SSTR2A. Grading of gastric NENs is performed by using the same system used for other gastroenteropancreatic NENs. Staging of gastric NETs is performed using NETspecific Union for International Cancer Control (UICC) TNM staging system; NEC and MiNEN are staged following the system used for adenocarcinomas.

#### **4.3 Small intestine and ampulla**

Neuroendocrine neoplasms (NENs) of the small intestine and ampulla, like other NEN of the GI tract, are epithelial neoplasms with neuroendocrine differentiation of the duodenum, jejunum, and ileum, are classified as follows: well differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). In the small intestine MiNENs have an exocrine component (commonly adenocarcinoma) and a neuroendocrine component (usually NEC), each accounting for ≥30% of the neoplasm. NETs are further subclassified based on hormone production, and include gastrinoma, somatostatin-producing neuroendocrine tumor, gangliocytic paraganglioma, NEC (further subtyped as small cell neuroendocrine carcinoma (SNEC) and large cell neuroendocrine carcinoma (LCNEC), and MiNEN. Duodenal NETs are located in part 1 or 2, with those in part 2 predominating in the ampullary region in 95% of cases. Somatostatin-producing NET, gangliocytic paraganglioma, and NEC are almost exclusively located in the ampullary region. Uniquely, jejuno-ileal NETs are often multifocal (2–100 tumors) in at least 30% of cases [22, 23], and those located in the upper jejunum tend to be large and locally infiltrative [24].