Doctor of Medicine(BRANCH-III)

DISSERTATION on

METASTATIC STATUS OF LYMPHNODES FROM RADICAL SURGICAL RESECTIONS - ROLE OF CYTOKERATIN AS AN ADJUVANT TO STANDARD STAINING METHOD

submitted in partial fulfillment of the requirements for the degree of

M.D. PATHOLOGY

THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY CHENNAI

TIRUNELVELI MEDICAL COLLEGE TIRUNELVELI MAY 2020

CERTIFICATE

This is to certify that the dissertation titled “METASTATIC STATUS OF

LYMPHNODES FROM RADICAL SURGICAL RESECTIONS-ROLE OF

CYTOKERATIN AS AN ADJUVANT TO STANDARD STAINING

METHOD” is a bonafide work done by Dr.S.AISWARYA NIVEDHITHA,

Post Graduate Student, Department of Pathology, Tirunelveli Medical College,

Tirunelveli – 627011, in partial fulfilment of the university rules and regulations for the award of MD DEGREE in PATHOLOGY BRANCH-III, under my guidance and supervision, during the academic period from 2017 to 2020.

DR. S.M.KANNAN, MS .MCh (Uro)., Dean, Tirunelveli Medical College, Tirunelveli-627011. CERTIFICATE

I hereby certify that this dissertation entitled “METASTATIC STATUS OF

LYMPHNODES FROM RADICAL SURGICAL RESECTIONS-ROLE OF

CYTOKERATIN AS AN ADJUVANT TO STANDARD STAINING

METHOD” is a record of work done by Dr.S.AISWARYA NIVEDHITHA, in the Department of Pathology, Tirunelveli Medical College, Tirunelveli, during her postgraduate degree course period from 2017- 2020. This work has not formed the basis for previous award of any degree.

Prof.DR.K.SWAMINATHAN. MD, Department of Pathology, Tirunelveli Medical College, Tirunelveli- 627011

Prof.DR.K. SHANTARAMAN.MD, Professor and Head, Department of Pathology, Tirunelveli Medical College Tirunelveli- 627011.

DECLARATION I solemnly declare that the dissertation titled “METASTATIC STATUS OF

LYMPHNODES FROM RADICAL SURGICAL RESECTIONS-ROLE OF

CYTOKERATIN AS AN ADJUVANT TO STANDARD STAINING METHOD” was done by me at Tirunelveli Medical College, Tirunelveli– 627011, during the period

September 2017 to august 2019 under the guidance and supervision of

Prof.DR.K.SWAMINATHAN, MD, to be submitted to The Tamil Nadu Dr. M.G.R.

Medical University towards the partial fulfilment of requirements for the award of MD

DEGREE in PATHOLOGY BRANCH-III.

Place : Tirunelveli Date :

Dr.S.AISWARYA NIVEDHITHA, Register No: 201713301 Post Graduate Student, Department of Pathology Tirunelveli Medical College, Tirunelveli – 627011.

ACKNOWLEDGEMENT I thank Professor Dr.S.M.KANNAN, M.S MCh, Dean, Tirunelveli Medical

College, for having permitted me to conduct the study and use the hospital resources in the study.

I express my heartfelt gratitude to Professor Dr. SHANTARAMAN. K. MD,

Professor and Head, Department of Pathology, for his inspiration, advice and guidance in making this work complete.

I am extremely thankful to Professors DR. SWAMINATHAN .K. M.D.,

DR.SURESH DURAI. J. M.D., DR.ARASI RAJESH, M.D, DR.VASUKI, M.D, my

Associate Professors DR.V.BHAGYALAKSHMI, M.D, DR.J.JOHNSY MERLA,

MD, Department of Pathology, for guiding me during the period of study.

I am extremely thankful to Assistant Professors Dr.Hidhaya Fathima M.D,

Dr.Sindhuja M.D, Dr.Mahalakshmi M.D, Dr.Dina Mary MD, Dr. Dharma Saranya

MD, Dr.Chandhru Mari MD, Department of Pathology, for guiding me academically and professionally during the period of study.

I also thank all the lab technicians and my fellow postgraduates for their cooperation which enormously helped me in the study. Without their humble cooperation, this study would not have been possible.

I thank GOD AND MY FAMILY, for blessing me not only in this study, but in all endeavours of my life.

CERTIFICATE – II

This is certify that this dissertation work title “METASTATIC STATUS OF

LYMPHNODES FROM RADICAL SURGICAL RESECTIONS-ROLE OF

CYTOKERATIN AS AN ADJUVANT TO STANDARD STAINING METHOD” of the candidate Dr.S.AISWARYA NIVEDHITHA with registration Number

201713301 for the award of M.D. Degree in the branch of PATHOLOGY (III).

I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion page and result shows 16 percentage of plagiarism in the dissertation.

Guide & Supervisor sign with Seal.

TABLE OF CONTENTS

Sl.No. CONTENT Page No.

1. INTRODUCTION 1

2. AIM & OBJECTIVES OF THE STUDY 3

3. REVIEW OF LITERATURE 4

4. METHODOLOGY 53

5. OBSERVATION AND RESULTS 59

6. DISCUSSION 85

7. CONCLUSION 89

8. BIBLIOGRAPHY

9. ANNEXURE

 MASTER CHART

LIST OF ABBREVIATIONS USED IN THIS STUDY ACB ADENOCARCINOMA BLADDER ACE ADENOCARCINOMA ENDOMETRIUM ALCL ANAPLASTIC LARGE CELL LYMPHOMA AMC ATYPICAL MEDULLARY CARCINOMA DAB DIAMINO BENZIDINE ENE EXTRA NODAL EXTENSION FNAC FINE NEEDLE ASPIRATION CYTOLOGY H&E HEMATOXYLIN AND EOSIN HEV HIGH ENDOTHELIAL VENULE IDC INVASIVE DUCTAL CARCINOMA IDLC INVASIVE DUCTO LOBULAR CARCINOMA IHC IMMUNOHISTOCHEMISTRY ILC INVASIVE LOBULAR CARCINOMA MDACCo MODERATEDLY DIFFERENTIATED ADENOCARCINOMA COLON MDACCx MODERATED LY DIFFERENTIATED ADENOCARCINOMA CERVIX MDACS MODERATEDLY DIFFERENTIATED ADENOCARCINOMA STOMACH MDSCCBM MODERATEDLY DIFFERENTIATED SQUAMOUS CELL CARCINOMA BUCCAL MUCOSA MDSCCL MODERATEDLY DIFFERENTIATED SQUAMOUS CELL CARCINOMA MDSCCT MODERATEDLY DIFFERENTIATED SQUAMOUS CELL CARCINOMA TONGUE MuADCS MUCINOUS ADENOCARCINOMA STOMACH NOS NOT OTHERWISE SPECIFIED PANCK PAN CYTOKERATIN PapCA/PTC PAPILLARY THYROID CARCINOMA PDACS POORLY DIFFERENTIATED ADENOCARCINOMA STOMACH PET POSITRON EMISSION TOMOGRAPHY SPAC SEROUS PAPILLARY CYSTADENOCARCINOMA SPECT SINGLE PHOTON EMISSION COMPUTED TOMOGRAPHY VEGF VASCULAR ENDOTHELIAL GROWTH FACTOR WDACCx WELL DIFFERENTIATED ADENOCARCINOMA CERVIX WDACE WELL DIFFERENTIATED ADENOCARCINOMA ENDOMETRIUM WDACI WELL DIFFERENTIATED ADENOCARCINOMA ILEUM WDACS WELL DIFFERENTIATED ADENOCARCINOMA STOMACH WDEACOv WELL DIFFERENTIATED ENDOMETRIOID ADENOCARCINOMA OVARY WDSCCA MODERATEDLY DIFFERENTIATED SQUAMOUS CELL CARCINOMA ALVEOLUS WDSCCBM WELL DIFFERENTIATED SQUAMOUS CELL CARCINOMA BUCCAL MUCOSA WDSCCF MODERATEDLY DIFFERENTIATED SQUAMOUS CELL CARCINOMA FOOT

INTRODUCTION

Cancer metastasis is a complex, multistep process that involves dissemination of cancer cells from the primary site to distant organs. Lymph nodes are initial sites for metastasis in many solid tumours[1].

Metastasis, the spread of a tumour cells from its primary lymph node

(LN) to distant organs, is the most fearsome aspect of cancer. Patients presenting with metastatic disease or those developing metastases after successful management of primary tumour carry a universally grave prognosis.

Metastasis is the cause of 90% of cancer deaths. Tumour cell invade either the blood or the lymphatic vessels to access the general circulation and then establish in other (visceral) tissues. Hence, nodal status is a significant predictor for survival of patients with malignant tumours[2].

Though surgical resection done early, some patients get recurrence and die of cancer. So it is our thought that those with an early stage of malignancy and died, might have got occult metastasis in their nodes at the time of initial diagnosis. Those occult metastasis can be identified by evaluating deeper levels of blocks by or IHC with epithelial markers such as cytokeratin[3].

Cytokeratin is a family of water insoluble intracytoplasmic structural proteins that are the dominant intermediate filament protein of epithelial and hair forming cell, also present in epithelial tumours, within a cell, form a dense network radiating from the nucleus to the plasma membrane which helps cells 1 to resist mechanic stress. Pan cytokeratin immunohistochemical stain that reacts to a wide range of keratins includes AE1/AE3, MNF116.

AE-3 is a broad spectrum anti pan-cytokeratin antibody cocktail, which differentiates epithelial tumours from non epithelial tumours. The pan cytokeratin antibody cocktail is a well-known broad spectrum immune histochemical epithelial marker for screening for epithelial differentiation in tumour and their metastases. Positive pan cytokeratin staining with the antibody in the lymph node or bone marrow can be useful for identification of metastasis[4].

Hence the present study is carried out with the aim to establish the utility and expression of immunohistochemistry marker (pan cytokeratin) in identifying occult metastasis in lymph nodes.

AIM

To determine the effectiveness of immunohistochemistry (Pan cytokeratin) as an adjuvant in detecting occult metastasis in lymph nodes

OBJECTIVES:

1. To Establish the role of cytokeratin in detecting occult metastases in

lymph nodes which are missed in routine H-E staining.

2. To understand their aid in accurate staging of the disease, thereby

determining further treatment plan of such patients. Thus, improving the

prognosis and survival of patients with malignancies.

REVIEW OF LITERATURE

LYMPHNODES - FUNCTION AND THEIR DISTRIBUTION:

The lymph node is one of the important anatomic components of the immune system[5, 6]. They are encapsulated and act as centres of antigen presentation, lymphocyte activation, differentiation and proliferation. These are facilitated by complex trafficking of cells and lymphatic flow via the structure

[7]. They generate mature, antigen-primed B and T cells, filter particles, including microbes, from the lymph by the action of macrophages. Normal young adult body has up to 450 lymph nodes, of which 60–70 are found in the head and neck, 100 in the thorax and as many as 250 in the pelvis and abdomen.

They are especially numerous in the neck, mediastinum, posterior abdominal wall, abdominal mesenteries, pelvis and proximal regions of the limbs (axillary and inguinal lymph nodes). Most of them lie close to the viscera, especially in the mesenteries.

Nodes in most of the regions drain the body’s surface, digestive system, or respiratory system. All these areas are having high-risk for the entry of foreign pathogens. Lymph nodes are abundant and accessible to palpation in the axilla, groin and femoral region, and neck. Deep sites that are not accessible for palpation include, those associated with the trachea and bronchi in the thorax, and with aorta and its branches in the abdomen.

MICROSTRUCTURE

Lymph nodes are small, oval or kidney-shaped bodies. They are 0.1–2.5 cm long, lying along the course of the lymphatic vessels. Each of which usually has a slight indentation on one side- the hilum, through which blood vessels enter and leave. And also the efferent lymphatic vessels leave the node via the hilum. Several afferent lymphatic vessels enter the capsule around the periphery of the lymph node. Lymph nodes have a cellular cortex and a medulla, which contains a network of minute lymphatic channels called sinuses through which lymph from the afferent lymphatics is filtered, to be collected at the hilum by the efferent lymphatics. The cortex is absent at the region of hilum, where the medulla reaches the surface. The capsule of lymph nodes are composed of collagen fibres, elastin fibres (particularly in the deeper layers) and few fibroblasts. Trabeculae of dense connective tissue extend radially into the node from the capsule. They are continuous with a network of fine type III collagen fibrils (reticulin), which branch and interconnect to form a very dense network in the cortex of the lymph node and providing attachment for various cells, such as dendritic cells, macrophages and lymphocytes. There are only fewer fibres in the germinal centres of follicles.

Figure -1 structure of lymph node; Adapted from - Gray’s anatomy,41st

edition.

VASCULAR SUPPLY

Arteries and veins supplying lymph nodes pass through the hilum, give off straight branches that traverse the medulla and send out minor branches as they do so. In the cortex of lymph nodes, arteries form dense arcades of arterioles and capillaries in numerous anastomosing loops, and then returning to highly branched venules and veins. Capillaries are especially profuse around the follicles, which contain only fewer vessels. Post capillary HEVs are numerous particularly in the para cortical zones. They form an important site of blood- borne lymphocyte extravasation into lymphoid tissue[8]. These specialized endothelial cells have receptors that bind intravascular lymphocytes as they pass by and facilitate their migration into the reticular meshwork[9]. Veins leave a

6 node through its principal trabeculae and capsule, and drain them and also the surrounding connective tissue.

LYMPHATIC TRUNKS AND DUCTS

All lymphatic vessels coalesce to form larger trunks or ducts, which then drain into the venous system at sites in the neck where the internal jugular veins join the subclavian veins to form the brachiocephalic veins.

 Lymph from the right side of the head and neck, the right upper limb, and

the right side of the thorax is carried by lymphatic vessels that connect

with veins on the right side of the neck.

 Lymph from all other regions of the body is carried by lymphatic vessels

that drain into veins on the left side of the neck.

HISTOLOGY

The three major regions of a lymph node are cortex, paracortex, and medulla[10]. The cortex is located just beneath the capsule and represents the compartment where most lymphoid follicles reside and is composed of mainly

B lymphocytes and also follicular dendritic cells. The medulla which is close to the hilum and grows in the form of cords. It is rich in lymph sinuses, arteries, and veins but contains only a minor lymphocytic component that is a combination of B and T lymphocytes. Appearance of the follicles varies according to their state of activity. Primary follicles appear as round aggregates

7 of small lymphocytes with little cytoplasm, secondary follicles appear following antigenic stimulation and they are characterized by the presence of germinal centers surrounded by mantle zones. The germinal center cells are mostly of B lymphocytes known as follicular center cells (centroblasts and centrocytes or small and large cleaved and noncleaved cells), macrophages, and follicular dendritic cells, but a population of T follicular–helper cells also present normally in this compartment. The germinal center contains pale-staining large cells on hematoxylin and eosin sections and that are normally highly proliferative and shows polarization toward the side of antigen stimulation. The surrounding mantle zone B lymphocytes are smaller in size.

Figure -2 Adapted fromWheater’s functional histology, sixth edition-

Barbara young, Geraldine O’Dowd

The Afferent lymphatic channels enter the convexity of the lymph node, and fluid drains into the peripheral subcapsular sinuses. The sinuses of the lymph node are lined by macrophages, and these cells filter out the debris from the fluid. The fluid percolates through the sinuses and emerges via an efferent lymphatic channel at the hilum of the node. Follicles in the cortex of the lymph node are the site of B-cell activation. The intervening paracortical areas are the main sites of T cells in the lymph node.

VARIOUS ROUTES OF METASTASIS

At an early stage of malignancy, malignant cells are confined to the primary site within the boundary of certain surrounding tissues. When the disease progresses, some cancer cells, as the result of genetic/ epigenetic predisposition, and the influence of environmental interaction/stimulation, and indeed the combination of these elements, become aggressive and start to breach the surrounding structure. These cells would either directly invade the surrounding tissue, or disseminate via lymphatic and haematogenous routes.

Direct invasion result in the spreading of cancer cells to surrounding tissues and adjacent organs. For example, local invasion of prostate cancer, can affect the erectile nerves, seminal vesicles, bladder and rectum nearby the prostate[11].

The lymphatic and vascular routes differ from cancer to cancer according to their primary sites of lesion, however, they frequently result in systemic spread of malignant cells to distant organs, such as bones, lung, and liver.

Both lymphatic and haematogenous dissemination occur, even during early stages of the malignancy, and are seen in a vast majority of the patients who have an advanced malignancy. Brain, bone, lung and liver are the most common haematogenous sites from certain solid tumours.[12, 13]

The process of metastasis is complex, which incorporates multiple cells, factors and stages. During the development and progression of primary malignancies, certain clones of malignant cells will have the required genotypic and phenotypic characteristics to enable themselves to interact with the local microenvironment. They release VEGF to initiate angiogenesis, and thus enhancing the blood flow to the tumour. The stromal cells are rich sources of protein factors that directly act on cancer cells thus helping the growth of tumours and dissemination of cancer cells. On the other hand, some of the stromal cell derived factors will directly promote angiogenesis, thus supporting the growth and spread of an aggressive tumour. An example of these stroma- derived protein factors are hepatocyte growth factor (HGF), which is a cytokine secreted by the stromal cells, which has been implicated in the angiogenesis and the dissemination of malignant cells[14] .The disruption of intercellular adhesion in the malignancy causes some malignant cells to detach from the mass called detachment, followed by these cells invading via the extracellular matrix, a process called invasion which incorporates the motility, migration of malignant cells and breakdown of extracellular matrix. Some malignant cells

10 will penetrate the blood vessels and enter the circulation called intravasation.

From this point, cells move away from the primary site and circulate in the blood where, they would encounter resistance by the immune system and the mechanical stresses of blood flow. Some malignant cells will eventually survive and adopt a process to leave the circulation, called as extravasation, in which cells adhere and penetrate the blood vessel again (virtual reversal of the intravasation process). Once the malignant cells escape from the circulation, they have to survive and finally develop a secondary malignancy at other site.

This complex process also needs the integration of multiple factors and events, such as invasion of malignancy, angiogenesis and the interaction between malignant cells and the local microenvironment at a distant site/organ.

LYMPH NODE METASTASIS OF MALIGNANCY:

Lymph nodes are the most common site of metastasis of malignancy and constitute the first clinical manifestation of the disease sometimes [15, 16].

Presence of malignant cells within efferent lymph vessels and/or extranodal adipose tissue, have prognostic significance.

Any malignant tumour can give rise to lymph node metastases, but the incidence varies depending upon the tumour type. It is common with carcinomas (epithelial malignancies), malignant melanomas, and germ cell tumours, rare with sarcomas and central nervous system tumours. Large cell lymphomas primary in an organ (such as stomach and thyroid) sometimes

11 involve the regional nodes in a pattern consistent with metastatic spread and the pattern of lymph node infiltration of ALCL may mimic a metastasis. An additional diagnosis to be considered in a lymph node involvement by metastatic tumour is malignant mesothelioma. Most of the primary tumours are located in the peritoneum than the pleura, regardless of the location of the lymph nodes[17]. Nodal metastases of squamous cell carcinoma have a particular tendency to undergo cystic changes. When these changes are prominent in a lymph node located in the neck, a mistaken diagnosis of branchial cleft cyst maybe made. The location of a lymph node involved by metastatic carcinoma gives important clues about the possible site of the primary tumour.

The majority of malignant tumours metastatic to upper cervical lymph nodes arise from the upper aerodigestive tract. The sites well known for harbouring small and clinically undetectable primaries in the presence of cervical lymphadenopathy are the nasopharynx and retrotonsillar pillar[18-20].

Midcervical nodes containing features of papillary carcinoma are usually of metastatic thyroid carcinoma, a possibility that becomes a virtual certainty in the presence of psammoma bodies. These papillary tumours may also originate from salivary gland, thymus or female genital tract. Squamous cell carcinomas present in lymph nodes of this region usually arise from upper aerodigestive tract, especially pharynx and larynx[21] .Most of the carcinomas metastatic to supraclavicular lymph nodes arise from the lung or breast. Other sources of

12 metastases to this group of lymph nodes, particularly if located on the left side, are carcinoma of stomach, pancreas, prostate, and testis[19, 22]. They reach the node via the terminal collecting lymphatic trunks. Those nodes involved by intra-abdominal carcinomas are referred as Virchow or Troisier nodes[23]. The majority of metastatic tumours in axillary nodes of adult females are breast carcinoma and malignant melanoma[24, 25]. Lung carcinoma should also be considered, especially in older patients with a history of smoking[26].

Metastasis in inguinal nodes are mostly of carcinomas of external genital organs or malignant melanomas of the lower extremities, but only rarely from the internal abdominal organs such as ovary, uterine cervix, and anal canal, even less commonly from the testis, unless direct extension to the scrotal skin has occurred[27].

Some of the benign conditions of lymph nodes that can mistakenly be interpreted as metastatic carcinoma. They include hyperplastic mesothelial cells[28], megakaryocytes[29], signet ring sinus histiocytosis[30], the related nodal muciphages and mucicarminophilic histiocytosis[31,32], florid anthracosis / anthracosilicosis[33].

METHODS OF DETECTION OF METASTASIS IN LYMPHNODES:

Non invasive radiological procedures are often done during the evaluation of malignancies. Lymph nodes are usually diagnosed as having metastasis by CT and MRI when their short axis is >10 mm in diameter [34].

But Such size criteria, shown to be uncertain [35]. The detection of metastasis

13 in unenlarged (occult) nodes are frequently low by PET and SPECT. Especially small nodal metastases < 5 mm are frequently missed by PET scan in breast malignancy[36].

The cytological evaluation of lymph nodes is a cost-effective simple procedure which may provide valuable information regarding the disease process, including both neoplastic and non-neoplastic conditions. Cytological evaluation of lymph nodes may be done either by fine-needle aspiration cytology (FNAC) or preparation of imprint or touch smears.

Fine-needle aspiration cytology has become a well-established method of diagnosis for metastasis to the lymph nodes[37]. This simple technique has gained wide acceptance since it offers several advantage to patients and physicians. The technique is relatively painless and minimally invasive, produces fast results and its accuracy can approach that of histopathology in providing a definite diagnosis[38]. It is now the first-line investigation technique for the diagnosis of significantly enlarged lymph node. This method is applicable both to the lesions that are easily palpable and also to the lesions which are deeply located under radiological guidance. The results of fine-needle aspiration (FNA) compare favourably with those of tissue biopsies in some situations, the aspirate has qualities of a micro biopsy [39]. However, FNAC is not a substitute for conventional histopathology, and it should be regarded as an essential component of preoperative/pretreatment diagnosis, when correlated clinico - radiologically and with other investigations[40].

Immunohistochemical techniques using anticytokeratin antibodies can identify the lymph node metastases which are missed on routine Haematoxylin and eosin staining of tissues ,since cytokeratin proteins are the essential constituents of the cytoskeleton proteins of both normal and malignant epithelial cells in the lymph nodes, they act as reliable markers for the epithelial origin of cells[41]. It is established that higher diagnostic accuracies can be achieved using multiple serial sections and by IHC [42, 43].

METASTASIS OF VARIABLE MALIGNANCIES TO REGIONAL

LYMPH NODES:

BREAST CARCINOMA

Grossly, all lymph nodes present in an axillary dissection specimen need to be identified and submitted for histopathological evaluation to identify metastases [44]. The total number of lymph nodes is an important prognostic factor. Gross examination by itself is unreliable for identifying the number of lymph nodes involved by malignancy, as their number is usually underestimated.

In general, invasive breast carcinoma is associated with increased risk of axillary lymph node metastases. Metastases to lymph nodes reach via afferent lymphatics and enter sinuses. Hence in nodes with partial involvement, the metastases particularly have a sinusoidal distribution. With more extensive involvement, the architecture of the lymph nodes will be progressively replaced.

The microscopic patterns of nodal metastases reflect the histologic types of the

15 primary breast malignancy, with exceptions in ≤ 5% of cases [45]. The recognition of blood vessel and lymphatic invasion in specimens is important, because the presence of either strongly correlates with the presence of nodal metastases [46]. The most common type of breast malignancy, representing up to 80% of all cases is infiltrating ductal carcinoma, not otherwise specified(NOS). This type is often accompanied by abundant fibrosis, both in the breast and in lymph node metastases. Nodal metastases also mostly reproduce the nests and cords pattern of the primary breast malignancy. Fibrosis around the carcinoma cells is moderate, however, thick bands of collagen can also occur occasionally. Cytologically, metastatic ductal epithelial cells vary from cuboidal, monomorphic and relatively bland to large and highly pleomorphic nuclei with frequent mitoses. The cytological features of malignancy in the lymph node strongly correlate with the grade of the primary breast malignancy. The response to the invading malignancy in the lymph node, in addition to the desmoplasia, can take the form of enlarged follicles with formation of reactive germinal centers, excessive histiocytosis /granuloma formation. Many variants of carcinoma of the breast exist, some of which less commonly metastasize to lymph nodes such as papillary, mucinous, tubular whereas others more frequently metastasize to lymph node such as inflammatory.

Lobular carcinoma metastasizing to lymph nodes is difficult to recognize in routinely stained H&E tissue sections. Invasive lobular carcinoma of the breast is composed of homogeneous small bland cells that does not form duct- like structures, instead the cells are arranged in Indian file pattern. They are small cells with scant cytoplasm with round or ovoid open nuclei, fine inconspicuous nucleoli and infrequent mitoses. Occasionally, they show an intracytoplasmic mucin-containing vacuoles and an eccentrically placed nuclei, which are typical of signet ring carcinoma cells. Focal signet ring cell differentiation is frequent in lobular breast carcinoma, although it rarely involves 50% or more of the total cell population. In the lymph nodes, lobular carcinoma metastatic cells can appear as dis cohesive and may not be associated with desmoplastic reaction. Extensively involved lymph nodes may resemble lymphoma at low power examination. At the other extreme, axillary lymph nodes with focal involvement of metastatic lobular carcinoma can be extremely difficult to recognize. Metastases are mostly distributed in the sinuses and sometimes deep in the cortex or the medulla, without initial involvement of the marginal sinus. When the metastases are sparse, they consist of single cells or arranged in small cell clusters that may closely resemble histiocytes.

Chemotherapy can change the cytological appearance of metastatic cells and also alters lymph nodes. Chemotherapy can cause carcinoma cells to develop foamy cytoplasm and closely resemble lipid-laden histiocytes.

However, the malignant cells usually retain their nuclear atypia, which is helpful for their identification and distinction from the histiocytes.

Chemotherapy depletes lymph nodes of the lymphoid tissue so that only the supporting framework of the node remains, with or without residual islands of the lymphoid tissue. Both the size and location of the breast cancer metastases within an individual axillary lymph node have prognostic significance. The prognosis is worse when lymph node metastases extend through the capsule into the perinodal adipose tissue called as extranodal extension. In general, presence of extranodal extension correlates with the size of metastases that is, being most likely in cases with larger metastases.

ADENOCARCINOMA STOMACH

The rich mucosal and submucosal lymphatic plexus of the stomach is often invaded by the tumour and from here it can spread to perigastric, periaortic, and celiac axis-related nodes[47]. Tumours which are present in the distal third of stomach show high incidence of hepatoduodenal nodes involvement [48]. The presence of mucosal lymphangiectasia has been found to be associated with regional lymph node metastases[49]. The incidence of detection of lymph node metastases is more influenced by the method used to search them, it is significantly higher with a comprehensive fat-clearing method[50,51] or if the microscopic sections are evaluated immunohistochemically using cytokeratin[52].

MUCINOUS ADENOCARCINOMA

Signet ring cell carcinoma cells tend to grow in singles and have bundant intracytoplasmic mucin distending the cells and pushing the nuclei to the periphery, imparting a signet ring cell appearance. Minor areas may have extracellular pools of mucin. Glands are not formed. Hence these cells can be missed, particularly at low-power magnification. Even at high-power magnification, these cells can be difficult to distinguish from histiocytes within the lymph node sinuses. Mucinous carcinomas usually have predominant areas of pools of mucin that may vary in size, some being visible macroscopically

[53, 54]. Some of the mucin pools may lost part / all of their cellular lining, and appear as entirely acellular. The residual cells which are lining the lakes of mucin may appear dysplastic, with only having moderately atypical nuclei.

It must be emphasized that immunohistochemistry is more needed in some lymph nodes for detecting the presence of occult micro metastases of signet ring cell carcinomas and mucinous carcinomas. This is particularly essential for diagnosing single cell metastases of signet ring cell carcinoma. Use of cocktail of keratin antibodies is more helpful for this purpose.

PAPILLARY CARCINOMA THYROID

Papillary carcinoma thyroid has a great propensity to metastasize to cervical nodes. Nodal metastases can occur in the form of well-developed papillae, even when the primary tumour in the thyroid gland having predominantly follicular pattern [55, 56]. As in the thyroid gland, the nuclei of

19 metastatic PTC are also more characteristic. Nodal metastases of thyroid carcinoma also tend to go for cystic degeneration, which sometimes can be so extensive, that it may resemble a branchial cleft cyst.

MALIGNANCIES OF FEMALE GENITAL TRACT:

ENDOMETRIAL CARCINOMA

The most common sites of extra uterine spread of endometrial

Adenocarcinoma particularly of endometrioid type are pelvic nodes and para aortic nodes and ovaries. Lymph node metastases can occur in approximately

5%–25% of clinically stage I tumours and in invasive high-grade tumours (even if they are superficial), and also in large sized and/or deeply invasive tumours regardless of their grade, in tumours which extends into cervix, and in tumours having vascular invasion[57, 58]. And serous carcinoma is having a particular propensity for lymph vessel permeation.

CERVICAL CARCINOMA

Cervical carcinoma spreads by direct extension to the vagina, endometrium and myometrial wall, parametria, the lower urinary tract and also to the uterosacral ligaments [59, 60]. Nodal metastases are also common. The pattern of lymph nodes involvement generally proceeds in a sequential fashion.

The first station is represented by the paracervical, obturator, hypogastric and external iliac groups and the second station by sacral, common iliac, aortic, and the inguinal groups[61]. Incidence of nodal involvement is directly related to the stage of the disease.

CARCINOMA OF OVARY

The incidence of nodal involvement in case of ovarian cancer reported in the literature is 10.6 –24% for stage I cancer, 23 –50% for stage II cancer, 53 –

74% for stage III cancer, and 65 – 75% for stage IV cancer[62-65]. It has been reported that lymph node involvement is more commonly seen with serous and clear cell malignancies than with other types [65, 66] and that low-grade malignancies are closely associated with nodal disease. The locations of lymph nodes involvement in ovarian cancer depend on histologic type of the primary malignancy. In case of serous tumour, the para-aortic lymph nodes, particularly above the inferior mesenteric artery are the site for the earliest nodal metastasis.

But, the likelihood of pelvic lymph nodes involvement is almost equal to that of the para-aortic lymph node involvement in cases of non-serous tumour.

CARCINOMA OF ORAL CAVITY

Metastases in case of malignancies of oral cavity occur primarily by lymphatic route, the distribution of lymph nodes involved by tumour depends on the location of the primary tumour[67]. The more anterior the location of primary tumour, the lower the position of the cervical lymph node involvement.

Primary carcinomas of base of the tongue and oropharynx tend to metastasize to upper cervical lymph nodes. These metastases are often cystic and frequently present before the detection of the primary malignancy. Metastases to the lymph nodes of posterior triangle - level V lymph nodes are rare, they found be involved in only about 6% of the oropharyngeal malignancies and 1% of the

21 oral malignancies[68]. Features of the primary carcinomas associated with the likelihood of lymph node metastases are location (higher for the oropharynx and posterior portion of tongue, middle for anterior portion of the tongue, and lower for floor of mouth, lip, buccal mucosa- cheek, gingiva and hard palate), high grade of microscopic differentiation, and depth of invasion[69,70]. Carcinomas of the lip associated with metastases tend to be wide, deep, and have high grade of differentiation, and associated with an inflammatory and desmoplastic response[71]. sometimes, the cervical lymph node metastases from these squamous cell carcinomas undergo cystic degeneration. This, along with the well differentiated nature of lesion, may easily lead to a mistaken diagnosis of branchial cyst with malignant transformation called as “branchial carcinoma”[72]. Another important peculiar morphological feature that squamous cell carcinoma can exhibit when metastasizing to cervical lymph nodes is that an extensive foreign body giant cell reaction around the clumps of keratin, without the viable malignant cells, this is particularly common if the neoplasm has been irradiated previously [73].

MIMICKERS OF METASTASIS IN LYMPHNODE

Inclusions of various types of benign tissue can also occur within the lymph nodes[74], and these inclusions can also be mistakenly diagnosed as metastasis.

These include the following:

1. Salivary gland tissue– It is a common finding in high cervical lymph nodes, and to be regarded as a normal event related to the embryology of this region [75]. Both the ducts and acini are usually present. These inclusions may also undergo neoplastic changes, Warthin tumour is the most common type, but many other types have also been reported, including benign mixed tumour

(pleomorphic adenoma), monomorphic adenoma, mucoepidermoid carcinoma, and acinic cell carcinoma.

2. Squamous epithelium - Microscopic cystic structures lined by well differentiated squamous epithelial cells are sometimes seen in the upper cervical lesion. They are thought to represent an anomaly, being composed of branchial pouch derivatives. The term “benign lympho epithelial cyst” is also sometimes applied to these structures. It is hypothesized that these formations are result from cystic dilatation of pre-existing epithelial inclusions as a result of their stimulation by the lymphoid component that surrounds them, a same pathogenesis also applies to multilocular thymic cysts, other cystic structures of the head and neck region, and also to Warthin tumour itself. Similar formations have also been described in peri pancreatic lymph nodes[76]. The obvious differential diagnosis is well differentiated squamous cell carcinoma metastasis, which particularly in the cervical region is notorious for its tendency to undergo marked cystic changes[77].

3. Thyroid follicles - They can be found in the capsular or sub capsular region of mid cervical lymph nodes in the absence of any pathologic changes in the thyroid gland. To differentiate it from metastatic thyroid carcinoma can be very difficult.

4. Decidual reaction - During pregnancy the decidual reaction can occur within the pelvic lymph nodes and they may mimic as metastatic carcinoma[78]. The decidual reaction can occur in the stromal cells of endometriosis foci or in hormonally receptive cells of this region, in a manner similar to that seen in peritoneal decidual reaction.

5. Mullerian-type epithelium - Glandular inclusions lined by cuboidal cells with a mullerian or coelomic appearance sometimes found in the capsule of the pelvic lymph nodes of females and sometimes within the lymph node itself

[79,80]. Their appearance and the pathogenesis is similar to those of the peritoneal lesions, usually known as endosalpingiosis. These nodal inclusions may be difficult to distinguish from the metastases originating in low-grade ovarian malignancies, as they may grow into the peripheral sinuses, and form papillae, sometimes be accompanied by psammoma bodies, and even proliferate as small sheets of cells[81]. Some authors suggested that some of these inclusions are actually metastases from the ovarian serous borderline tumours[82]. Morphologically similar inclusions have also been seen in the mediastinal lymph nodes of males and axillary lymph nodes of females.

Glandular inclusions of similar appearance but surrounded by endometrial-type

24 stroma in the lymph node occur less frequently and they represent nodal endometriosis.

6. Nevus cells- Clusters of normal-appearing nevus cells occasionally may found in the capsule of lymph nodes, without involvement of the parenchyma of the lymph node. Most of the such reported cases have found to be occurred in the axillary lymph nodes[83]. A related lesion is the blue nevus that has been reported in the capsule of lymph node [84]. The morphologic features of these formations may mimic metastatic malignant melanoma deposits.

7. Mesothelial cells – Rarely mesothelial cells are found within the lymph nodes in the apparent absence of a malignant mesothelioma[28, 85, 86]. The differential diagnosis is with metastatic malignant mesothelioma from an occult primary which may be present in the peritoneal cavity / pleura[17].

8. Breast tissue –Sometimes normal mammary lobules may present within the axillary lymph nodes[87-89]. And slightly more common occurrence in axillary lymph nodes is that presence of tubules lined by a single layer of cuboidal cells, sometimes with a hobnail appearance located in the lymph node capsule or immediately beneath it. These formations are similar to the mullerian-type epithelial inclusions which may occur in the pelvic lymph nodes. Hence some of these cases occur in patients with breast malignancy, it may interfere with the diagnosis of metastatic tumour [90, 91]. Epithelial inclusions which are present in the axillary lymph nodes of females can be classified into three major groups, those composed exclusively of glandular structures, those composed only of

25 squamous cysts, and those containing both glandular and squamous epithelium[92].

ROLE OF CYTOKERATIN IN DETECTING OCCULT METASTASIS

Keratins are the main cytoskeletal component of epithelial cells and the most diverse group of intermediate filaments. Fifty-four functional keratin genes have been identified in humans [93]. These genes have been subdivided into 28 type I genes and 26 type II genes that form 2 clusters of 27 genes each on chromosome 17q12-q21 and 12q11-q13, respectively. Keratins have been subdivided into 2 major sequence types corresponding to class I and class II of the current classification of intermediate filaments [94]. Type I is comprised of

17 epithelial and 11 hair keratins, and type II is composed of 20 epithelial and 6 hair keratins . Keratins are resistant to degradation, show great fidelity of expression, and are very antigenic. All keratins have a common structure, which consists of a central alpha-helical rod domain of 310 amino acid residues that is flanked on either side by head and tail domains. The head domain is the amino terminus, while the tail domain is the carboxyl terminus. Specific keratins are expressed in different epithelia. Stratified squamous epithelia express mostly keratins 1 to 6 and 9 to 17, while keratins 7, 8, 18, 19, and 20 are identified in simple epithelia. Of the latter, keratins 8, 18, and 19 are the most abundant in malignancies [95].

Although it was originally believed that keratin expression was restricted to epithelial cells, subsequent studies demonstrated their presence in subsets of

26 several types of mesenchymal cells, including smooth muscle cells of the myometrium and atherosclerotic plaques, virally transformed fibroblasts, and subsets of cultured smooth muscle cells, subserosal connective tissue cells, normal fetal myocardium, vascular endothelial cells, and interstitial reticulum cells of lymphoid tissue [96].

AE1/AE3 is the broad-spectrum keratin antibody cocktail that is probably the most commonly used in surgical pathology. It is composed of the mouse monoclonal antibody AE1 that recognizes the acidic (type I) keratins 10, 14, 15,

16, and 19, and AE3 that reacts with the basic (type II) keratins 1, 2, 3, 4, 5, 6,

7, and 8 [97]. Both clones were generated using epidermal keratin as immunogen. A drawback of this cocktail is that it can cross-react with other intermediate filaments, particularly glial fibrillary acidic protein and, thus, may give a false-positive staining result[98]. Another shortcoming of this cocktail is that it does not react with keratin 18, which is expressed in a wide variety of carcinomas. Therefore, it is important to keep in mind that a negative staining for AE1/ AE3 should not be regarded as sufficient evidence to rule out the possibility of carcinoma. The carcinomas that are most frequently negative for

AE1/AE3 are hepatocellular carcinomas [99], adrenal cortical carcinomas [99], some renal cell carcinomas (clear cell and chromophobe) [100], and renal oncocytomas [100, 101]. To increase the sensitivity of the AE1/ AE3 cocktail for detecting epithelial differentiation, some commercial sources have added other clones to this cocktail, such as 5D3, which reacts with keratins 8 and 18.

The recognition of epithelial differentiation in well- or moderately differentiated carcinomas does not pose significant diagnostic difficulty on routine histologic preparations since evidence of such differentiation (e.g. keratinization or glandular formation) is usually apparent. In those instances in which the neoplasm shows little or no evidence of epithelial differentiation, immunohistochemical studies using a variety of broad-spectrum epithelial markers are often necessary to either confirm or rule out the epithelial nature of the tumour[4].

Occult nodal metastases which are not identified by the routinely used method of examining a single haematoxylin and eosin (HE) stained slides per lymph node have been detected for years[102,103], but their prognostic significance is not well established[104]. Therefore, those occult metastases which are missed on routine methods can be identified either by evaluating further deeper sections of the blocks, or by using a more efficient method of detecting malignant cells. IHC can be used for the latter purpose using epithelial markers.

All cytokeratin positive cells should not be considered as metastatic deposits, since some instance, artefactually displaced malignant cells or normal components of nodes (Interstitial reticulum cells) may also show positive with cytokeratin antibodies [105]. And Plasma cells also show positivity with

CAM5.2 & Pan ck [106]. Occasionally, cells compatible with histiocyte may also show positivity with cytokeratin. Certainly epithelial inclusions of the

28 lymph nodes also show positivity with cytokeratin[88]. Therefore, morphology should not be neglected with the presence of cytokeratin positivity, which will help to differentiate between the malignant cells and others. So if any doubt exist as to the nature of cytokeratin positive cells, it should not be called as metastases in line with the rules of the TNM staging of malignancies[107].

TNM STAGING OF VARIOUS EPITHELIAL MALIGNANCIES[108]:

DEFINITIONS OF AJCC TNM- BREAST Definition of Primary Tumour (T) – Clinical and Pathological TX - Primary tumour cannot be assessed

TO - No evidence of primary tumour

Tis (DCIS)- Ductal carcinoma in situ

Tis (Paget) - Paget disease of the nipple not associated with invasive carcinoma and/or carcinoma in situ (DCIS) in the underlying parenchyma.

TI - Tumour < 20 mm in greatest dimension

T2 -Tumour > 20 mm but < 50 mm in greatest dimension

T3 -Tumour > 50 mm in greatest dimension

T4 -Tumour of any size with direct extension to the chest wall and/or to the skin

T4a - Extension to the chest wall

T4b - Ulceration and/or ipsilateral macroscopic satellite nodules and/or edema

(including peau d ’orange) of the skin

T4c - Both T4a and T4b are present

T4d - Inflammatory carcinoma

Definition of Regional Lymph Nodes -Pathological (pN) pNX - Regional lymph nodes cannot be assessed pNO - No regional lymph node metastasis identified pN0(i+) - ITCs only (malignant cell clusters no larger than 0.2 mm) pN1- Micro metastases or metastases in 1-3 axillary lymph nodes. pN2 - Metastases in 4 -9 axillary lymph nodes pN3 - Metastases in 10 or more axillary lymph nodes

Definition of Distant Metastasis (M)

M0 - No clinical or radiographic evidence of distant metastases

MI - Distant metastases detected by clinical and radiographic means and/or histologically proven metastases larger than 0.2 mm (pM)

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

Tis N0 M0 0

T1 N0 M0 IA

T0 N1mi M0 IB

T1 N1mi M0 IB

T0 N1 M0 IIA

T1 N1 M0 IIA

T2 N0 M0 IIA

T2 N1 M0 IIB

T3 N0 M0 IIB

T0 N2 M0 IIIA

T1 N2 M0 IIIA

T2 N2 M0 IIIA

T3 N1 M0 IIIA

T3 N2 M0 IIIA

T4 N0 M0 IIIB

T4 N1 M0 IIIB

T4 N2 M0 IIIB

Any T N3 M0 IIIC

Any T Any N M1 IV

DEFINITIONS OF AJCC TNM -STOMACH

Definition of Primary Tumour (T)

TX - Primary tumour cannot be assessed

TO - No evidence of primary tumour

Tis - Carcinoma in situ: intraepithelial tumour without invasion of the lamina propria, high-grade dysplasia

TI - Tumour invades the lamina propria, muscularis mucosae, or submucosa

T1a - Tumour invades the lamina propria or muscularis mucosae

Tlb - Tumour invades the submucosa

T2 -Tumour invades the muscularis propria

T3 -Tumour penetrates the sub serosal connective tissue without invasion of the visceral peritoneum or adjacent structures

T4 -Tumour invades the serosa (visceral peritoneum) or adjacent structures

T4a Tumour invades the serosa (visceral peritoneum)

T4b Tumour invades adjacent structures/organs

Definition of Regional Lymph Node (N)

NX - Regional lymph node(s) cannot be assessed

NO -No regional lymph node metastasis

N1 -Metastasis in one or two regional lymph nodes

N2 -Metastasis in three to six regional lymph nodes

N3 -Metastasis in seven or more regional lymph nodes

N3a -Metastasis in seven to 15 regional lymph nodes

N3b -Metastasis in 16 or more regional lymph nodes

Definition of Distant Metastasis (M)

M0-No distant metastasis

MI -Distant metastasis

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

Tis N0 M0 0

T1 N0 M0 IA

T1 N1 M0 IB

T2 N0 M0 IB

T1 N2 M0 IIA

T2 N1 M0 IIA

T3 N0 M0 IIA

T1 N3a M0 IIB

T2 N2 M0 IIB

T3 N1 M0 IIB

T4a N0 M0 IIB

T2 N3a M0 IIIA

T3 N2 M0 IIIA

T4a N1 M0 IIIA

T4a N2 M0 IIIA

T4a N0 M0 IIIA

T4b N3b M0 IIIB

T1 N3b M0 IIIB

T2 N3a M0 IIIB

T3 N3a M0 IIIB

T4a N1 M0 IIIB

T4b N2 M0 IIIB

T4b N3b M0 IIIC

T3 N3b M0 IIIC

T4a N3a M0 IIIC

T4b N3b M0 IIIC

Any T Any N M1 IV

DEFINITIONS OF AJCC TNM- SMALL INTESTINE

Definition of Primary Tumour (T)

TX -Primary tumour cannot be assessed

TO -No evidence of primary tumour

Tis -High-grade dysplasia/carcinoma in situ

TI -Tumour invades the lamina propria or submucosa

T1a -Tumour invades the lamina propria

T1b -Tumour invades the submucosa

T2 -Tumour invades the muscularis propria

T3 -Tumour invades through the muscularis propria into the subserosa, or extends into nonperitonealized perimuscular tissue (mesentery or retro peritoneum) without serosal penetration

T4 -Tumour perforates the visceral peritoneum or directly invades other organs or structures

Definition of Regional Lymph Node (N)

NX -Regional lymph nodes cannot be assessed

NO -No regional lymph node metastasis

N1 -Metastasis in one or two regional lymph nodes

N2 -Metastasis in three or more regional lymph nodes

Definition of Distant Metastasis (M)

M0-No distant metastasis

M1 -Distant metastasis present

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

Tis N0 M0 0

T1-2 N0 M0 I

T3 N0 M0 IIA

T4 N0 M0 IIB

Any T N1 M0 IIIA

Any T N2 M0 IIIB

Any T Any N M1 IV

DEFINITIONS OF AJCC TNM-COLON

Definition of Primary Tumour (T)

TX -Primary tumour cannot be assessed

TO -No evidence of primary tumour

Tis -Carcinoma in situ, intramucosal carcinoma

TI -Tumour invades the submucosa

T2 -Tumour invades the muscularis propria

T3 -Tumour invades through the muscularis propria into pericolorectal tissues

T4 -Tumour invades the visceral peritoneum /invades or adheres to adjacent organ or structure

T4a -Tumour invades through the visceral peritoneum

T4b Tumour directly invades or adheres to adjacent organs or structures

Definition of Regional Lymph Node (N)

NX -Regional lymph nodes cannot be assessed

NO -No regional lymph node metastasis

N1 -One to three regional lymph nodes are positive

N1a -One regional lymph node is positive

N1b -Two or three regional lymph nodes are positive

Nlc -No regional lymph nodes are positive, but there are tumour deposits in the subserosa/mesentery/nonperitonealized pericolic/perirectal/mesorectal tissues.

N2 -Four or more regional nodes are positive

N2a -Four to six regional lymph nodes are positive

N2b -Seven or more regional lymph nodes are positive

Definition of Distant Metastasis (M)

M0-No distant metastasis by imaging, no evidence of tumour in distant sites or organs

M1 -Metastasis to one or more distant sites/organs/peritoneal metastasis is identified

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

Tis N0 M0 0

T1,T2 N0 M0 I

T3 N0 M0 IIA

T4a N0 M0 IIB

T4b N0 M0 IIC

T1-T2 N1/N1c M0 IIIA

T1 N2a M0 IIIA

T3-T4a N1/N1c M0 IIIB

T2-T3 N2a M0 IIIB

T1-T2 N2b M0 IIIB

T4a N2a M0 IIIC

T3-T4a N2b M0 IIIC

T4b N1-N2 M0 IIIC

Any T Any N M1 IV

DEFINITIONS OF AJCC TNM-ORAL CAVITY

Definition of Primary Tumour (T)

TX -Primary tumour cannot be assessed

Tis -Carcinoma in situ

TI -Tumour< 2 cm, < 5 mm depth of invasion (DOI)

T2 -Tumour < 2 cm, DOI > 5 mm and < 1 0 mm or tumour > 2 cm but < 4 cm, and < 10 mm DOI

T3 -Tumour>4 cm or any tumour> 10 mm DOI

T4 -Moderately advanced or very advanced local disease

T4a -Moderately advanced local disease

Lip-Tumour invades through cortical bone or involves the inferior alveolar nerve, floor of mouth/skin of face.

Oral cavity-Tumour invades adjacent structures only (e.g., through cortical bone of the mandible or maxilla, or involves the maxillary sinus or skin of the face).

T4b -Very advanced local disease Tumor invades masticator space, pterygoid plates, or skull base and/or encases the internal carotid artery

Pathological N (pN)

NX -Regional lymph nodes cannot be assessed

NO -No regional lymph node metastasis

NI -Metastasis in a single ipsilateral lymph node 3 cm or smaller and ENE(-)

N2 -Metastasis in a single ipsilateral lymph node 3 cm or smaller and ENE(+) or larger than 3 cm but not larger than 6 cm and ENE(-);

39 or metastases in multiple ipsilateral lymph nodes, none larger than 6 cm and

ENE(-); or in bilateral or contralateral lymph nodes,none larger than 6cm and ENE(-)

N3 -Metastasis in a lymph node larger than 6cm and ENE(-) or in a single ipsilateral node larger than 3 cm and ENE(+) or multiple ipsilateral, contralateral or bilateral nodes any with ENE(+)

Definition of Distant Metastasis (M)

M0-No distant metastasis

M1 -Distant metastasis

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

T1 N0 M0 I

T2 N0 M0 II

T3 N0 M0 III

T1,2,3 N1 M0 III

T4a N0,N1 M0 IVA

T1,2,3,4a N2 M0 IVA

Any T N3 M0 IVB

T4b Any N M0 IVB

Any T Any N M1 IVC

DEFINITIONS OF AJCC TNM-ENDOMETRIUM

Definition of Primary Tumour (T)

TX - Primary tumour cannot be assessed

T0 -No evidence of primary tumour

TI - Tumour confined to the corpus uteri, including endocervical glands

T1a - Tumour limited to the endometrium or invading less than half the myometrium

Tlb - Tumour invading one half or more of the myometrium

T2 - Tumour invading the stromal connective tissue of the cervix but not extending beyond the uterus

T3 - Tumour involving serosa, adnexa, vagina/ parametrium

T3a -Tumour involving the serosa and/or adnexa

T3b -Vaginal involvement or parametrial involvement

T4 -Tumour invading the bladder mucosa and/or bowel mucosa

Definition of Regional Lymph Node (N)

NX -Regional lymph nodes cannot be assessed

N0 -No regional lymph node metastasis

N1 -Regional lymph node metastasis to pelvic lymph nodes

N2-Regional lymph node metastasis to para-aortic lymph nodes, with or without

positive pelvic lymph nodes

M Category FIGO Stage M Criteria

M0-No distant metastasis

MI -Distant metastasis

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

T1 N0 M0 I

T1a N0 M0 IA

T1b N0 M0 IB

T2 N0 M0 II

T3 N0 M0 III

T3a N0 M0 IIIA

T3b N0 M0 IIIB

T1-T3 N1 M0 IIIC1

T1-T3 N2 M0 IIIC2

T4 Any N M0 IVA

Any T Any N M1 IVB

DEFINITIONS OF AJCC TNM-CERVIX

Definition of Primary Tumour (T)

TX - Primary tumour cannot be assessed

T0 -No evidence of primary tumour

TI-Cervical carcinoma confined to the uterus

T1a-Invasive carcinoma diagnosed only by microscopy. Stromal invasion with a maximum depth of 5.0 mm measured from the base of the epithelium and a

43 horizontal spread of 7.0 mm or less.

Tlb -Clinically visible lesion confined to the cervix or microscopic lesion greater than Tla. Includes all macroscopically visible lesions, even those with superficial invasion.

T2 -Cervical carcinoma invading beyond the uterus but not to the pelvic wall or to lower third of the vagina

T2a -Tumour without parametrial invasion

T2b -Tumour with parametrial invasion

T3 -Tumour extending to the pelvic side wall and/or involving the lower third of the vagina and/or causing hydronephrosis or nonfunctioning kidney

T4 -Tumour invading the mucosa of the bladder or rectum and/or extending beyond the true pelvis

Definition of Regional Lymph Node (N)

NX -Regional lymph nodes cannot be assessed

NO -No regional lymph node metastasis

N0(i+) -Isolated tumour cells in regional lymph node(s) no greater than 0.2 mm

NI -Regional lymph node metastasis

Definition of Distant Metastasis (M)

M0 -No distant metastasis

MI-Distant metastasis

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

T1 Any N M0 I

T1a Any N M0 IA

T1b Any N M0 IB

T2 Any N M0 II

T2a Any N M0 IIA

T2b Any N M0 IIB

T3 Any N M0 III

T3a Any N M0 IIIA

T3b Any N M0 IIIB

T4 Any N M0 IVA

Any T Any N M1 IVB

DEFINITIONS OF AJCC TNM-OVARY

Definition of Primary Tumour (T)

TX -Primary tumour cannot be assessed

T0 -No evidence of primary tumour

T1 -Tumour limited to ovaries (one or both)

T1a -Tumour limited to one ovary (capsule intact), no tumour on ovarian surface, no malignant cells in ascites or peritoneal washings

T1b -Tumour limited to both ovaries (capsules intact), no tumour on ovarian surface, no malignant cells in ascites or peritoneal washings

T1c -Tumour limited to one or both ovaries with any of the following

T1c1 -Surgical spill

T1c2 -Capsule ruptured before surgery or tumour on ovarian or fallopian tube surface

T1c -Malignant cells in ascites or peritoneal washings

T2 -Tumour involves one or both ovaries with pelvic extension below pelvic brim

T2a –Extension and/or implants on the uterus and/or tube(s)

T2b -Extension to and/or implants on other pelvic tissues

T3 -Tumour involves one or both ovaries with microscopically confirmed peritoneal metastasis outside the pelvis and/or metastasis to the retroperitoneal lymph nodes

T3a -Microscopic extrapelvic (above the pelvic brim) with or without positive retroperitoneal lymph nodes

T3b -Macroscopic peritoneal metastasis beyond pelvis 2 cm or less in greatest dimension with or without metastasis to the retroperitoneal lymph nodes

T3c - Macroscopic peritoneal metastasis beyond the pelvis more than2 cm in greatest dimension with or without metastasis to the retroperitoneal lymph nodes (includes extension to capsule of liver and spleen without parenchymal involvement of either organ)

Definition of Regional Lymph Node (N)

NX -Regional lymph nodes cannot be assessed

N0 -No regional lymph node metastasis

N0(i+) -Isolated tumour cells in regional lymph node(s) no greater than 0.2 mm

N1 -Positive retroperitoneal lymph nodes only (histologically confirmed)

N1a -Metastasis up to 10 mm in greatest dimension

N1b -Metastasis more than 10 mm in greatest dimension

Definition of Distant Metastasis (M)

M0-No distant metastasis

M1 -Distant metastasis including pleural effusion with positive cytology(M1a) liver or splenic parenchymal metastasis, metastasis to extra-abdominal organs (including inguinal lymphnodes and lymph nodes outside the abdominal cavity ): and transmural involvement of intestine(M1b)

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

T1 N0 M0 I

T1a N0 M0 IA

T1 N0 M0 IB

T1 N0 M0 IC

T2 N0 M0 II

T2 N0 M0 IIA

T2 N0 M0 IIB

T1/T2 N1 M0 IIIA1

T3 N0/N1 M0 IIIA2

T3 N0/N1 M0 IIIB

T3 N0/N1 M0 IIIC

Any T Any N M1 IV

Any T Any N M1a IVA

Any T Any N M1b IVB

DEFINITIONS OF AJCC TNM-THYROID

Definition of Primary Tumour (T)

TX -Primary tumour cannot be assessed

TO -No evidence of primary tumour

TI -Tumour <2 cm limited to the thyroid

T2 -Tumour >2 cm but <4 cm limited to the thyroid

T3 -Tumour >4 cm limited to the thyroid(T3a), or gross extra thyroidal extension invading only strap muscles(T3b)

T4 -Includes gross extra thyroidal extension

T4a -Gross extra thyroidal extension invading subcutaneous soft tissues, larynx, trachea, esophagus, or recurrent laryngeal nerve

T4b -Gross extra thyroidal extension invading prevertebral fascia or encasing the carotid artery or mediastinal vessels.

Definition of Regional Lymph Node (N)

NX -Regional lymph nodes cannot be assessed

N0-No evidence of loco regional lymph node metastasis

N1 -Metastasis to regional nodes

N1a-Metastasis to level VI or VII lymph nodes. This can be unilateral/bilateral

N1b -Metastasis to levels I. II. III. IV, or V.This unilateral, bilateral, or contralateral lateral neck lymph nodes or retropharyngeal lymph nodes

Definition of Distant Metastasis (M)

M0 -No distant metastasis

M1 -Distant metastasis

AJCC PROGNOSTIC STAGE GROUPS

AGE(Years) T N M STAGE

<55 Any T Any N M0 I

<55 Any T Any N M0 II

≥55 T1 N0/NX M0 I

≥55 T1 N1 M0 II

≥55 T2 N0/NX M0 I

≥55 T2 N1 M0 II

≥55 T3a/T3b Any N M0 II

≥55 T4a Any N M0 III

≥55 T4b Any N M0 IVA

≥55 Any T Any N M1 IVB

DEFINITIONS OF AJCC TNM-BLADDER

Definition of Primary Tumour (T)

TX -Primary tumour cannot be assessed

T0-No evidence of primary tumour

Ta -Non-invasive papillary carcinoma

Tis -Urothelial carcinoma in situ: "fiat tumour”

TI -Tumour invades lamina propria (subepithelial connective tissue)

T2 -Tumour invades muscularis propria

50 pT2a -Tumour invades superficial muscularis propria (inner half) pT2b -Tumour invades deep muscularis propria (outer half)

T3 -Tumour invades perivesical soft tissue pT3a -Microscopically pT3b -Macroscopically (extravesical mass)

T4 -Extravesical tumor directly invades any of the following: prostatic stroma. seminal vesicles, uterus. vagina, pelvic wall. abdominal wall

T4a -Extravesical tumor invades directly into prostaticstroma. uterus, vagina

T4b -Extravesical tumor invades pelvic wall, abdominal wall

Definition of Regional Lymph Node (N)

NX -Lymph nodes cannot be assessed

N0-No lymph node metastasis

N1 -Single regional lymph node metastasis in the true pelvis (perivesical, obturator. internal and external iliac, or sacral lymph node)

N2 -Multiple regional lymph node metastasis in the true pelvis

N3 Lymph node metastasis to the common iliac lymph nodes

Definition of Distant Metastasis (M)

M0-No distant metastasis

M1 -Distant metastasis

M1a -Distant metastasis limited to lymph nodes beyond the common iliacs

MIb -Non-lymph-node distant metastases

AJCC PROGNOSTIC STAGE GROUPS

T N M STAGE

Ta N0 M0 0a

Tis N0 M0 0is

T1 N0 M0 I

T2a N0 M0 II

T2b N0 M0 II

T3a,T3b,T4a N0 M0 IIIA

T1-T4a N1 M0 IIIA

T1-T4a N2,N3 M0 IIIB

T4b N0 M0 IVA

Any T Any N M1a IVA

Any T Any N M1b IVB

MATERIALS AND METHODS

Present study is a prospective study to determine the number of lymph nodes positive for metastasis more accurately than with the routine hematoxylin and eosin staining, with the help of immunohistochemistry. The study was done in the department of pathology, Tirunelveli medical college over a period of

2017-2019. Study material includes about 50 cases of radical resections diagnosed as malignancy. Since it is a prospective study, cases of radical resections from 2017 to 2019 were collected. Among those cases, which were diagnosed as epithelial malignancies on H&E were separated and chosen for study. Out of 50 cases,20 cases belongs to breast malignancy, 8 cases belongs to gastric malignancy, 8 cases belongs to uterine malignancies including cervix, 3 cases belongs to ovary,11 cases belongs to others (including malignancies of thyroid (1 case), oral cavity (6cases),bladder(1case),foot(1case),ileum(1case), colon(1case)). Grading was performed for those cases on H&E slides as grade 1, grade 2, grade 3, low grade/ high grade, and as grading could not be done for cases such as invasive lobular carcinoma and papillary carcinoma thyroid. Followed by TNM staging was done for those cases after identifying the lymph node metastasis with slides stained using H&E method. And the nodes which were negative for metastasis on H&E slides, were taken for immunohistochemistry using pan cytokeratin.

Inclusion criteria

Patients diagnosed with malignancies by radiology, cytology and histopathology and then underwent radical surgery with lymph nodes harvesting and diagnosed as epithelial malignancy on histopathological examination.

Exclusion criteria

1. Cases diagnosed as in situ malignancy on histopathological examination.

2. Radical resection specimens which are diagnosed as malignancy of nonepithelial origin.

3. Autolysed specimen

Materials Required

1.Haematoxylin and eosin stained tissue sections of tumour and lymph nodes sections from radical resections diagnosed as epithelial malignancy.

2. Blocks which contains formalin fixed paraffin embedded lymph node tissue which were diagnosed as negative for metastasis by H&E staining.

3.Postively charged slides for holding tissue sections for IHC

4.Chemicals for preparing antigen retrieval solutions and wash buffers

5.Microoven for antigen retrieval.

6. Kit for performing immunohistochemistry which includes primary antibody (Panck) and universal kit

7. Microscope to identify the lymph nodes which were negative for metastasis in haematoxylin and eosin stained slides and also to identify the occult metastasis which are detected in the IHC slides.

METHODOLOGY

I. Collection of donor blocks and slides

The haematoxylin and eosin stained sections and formalin fixed paraffin embedded blocks of lymph nodes which are negative for metastasis are collected.

The lymph nodes of following cases were included,

1.Invasive ductal carcinoma ,lobular carcinoma and atypial medullary carcinoma of breast

2. Adenocarcinoma of stomach, small intestine, colon, urinary bladder

3. Adenocarcinoma of endometrium, cervix, ovary

4. Squamous cell carcinoma of buccal mucosa, tongue, alveolus, lip, foot

5. Papillary carcinoma of thyroid.

II.Preparation of haematoxylin and eosin slides

Formalin fixed paraffin embedded blocks were made. Sections of 2-4 micron thickness were made and routine staining with hematoxylin and eosin was done. Lymph nodes which were negative for metastasis were selected after examining the slides.

III.Immunohistochemistry

1.Section cutting

Sections were taken at 5 microns thickness on the surface of the APES

(3-aminopropyltriethoxysilane) coated slides. This was followed by incubation of slides at 58-60°c for one hour.

2.Antigen retrieval solution

We used antigen retrieval solution and a wash buffer as instructed by the manufacturer (PATH INSITU).

1 Tris EDTA at a pHof 9.

2 Tris wash buffer at pH of 7.6.

3.Antigen retrieval method

Many methods have been performed for antigen retrieval which includes

Microwave method, water bath, autoclave, proteolytic enzyme and pressure cooker method. In our institution we followed antigen retrieval by using microwave method as it produces even heating and less time consuming with lesser disadvantages as compared to other methods.

4. immunohistochemistry procedure as prescribed by manufacturer

1. Xylene wash - 3 changes each for 10minutes.

2. Followed by rehydration using graded alchohol containing 100%, 80%, 70% each for five minutes.

3. Rinsed in distilled water - 2minutes.

4. Followed by Antigen retrieval in Tris-EDTA buffer using microwave method for 15 – 20 minutes.

5.Followed by Cooling for 15minutes.

6. Washed in TBS wash buffer- 3 changes each for 5minutes.

7. Treated with endogenous peroxide for 7-10minutes.

8. Washed in TBS wash buffer- 3 changes each for 10 minutes.

9.Then Application of primary antibody (Pan cytokeratin) for 1 hour.

10. Washed in TBS wash buffer- 3 changes each for 10minutes.

11. Followed by Addition of Target binder for 15 minutes.

12. Washed in TBS wash buffer- 3 changes each for 10minutes.

13.Followed by HRP POLYMERASE for 15 minutes.

14. Washed in TBS wash buffer- 3 changes each for 10minutes.

15.Followed by Application of Diamino-benzidine tetrachloride(DAB) chromogen along with DAB buffer for 5 minutes.

16. Rinsed in distilled water – 2 changes.

17. Counterstaining using Harris Haematoxylin – 1dip/30seconds for each slides.

18. Wash it in running tap water.

19.Followed by xylene – 2 changes each for 5 minutes.

20. Dehydration in 100% alcohol for 5 minutes.

21. Then Mount the slides with Dextrene phthalate xylene

22. Observation under light microscope.

5. Examination of IHC slides:

After immunohistochemistry using Pan cytokeratin, those slides were examined under all the magnification with the help of light microscopy to detect occult metastasis in the lymph node.

Interpretation of pan cytokeratin immunostaining:

Immunostaining of pan cytokeratin was interpreted as positive/negative.

Positive staining was defined as positive staining of metastatic malignant cells.

Positive staining in the foci in question, was taken as malignant metastatic cells, negative staining of an entire suspicious focus was taken as non malignant. The

Pattern of positive staining were mostly resemble the pattern of primary malignancy. Followed by revised staging was done with an additional lymph nodes detected using IHC.

OBSERVATION AND RESULTS

Software used: Graph Pad Prism version 5.0

STATISTICAL ANALYSIS:

The data were coded and entered in MS-excel office 2010. The data were analyzed using Graph Pad Prism version 5. The categorical data were represented as n(%). Sensitivity and specificity were calculated by constructing the 2 X 2 table. Fisher’s exact test was used to compare the frequency between the groups. p<0.05 was considered statistically significant.

Table 1. Description of Age category of the patients in the study.

S.No Age category n %

1 <40 years 4 8

2 40-60 years 26 52

3 >60 years 20 40

Data are expressed as n with %. The total N=50.

The above table and chart explains that ,Among 50 cases 4 cases(8%) are below 40 years,26 cases (52%)are between 40 – 60 years,and 20 cases(40%) are above 60 years .

Table 2. Description of gender category of the patients in the study.

S.No Gender n %

1 Male 11 22

2 Female 39 78

Data are expressed as n with %. The total N=50.

The above table and chart explains that, among 50 cases,11 are males

(22%),39 are females(78%)

Table 3. Frequency distribution of type of organ of taken for section in the

study.

S.No Type of organ n %

1 Breast 20 40

2 Stomach 8 16

3 Uterus 11 22

4 Others 11 22

Data are expressed as n with %. The total N=50

The above table and chart shows that, distribution of organ types among

50 cases. Out of 50 cases,20 cases belongs to breast(40%), 8 cases belongs to stomach(16%), 11 cases belongs to uterus(22%), 11 cases belongs to others(22%).Others-including thyroid (1 case),oral cavity including tongue(6cases),bladder(1case), foot(1case),ileum(1case),colon(1case). 62

Table 4. Comparison of frequency distribution of age category with respect

to gender in the study.

Male Female Chi- S. Age square Df P value No category n % n % value

1 <40 years 1 9.1 3 7.7 0.886 2 40- 60 years 5 45.5 21 53.8 0.242 2 (NS) 3 >60 years 5 45.5 15 38.5

Data are expressed as n with %. Fisher’s exact test was used to compare the frequencies between the gender. NS= Not significant.

 The above table and chart explains, frequency distribution of age with

respect to gender, Among 50 cases ,

 4 cases are below 40 years of which 1 male(9.1%),3 are females(7.7%)

 26 cases are between 40 -60 years of which 5 are males(45.5%),21 are

females(53.8%)

 20 cases are above 60 years of which 5 are males(45.5%),15 are

females(38.5%)

Table 5. Comparison of frequency distribution of age category with Ca-

organ observed in the study.

40- 60 >60 Chi- <40 years S. Type of years years squar Df P value No organ e N % n % N % value

1 Breast 2 50 12 46.1 6 30

Stomac 0.585 2 0 0 3 11.5 5 25 h 4.686 2 (NS)

3 Uterus 1 25 4 15.4 6 30

4 Others 1 25 7 27 3 15

Data are expressed as n with %. Fisher’s exact test was used to compare the frequencies between the gender. NS= Not significant.

 The above table and chart explains, comparison between distribution of

age category with organ.

 4 cases are below 40 years, of which 2 cases belongs to breast(50%), 1

case belong to uterus(25%),1 belong to others(25%), no stomach case

(0%)belongs to this category

 26 cases are between 40 -60 years of which 12 cases belongs to

breast(46.1%),3 belongs to stomach(11.5%),4 belongs to uterus(15.4%),

7 belongs to others(27%).

 20 cases are above 60 years, of which 6 cases belongs to

breast(30%),5cases belongs to stomach(25%),6cases belongs

uterus(30%), 3cases belongs others(15%).

Table 6. Frequency distribution of type of procedure done on the patients

with Ca in the study.

S.No Procedure done n %

1 Modified radical mastectomy 20 40

2 Total hysterectomy with bilateral oopherectomy 7 14

3 Partial gastrectomy with D1 lymphadenectomy 6 12

4 Wertheim's Radical Hysterectomy 4 8

5 Composite resection with MRND 4 8

6 Total gastrectomy with D2 lymphadenectomy 2 4

7 Others 7 14

 Data are expressed as n with %. The total N=50

 The above table explains distribution of types of procedures among 50

cases studied,

 20 cases are belongs to Modified radical mastectomy(40%)

 7 cases are belongs to Total hysterectomy with bilateral

oophorectomy(14%)

 6 cases are belongs toPartial gastrectomy with D1

lymphadenectomy(12%)

 4 cases are belongs toWertheim's Radical Hysterectomy(8%)

 4 cases are belongs to Composite resection with MRND(8%)

 2 cases are belongs to Total gastrectomy with D2 lymphadenectomy(4%)

 7 cases are belongs to category of others(14%) including, radical

cystoprostatectomy,

 Total thyroidectomy, glossectomy, hemicolectomy, foot amputation ,lip

excision ,ileal resection

Table 7. Frequency distribution of type of regional lymphnodes harvested

in the patients.

S.No Procedure done N %

1 Axillary lymph nodes 20 40

2 Pelvic lymph nodes 12 24

3 Gastric lymph nodes 8 16

4 Cervical lymph nodes 6 12

5 Mesenteric nodes 2 4

6 Inguinal lymph nodes 1 2

7 Para-tracheal nodes 1 2

Data are expressed as n with %. The total N=50

 The above table and chart explains distribution of type of regional lymph

nodes among 50 cases studied,

 20cases are belongs to axillary group of lymph nodes(40%)which

contributes to maximum of lymph nodes in the study

 12cases are belongs topelvic group of lymph nodes(24%)

 8cases are belongs togastric group of lymph nodes(16%)

 6cases are belongs tocervical group of lymph nodes(12%)

 2cases are belongs tomesenteric group of lymph nodes(4%)

 1cases are belongs toinguinal group of lymph nodes(2%)

 1cases are belongs to para-tracheal group of lymph nodes(2%)

Table 8. Frequency distribution of type of diagnosis arrived as per

Histopathological examination in the study.

S.No DIagnosis n %

1 ACB 1 2

2 ACE 3 6

3 AMC 2 4

4 IDC 15 30

5 IDLC 1 2

6 ILC 2 4

7 MDACCo 1 2

8 MDACCx 1 2

9 MDACS 3 6

10 MDSCCBM 1 2

11 MDSCCL 1 2

12 MDSCCT 1 2

13 MuADCS 1 2

14 PapCA 1 2

15 PDACS 2 4

16 SPAC 2 4

17 WDACCx 2 4

18 WDACE 2 4

19 WDACI 1 2

20 WDACS 2 4

21 WDEACOv 1 2

22 WDSCCA 1 2

23 WDSCCBM 2 4

24 WDSCCF 1 2

 Data are expressed as n with %. The total N=50.

 The above table explains distribution of diagnosis among 50 cases

studied,

 Among 50 cases,

 15 cases are dignosed as IDC(30%)

 1 case diagnosed as IDLC

 2 cases are diagnosed as ILC

 1 case diagnosed as adenocarcinoma bladder

 3 cases are diagnosed as adenocarcinoma endometrium

 2 cases are diagnosed as atypical medullary carcinoma

 1 case diagnosed as moderately differentiated adenocarcinoma colon

 1 case diagnosed as moderately differentiated adenocarcinoma cervix

 3 cases are diagnosed as moderately differentiated adenocarcinoma

stomach

 2 cases are diagnosed as well differentiated adenocarcinoma cervix

 2 cases are diagnosed as well differentiated adenocarcinoma

endometrium

 1 case diagnosed as well differentiated adenocarcinoma ileum

 2 case diagnosed as well differentiated adenocarcinoma stomach

 1 case diagnosed as well differentiated endometrioid adenocarcinoma-

ovary

 1 case diagnosed as well differentiated SCC – Alveolus

 2 cases are diagnosed as well differentiated SCC – buccal mucosa

 1 case diagnosed as moderately differentiated SCC – buccal mucosa

 1 case diagnosed as moderately differentiated SCC – lip

 1 case diagnosed as moderately differentiated SCC – tongue

 1 case diagnosed as mucinous adenocarcinoma stomach

 1 case diagnosed as papillary carcinoma thyroid

 2 cases are diagnosed as poorly differentiated adenocarcinoma stomach

 2 cases are diagnosed as serous papillary cystadenocarcinoma of ovary

 1 case diagnosed as well differentiated SCC- foot

Table 9. Frequency distribution of type of differentiation (in Grades)

observed in the samples in the study.

S.No Type of grading N %

1 Grading could not be done 3 6

2 Grade1 14 28

3 Grade 2 28 56

4 Grade 3 3 6

5 High Grade 1 2

6 Low Grade 1 2

Data are expressed as n with %. The total N=50

 The above table and chart explains distribution of grades among 50 cases

studied,

 14 cases are of grade 1(28%)

 28 cases are of grade 2(56%)- contributes to the maximum of cases in the

study

 3 cases are of grade 3(6%)

 1 case is of high grade(2%)

 1 case is of low grade(2%)

 For 3 cases – grading could not be done(6%)

Table 10. Description statistics of lymph node assessment done in the study

(overall)

S.No Parameter n %

1 Total nodes assessed 498 100

By H & E method -nodes classified as (n=498)

2 positive for Ca 54 10.8

Negative for Ca 444 89.2

By PANCK method-nodes classified as (n=444)

3 positive for Ca 14 3.15

Negative for Ca 430 96.85

Data are expressed as n with %.

 Out of 498 lymph nodes collected from 50 cases studied,54 nodes were

found to be positive for metastasis and 444 nodes were found to be

negative for metastasis by H&E method, all those 444 negative nodes

were subjected to IHC, among that 14 nodes were found to be positive for

metastasis.

 Hence, by PANCK-method an additional 3.15% of lymph nodes can be

identified as to be positive for metastasis.

Table 11. Diagnostic statistics of H & E and PANCK method observed in

the study (overall).

Carcinoma S.No Method Sensitivity Specificity Accuracy Positive Negative

H & E Positive 54 0 1 79.4 100 97.2% method Negative 14 430

HE+ Positive 68 0

2 PANCK 100 100 100% Negative 0 430 method

 This table and chart explains that, sensitivity of H&E Method is found be

79.4% and specificity 100%

 Sensitivity and specificity of Pan ck method along with H&E method is

found to be 100%

Table 12. Description statistics for lymph done assessment done in the

study for Ca with respect to the organs.

Ca Positive Ca Negative Additional

S. detection Organ Method No n % n % achieved by

PANCK

Breast H &E 37 16.6 186 83.4

1 (n=223 H&E + 3.5% 45 20.1 178 79.9 nodes) PANCK

Uterus H &E 2 1.7 116 98.3

2 (n=118 H&E + 0% 2 1.7 116 98.3 nodes) PANCK

Stomach H &E 2 5.1 37 94.9

3 (n=39 H&E + 10.3% 6 15.4 33 84.6 nodes) PANCK

H &E 13 11 105 89 Others 4 H&E + 1.7% (n=118) 15 12.7 103 87.2 PANCK

 The above table and chart explains that, additional detection achieved by

panck is 3.5% for breast malignancies,10.3% for gastric

malignancies,1.7% for other malignancies (scc of lip).

 No additional detection achieved for uterine malignancies

Table 13. Comparative description of grading of Ca in lymph nodes

between the H&E and PANCK method in the study.

Ca Positive Ca Negative Additional

S. detection Grade Method No n % N % achieved by

PANCK

Grade 1 H &E 4 3.6 107 96.4

1 (n=111 H&E + 0% 4 3.6 107 96.4 nodes) PANCK

Grade 2 H &E 46 16 241 84

2 (n=287 H&E + 3.5% 56 19.5 231 80.5 nodes) PANCK

Grade 3 H &E 1 5.3 19 94.7

3 (n=20 H&E + 15.8% 4 21.1 16 78.9 nodes) PANCK

Grade 2 and H &E 47 15.3 260 84.7

3 4 H&E + 4.3% (n=307 60 19.6 247 80.4 PANCK nodes)

 The above table and chart explains that additional detection achieved by

panck for grade 2 malignancies is 3.5%,whereas for grade 3 malignancies

is 15.8%,for grade 2 and 3 malignancies together is 4.3%.

 Hence there is a significant increase in metastatic lymph node detection

by panck for grade 3 malignancies (high grade/poorly differentiated

tumours).

Table 14. Frequency distribution of modification of staging based on

PANCK findings observed in the study.

Stage modified due to PANCK S. Organ finding No Observation n %

No 16 80 1 Breast (n=20) Yes 4 20

No 6 75 2 Stomach (n=8) Yes 2 25

No 11 100 3 Uterus (n=11) Yes 0 0

No 11 100 4 Others (n=11) Yes 0 0

No 44 88 5 Overall (n=50) Yes 6 12

 Data are expressed as n with %. The above table explains that, modification

of TNM stage after using PANCK.

 Among 50 cases studied ,TNM stage is modified for 6 cases, among which

4/20 cases of breast malignancies(1/2 of invasive lobular carcinoma, 3/18 of

invasive ductal carcinoma) and 2/8 cases of gastric malignancies.

Table 15. Frequency distribution of modification of staging based on

PANCK findings observed in the study.

H&E H&E pvalue

(POSITIVE) (NEGATIVE)

PAN CK 54 14 (POSITIVE) <0.00001(S) PAN CK 0 430 (NEGATIVE)

 P value = S-significant

 The above table explains that, out of 498 nodes, 54 nodes are detected as

positive for metastasis by H&E method, whereas additional 14 nodes

which have been reported as negative on H&E are detected as positive for

metastasis by PANCK method.

 Fisher’s exact test was used to compare the efficiency of both test,and

showed p value of about <0.00001 which is statistically significant.

DISCUSSION

Overall Epithelial malignancy is the most common form of malignancy.

Because of the progression in the surgical treatment available at present, mortality is related mostly to early metastasis, which is often occult at the time of initial diagnosis. For those patients having no evidence of systemic metastases when the primary malignant tumour is resected, staging is done using the parameters such as tumour size & involvement of lymph nodes. Based on that only, the decision is made as to whether to give or not the systemic adjuvant therapy to prevent metastatic relapse of the disease. Since the undetected metastases can contribute to the failure of primary treatment, the identification of occult metastases in such patients with early stage of cancer could have a significant clinical impact on the prognosis and optimal therapy for patients with malignancy [109]. Hence the immunohistochemistry has a significant role in identifying such metastasis, it can be used as an adjuvant method to standard H&E staining method.

The fact is proved in our study also, by giving increased detection rate of lymph nodes metastasis (overall) by 3.15% using IHC (pan cytokeratin) and we got significant p value of <0.00001.

In this study, sensitivity and specificity of H&E method was 79.4% and

100% respectively, for Pan cytokeratin both were 100%.

Invasive lobular carcinoma of breast is known to produce lymph node metastasis that can be difficult to identify on H&E stained slides, as it is

85 composed of single non cohesive cells of a similar size mimicking lymphocytes. Occult metastases‐ of this type of malignancy detected by IHC have been reported to be more common than the occult metastases from invasive ductal carcinomas of breast[3]

C. A. Wells, A. Heryet et al[110] studied 45 cases of breast malignancy, in all those cases lymph nodes were reported as free of metastatic tumour by H

& E. In 4/12 cases of lobular carcinoma and in 3/33 cases of ductal carcinoma, occult metastases were revealed by IHC staining, which shows an increase in detection rate of 9% for invasive ductal carcinoma and 33% for invasive lobular carcinoma.

M. Trojani, I. de Mascarel et al[111] also reported that Positive staining of metastatic malignant cells with immunohistochemistry were more common in case of invasive lobular carcinoma than invasive ductal carcinoma of breast.

Berry N, Jones DB et al [112] reported that the use of IHC resulted in an increased detection of metastases in both invasive ductal carcinoma (13.1%) and invasive lobular carcinoma (37.5%),thus suggesting more common in lobular carcinoma than ductal carcinoma and also shows overall increase of

17.3% of metastatic lymph nodes.

In our study also there is more detection of occult metastasis in lymph nodes of carcinoma breast by 3.5% using IHC (pan ck). And also showed that more detection rate for lobular carcinoma (1/2) than invasive ductal carcinoma

(3/18), by immunohistological staining, representing an increase in detection rate of 50% for lobular and of 16.6% for the ductal carcinoma cases.

Multivariate analysis demonstrated that the total number of metastatic lymph nodes is the most important prognostic factor in node-positive gastric malignancies[113].

Hence the IHC staining using cytokeratin can be of more useful in detecting accurate number of involved nodes by identifying their occult metastases of patients with gastric malignancies.

Hye Seung Lee, Min A Kim et al [114] reported that presence of occult

Lymph node metastases using IHC in 58 of 280 node-negative cases of gastric carcinomas (20.7%)

Marina Alessandra ,Marcus Fernando Kodama Pertille et al [115] also showedthe presence of occult lymph node metastasis by IHC in 24 lymph nodes from 12 patients of stomach cancer (30%).

In our study also there is an increase in the detection of number of lymph nodes involved by metastasis using IHC for gastric carcinoma by 10.3% and moderately differentiated SCC of lip by 1.7%.

But in our study there is no additional detection of lymph node metastasis for other malignancies including uterine, and ovarian malignancies.

Monisha Choudhury, Sapna Agrawal et al [116]observed that Occult lymph node metastases detected by IHC (pan ck) in 100% of the grade 3,

22.22% of grade 2 tumours, and 10.53% of grade 1 tumours. Thus establishing the fact that with increasing tumour grade there is an increase in probability of occult metastases detection by immunohistochemistry.

This is also supported by Viale et al [117] who reported 22.7, 32.2, and

38% metastases detection rates in grade 1, 2, and 3 tumors, respectively.

In our study also we found to have an additional detection of occult metastasis by 3.15% for grade 2 tumours and 15.8% for grade 3 tumours using IHC. Thus supporting the same fact.

In our study, after the additional detection of metastatic lymph nodes, stage has been changed for 6 cases out of 50 cases studied, among which 4 belongs to breast carcinoma and 2 belongs to adenocarcinoma of stomach.

CONCLUSION

1. The ideal approach in the use of immunohistochemical markers is to use

it as an adjuvant to the examination of standard hematoxylin-eosin slides,

as an occult metastasis are readily highlighted. It leads to categorise the

additional metastatic nodal load (p value <0.00001).

2. Immunohistochemistry for ck reveals a higher rate detection of occult

lymph node metastases among the invasive lobular breast carcinomas

than among the invasive ductal breast carcinomas. Hence this method can

be used in the evaluation of nodes in invasive lobular carcinoma.

3. It is found that, with increase in the tumour grade there is an increase

in probability of occult metastases which can be detected by

immunohistochemistry. So this method can be used for high grade

tumours than low grade tumours.

4. A routine screening of lymph nodal slides with Pan ck – IHC could

highlight the nodal status exactly, so that improved therapeutic decision

can be made.

BIBLIOGRAPHY

1. Pereira, E.R., et al. The lymph node microenvironment and its role in the progression of metastatic cancer. in Seminars in cell & developmental biology. 2015. Elsevier. 2. Kapoor, C., et al., Lymph node metastasis: A bearing on prognosis in squamous cell carcinoma. Indian journal of cancer, 2015. 52(3): p. 417. 3. Cserni, G., et al., The value of cytokeratin immunohistochemistry in the evaluation of axillary sentinel lymph nodes in patients with lobular breast carcinoma. Journal of clinical pathology, 2006. 59(5): p. 518-522. 4. Ordóñez, N.G., Broad-spectrum immunohistochemical epithelial markers: a review. Human pathology, 2013. 44(7): p. 1195-1215. 5. Stein, H., et al., Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin's lymphomas. Journal of Histochemistry & Cytochemistry, 1980. 28(8): p. 746-760. 6. Jaffe, E.S., et al., Hematopathology e-book. 2016: Elsevier Health Sciences. 7. Girard, J.-P., C. Moussion, and R. Förster, HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes. Nature Reviews Immunology, 2012. 12(11): p. 762. 8. De Bruyn, P. and Y. Cho, Structure and function of high endothelial postcapillary venules in lymphocyte circulation, in Reaction Patterns of the lymph node. 1990, Springer. p. 85-101. 9. Sasaki, K., et al., Ultrastructural localization of the intercellular adhesion molecule (ICAM 1) on the cell surface of high endothelial venules in lymph nodes. The Anatomical Record: An Official Publication of the American Association of Anatomists,‐ 1996. 244(1): p. 105-111. 10. Willard-Mack, C.L., Normal structure, function, and histology of lymph nodes. Toxicologic pathology, 2006. 34(5): p. 409-424. 11. Martin, T.A., et al., Cancer invasion and metastasis: molecular and cellular perspective, in Madame Curie Bioscience Database [Internet]. 2013, Landes Bioscience. 12. Bubendorf, L., et al., Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Human pathology, 2000. 31(5): p. 578-583. 13. Nguyen, D.X., P.D. Bos, and J. Massagué, Metastasis: from dissemination to organ-specific colonization. Nature Reviews Cancer, 2009. 9(4): p. 274. 14. Jiang, W.G., et al., Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Critical reviews in oncology/hematology, 2005. 53(1): p. 35-69. 15. Didolkar, M., et al., Metastatic carcinomas from occult primary tumors: A study of 254 patients. Annals of surgery, 1977. 186(5): p. 625. 16. Haagensen, C.D., et al., The lymphatics in cancer. 1972: Saunders Philadelphia. 17. Sussman, J. and J. Rosai, Lymph node metastasis as the initial manifestation of malignant mesothelioma. Report of six cases. The American journal of surgical pathology, 1990. 14(9): p. 819-828. 18. Batsakis, J.G., The pathology of head and neck tumors: the occult primary and metastases to the head and neck, Part 10. Head & neck surgery, 1981. 3(5): p. 409-423. 19. Lindberg, R., Distribution of cervical lymph node metastases from squamous cell carcinoma of the upper respiratory and digestive tracts. Cancer, 1972. 29(6): p. 1446-1449. 20. Mancuso, A.A. and W.N. Hanafee, Elusive head and neck carcinomas beneath intact mucosa. The Laryngoscope, 1983. 93(2): p. 133-139. 21. Silverman, C. and J.E. Marks. Metastatic cancer of unknown origin: epidermoid and undifferentiated carcinomas. in Seminars in oncology. 1982. 22. Markman, M., Metastatic adenocarcinoma of unknown primary site: Analysis of 245 patients seen at the johns hopkins hospital from 1965 1979. Medical and pediatric oncology, 1982. 10(6): p. 569-574. 23. Cervin, J.R., et al., Virchow's node revisited. Analysis ‐ with clinicopathologic correlation of 152 fine-needle aspiration biopsies of supraclavicular lymph nodes. Archives of pathology & laboratory medicine, 1995. 119(8): p. 727-730. 24. Copeland, E.M. and C.M. McBride, Axillary metastases from unknown primary sites. Annals of surgery, 1973. 178(1): p. 25. 25. Feigenberg, Z., M. Zer, and M. Dintsman, Axillary metastases from an unknown primary source. Israel journal of medical sciences, 1976. 12(10): p. 1153-1158. 26. Clary, C.F., et al., Metastatic carcinoma: The lung as the site for the clinically undiagnosed primary. Cancer, 1983. 51(2): p. 362-366. 27. Zaren, H.A. and E.M. Copeland III, Inguinal node metastases. Cancer, 1978. 41(3): p. 919-923. 28. Argani, P. and J. Rosai, Hyperplastic mesothelial cells in lymph nodes: report of six cases of a benign process that can simulate metastatic involvement by mesothelioma or carcinoma. Human pathology, 1998. 29(4): p. 339-346. 29. Hoda, S.A., et al., Megakaryocytes mimicking metastatic breast carcinoma: A potential trap in the interpretation of sentinel lymph nodes. Archives of pathology & laboratory medicine, 2002. 126(5): p. 618-620. 30. Guerrero Medrano, J., R. Delgado, and J. Albores Saavedra, Signet ring sinus histiocytosis: a reactive disorder that mimics metastatic adenocarcinoma.‐ Cancer: Interdisciplinary International‐ Journal of‐ the American Cancer Society, 1997. 80(2): p. 277-285. 31. De Petris, G., R. Lev, and S. Siew, Peritumoral and nodal muciphages. The American journal of surgical pathology, 1998. 22(5): p. 545-549. 32. Groisman, G.M., et al., Mucicarminophilic histiocytosis (benign signet- ring cells) and hyperplastic mesothelial cells: two mimics of metastatic carcinoma within a single lymph node. Archives of pathology & laboratory medicine, 1998. 122(3): p. 282. 33. Argani, P., R. Ghossein, and J. Rosai, Anthracotic and anthracosilicotic spindle cell pseudotumors of mediastinal lymph nodes: Report of five cases of a reactive lesion that simulates malignancy. Human pathology, 1998. 29(8): p. 851-855. 34. Jager, G.J., et al., Pelvic adenopathy in prostatic and urinary bladder carcinoma: MR imaging with a three-dimensional TI-weighted magnetization-prepared-rapid gradient-echo sequence. AJR. American journal of roentgenology, 1996. 167(6): p. 1503-1507. 35. Anzai, Y., et al., Evaluation of neck and body metastases to nodes with ferumoxtran 10–enhanced MR imaging: phase III safety and efficacy study. Radiology, 2003. 228(3): p. 777-788. 36. Guller, U., et al., Is positron emission tomography an accurate non- invasive alternative to sentinel lymph node biopsy in breast cancer patients? Journal of the National Cancer Institute, 2003. 95(14): p. 1040- 1043. 37. Steel, B.L., M.R. Schwartz, and I. Ramzy, Fine needle aspiration biopsy in the diagnosis of lymphadenopathy in 1,103 patients. Role, limitations and analysis of diagnostic pitfalls. Acta cytologica, 1995. 39(1): p. 76-81. 38. Frable, W.J., Fine-needle aspiration biopsy: a review. Human Pathology, 1983. 14(1): p. 9-28. 39. Lee, R., et al., Lymph node examination by fine needle aspiration in patients with known or suspected malignancy. Acta cytologica, 1987. 31(5): p. 563-572. 40. Mehdi, G., et al., Cytological evaluation of enlarged lymph nodes in metastatic disease: A hospital-based assessment. Clinical Cancer Investigation Journal, 2015. 4(2): p. 152. 41. Maehara, Y., et al., Clinical significance of occult micrometastasis in lymph nodes from patients with early gastric cancer who died of recurrence. Surgery, 1996. 119(4): p. 397-402. 42. Pargaonkar, A.S., et al., Evaluation of immunohistochemistry and multiple- level sectioning in sentinel lymph nodes from patients with breast cancer. Archives of pathology & laboratory medicine, 2003. 127(6): p. 701-705. 43. Matsuda, J.-i., et al., Significance of metastasis detected by molecular techniques in sentinel nodes of patients with gastrointestinal cancer. Annals of surgical oncology, 2004. 11(3): p. 250S-254S. 44. Singletary, S.E. and J.L. Connolly, Breast cancer staging: working with the sixth edition of the AJCC Cancer Staging Manual. CA: a cancer journal for clinicians, 2006. 56(1): p. 37-47. 45. Sharkey, F.E. and A. Greiner, Morphologic identity of primary tumor and axillary metastases in breast carcinoma. A quantitative study. Archives of pathology & laboratory medicine, 1985. 109(3): p. 256-259. 46. Lauria, R., et al., The prognostic value of lymphatic and blood vessel invasion in operable breast cancer. Cancer, 1995. 76(10): p. 1772-1778. 47. Lehnert, T., R.A. Erlandson, and J.J. Decosse, Lymph and blood capillaries of the human gastric mucosa: a morphologic basis for metastasis in early gastric carcinoma. Gastroenterology, 1985. 89(5): p. 939-950. 48. Wu, C.W., et al., Lymph node metastasis from carcinoma of the distal one third of the stomach. Cancer, 1994. 73(12): p. 3109-3114. 49. Mak, K.-l., et al., Mucosal lymphangiectasia in gastric adenocarcinoma.‐ Archives of pathology & laboratory medicine, 1996. 120(1): p. 78-80. 50. Dias, A.R., et al., Carnoy’s solution increases the number of examined lymph nodes following gastrectomy for adenocarcinoma: a randomized trial. Gastric Cancer, 2016. 19(1): p. 136-142. 51. Lavy, R., et al., A comparative study on two different pathological methods to retrieve lymph nodes following gastrectomy. International Journal of Surgery, 2014. 12(7): p. 725-728. 52. Fukagawa, T., et al., Immunohistochemically detected micrometastases of the lymph nodes in patients with gastric carcinoma. Cancer, 2001. 92(4): p. 753-760. 53. Symonds, D.A. and A.L. Vickery Jr, Mucinous carcinoma of the colon and rectum. Cancer, 1976. 37(4): p. 1891-1900. 54. Connelly, J., S. Robey-Cafferty, and K. Cleary, Mucinous carcinomas of the colon and rectum. An analysis of 62 stage B and C lesions. Archives of pathology & laboratory medicine, 1991. 115(10): p. 1022-1025. 55. Lam, A.K.-Y., C.-Y. Lo, and K.S.-L. Lam, Papillary carcinoma of thyroid: a 30-yr clinicopathological review of the histological variants. Endocrine pathology, 2005. 16(4): p. 323-330. 56. Carcangiu, M.L., et al., Papillary carcinoma of the thyroid. A clinicopathologic study of 241 cases treated at the University of Florence, Italy. Cancer, 1985. 55(4): p. 805-828. 57. Boronow, R., et al., Surgical staging in endometrial cancer: clinical- pathologic findings of a prospective study. Obstetrics and gynecology, 1984. 63(6): p. 825-832. 58. Creasman, W.T., et al., Surgical pathologic spread patterns of endometrial cancer: a Gynecologic Oncology Group study. Cancer, 1987. 60(S8): p. 2035-2041. 59. Benedetti Panici, P., et al., Early cervical carcinoma: the natural history of lymph node involvement redefined on the basis of thorough parametrectomy‐ and giant section study. Cancer, 2000. 88(10): p. 2267- 2274. 60. Uçlmakli, A., W.A. Bonney Jr, and A. Palladino, The nonlymphatic metastases of carcinoma of the uterine cervix. A prospective analysis based on laparotomy. Cancer, 1978. 41(3): p. 1027-1033. 61. Henriksen, E., The lymphatic spread of carcinoma of the cervix and of the body of the uterus: a study of 420 necropsies. American journal of obstetrics and gynecology, 1949. 58(5): p. 924-942. 62. Morice, P., et al., Lymph node involvement in epithelial ovarian cancer: analysis of 276 pelvic and paraaortic lymphadenectomies and surgical implications. Journal of the American College of Surgeons, 2003. 197(2): p. 198-205. 63. Onda, T., et al., Assessment of metastases to aortic and pelvic lymph nodes in epithelial ovarian carcinoma: a proposal for essential sites for lymph node biopsy. Cancer: Interdisciplinary International Journal of the American Cancer Society, 1996. 78(4): p. 803-808. 64. Burghardt, E., et al., Patterns of pelvic and paraaortic lymph node involvement in ovarian cancer. Gynecologic oncology, 1991. 40(2): p. 103-106. 65. Di Re, F., et al., 13 Pelvic and para-aortic lymphadenectomy in cancer of the ovary. Bailliere's clinical obstetrics and gynaecology, 1989. 3(1): p. 131-142. 66. Petru, E., et al., Lymphadenectomy in stage I ovarian cancer. American journal of obstetrics and gynecology, 1994. 170(2): p. 656-662. 67. Shah, J.P., F.C. Candela, and A.K. Poddar, The patterns of cervical lymph node metastases from squamous carcinoma of the oral cavity. Cancer, 1990. 66(1): p. 109-113. 68. Davidson, B.J., et al., Posterior triangle metastases of squamous cell carcinoma of the upper aerodigestive tract. The American Journal of Surgery, 1993. 166(4): p. 395-398. 69. Frierson Jr, H.F. and P.H. Cooper, Prognostic factors in squamous cell carcinoma of the lower lip. Human pathology, 1986. 17(4): p. 346-354. 70. Umeda, M., et al., Lymph node metastasis in squamous cell carcinoma of the oral cavity: correlation between histologic features and the prevalence of metastasis. Head & neck, 1992. 14(4): p. 263-272. 71. Quaedvlieg, P., et al., Histopathological characteristics of metastasizing squamous cell carcinoma of the skin and lips. Histopathology, 2006. 49(3): p. 256-264. 72. McHugh, J.B., Association of Cystic Neck Metastases and Human Papillomavirus–Positive Oropharyngeal Squamous Cell Carcinoma. Archives of pathology & laboratory medicine, 2009. 133(11): p. 1798- 1803. 73. Safaii, H. and H.A. Azar, Keratin granulomas in irradiated squamous cell carcinoma of various sites. Cancer research, 1966. 26(3 Part 1): p. 500- 508. 74. Longo, S., Benign lymph node inclusions. Human pathology, 1976. 7(3): p. 349-354. 75. Brown, R., R. Gaillard, and J. Turner, The significance of aberrant or heterotopic parotid gland tissue in lymph nodes. Annals of surgery, 1953. 138(6): p. 850. 76. Arai, T., et al., Epidermal inclusions in abdominal lymph nodes: Report of two cases studied immunohistochemically. Pathology International, 1992. 42(2): p. 126-129. 77. Micheau, C., Y. Cachin, and B. Caillou, Cystic metastases in the neck revealing occult carcinoma of the tonsil. A report of six cases. Cancer, 1974. 33(1): p. 228-233. 78. Covell, L.M., A.J. Disciulio, and R.C. Knapp, Decidual change in pelvic lymph nodes in the presence of cervical squamous cell carcinoma during pregnancy. American Journal of Obstetrics & Gynecology, 1977. 127(6): p. 674-676. 79. Koss, L.G., Miniature adenoacanthoma arising in an endometriotic cyst in an obturator lymph node. Report of first case. Cancer, 1963. 16(11): p. 1369-1372. 80. Maaßen, n.V. and K. Hiller, Glandular inclusions in lymph nodes: pattern of distribution and metaplastic transformation. Archives of gynecology and obstetrics, 1994. 255(1): p. 1-8. 81. Ehrmann, R., J. Federschneider, and R. Knapp, Distinguishing lymph node metastases from benign glandular inclusions in low-grade ovarian carcinoma. American Journal of Obstetrics & Gynecology, 1980. 136(6): p. 737-746. 82. Moore, W.F., et al., Some mullerian inclusion cysts in lymph nodes may sometimes be metastases from serous borderline tumors of the ovary. The American journal of surgical pathology, 2000. 24(5): p. 710-718. 83. Johnson, W.T. and E.B. Helwig, Benign nevus cells in the capsule of lymph nodes. Cancer, 1969. 23(3): p. 747-753. 84. Azzopardi, J., C. Ross, and G. Frizzera, Blue naevi of lymph node capsule. Histopathology, 1977. 1(6): p. 451-461. 85. Brooks, J.S., V.A. Livolsi, and G.G. Pietra, Mesothelial cell inclusions in mediastinal lymph nodes mimicking metastatic carcinoma. American journal of clinical pathology, 1990. 93(6): p. 741-748. 86. Parkash, V., M. Vidwans, and D. Carter, Benign mesothelial cells in mediastinal lymph nodes. The American journal of surgical pathology, 1999. 23(10): p. 1264. 87. Edlow, D.W. and D. Carter, Heterotopic epithelium in axillary lymph nodes: report of a case and review of the literature. American Journal of Clinical Pathology, 1973. 59(5): p. 666-673. 88. Maiorano, E., et al., Ectopic breast tissue as a possible cause of false- positive axillary sentinel lymph node biopsies. The American journal of surgical pathology, 2003. 27(4): p. 513-518. 89. Turner, D. and R. Millis, Breast tissue inclusions in axillary lymph nodes. Histopathology, 1980. 4(6): p. 631-636. 90. Fisher, C., S. Hill, and R. Millis, Benign lymph node inclusions mimicking metastatic carcinoma. Journal of clinical pathology, 1994. 47(3): p. 245- 247. 91. Holdsworth, P., J. Hopkinson, and S. Leveson, Benign axillary epithelial lymph node inclusions—a histological pitfal. Histopathology, 1988. 13(2): p. 226-228. 92. Fellegara, G., M.L. Carcangiu, and J. Rosai, Benign epithelial inclusions in axillary lymph nodes: report of 18 cases and review of the literature. The American journal of surgical pathology, 2011. 35(8): p. 1123-1133. 93. Schweizer, J., et al., New consensus nomenclature for mammalian keratins. J Cell Biol, 2006. 174(2): p. 169-174. 94. Herrmann, H., et al., Functional complexity of intermediate filament cytoskeletons: from structure to assembly to gene ablation, in International review of cytology. 2002, Elsevier. p. 83-175. 95. Moll, R., et al., The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell, 1982. 31(1): p. 11-24. 96. Brown, D., et al., Cytokeratin expression in smooth muscle and smooth muscle tumours. Histopathology, 1987. 11(5): p. 477-486. 97. Bartek, J., et al., Differential expression of keratin 19 in normal human epithelial tissues revealed by monospecific monoclonal antibodies. The Histochemical journal, 1986. 18(10): p. 565-575. 98. Kriho, V.K., et al., Keratin expression in astrocytomas: an immunofluorescent and biochemical reassessment. Virchows Archiv, 1997. 431(2): p. 139-147. 99. Christensen, W., J. Boitnott, and F. Kuhajda, Immunoperoxidase staining as a diagnostic aid for hepatocellular carcinoma. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc, 1989. 2(1): p. 8-12. 100. Allory, Y., et al., Profiling and classification tree applied to renal epithelial tumours. Histopathology, 2008. 52(2): p. 158-166. 101. Bonsib, S., C. Bromley, and D. Lager, Renal oncocytoma: diagnostic utility of cytokeratin-containing globular filamentous bodies. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc, 1991. 4(1): p. 16-23. 102. Saphir, O. and G.D. Amromin, Obscure axillary lymph node metastasis in carcinoma of the breast. Cancer, 1948. 1(2): p. 238-241. 103. Pickren, J.W., Significance of occult metastases. A study‐ of breast cancer. Cancer, 1961. 14(6): p. 1266-1271. 104. Cserni, G., et al., Pathological work-up of sentinel lymph nodes in breast cancer. Review of current data to be considered for the formulation of guidelines. European Journal of Cancer, 2003. 39(12): p. 1654-1667. 105. Linden, M.D. and R.J. Zarbo, Cytokeratin immunostaining patterns of benign, reactive lymph nodes: applications for the evaluation of sentinel lymph node specimen. Applied Immunohistochemistry & Molecular Morphology, 2001. 9(4): p. 297-301. 106. Xu, X., et al., Undesirable cytokeratin immunoreactivity of native nonepithelial cells in sentinel lymph nodes from patients with breast carcinoma. Archives of pathology & laboratory medicine, 2000. 124(9): p. 1310-1313. 107. Wittekind, C., et al., TNM supplement: a commentary on uniform use. 2019: John Wiley & Sons. 108. Amin, M.B. and S.B. Edge, AJCC cancer staging manual. 2017: Springer. 109. Pantel, K., R.J. Cote, and Ø. Fodstad, Detection and clinical importance of micrometastatic disease. Journal of the National Cancer Institute, 1999. 91(13): p. 1113-1124. 110. Wells, C., et al., The immunocytochemical detection of axillary micrometastases in breast cancer. British journal of cancer, 1984. 50(2): p. 193. 111. Trojani, M., et al., Micrometastases to axillary lymph nodes from carcinoma of breast: detection by immunohistochemistry and prognostic significance. British journal of cancer, 1987. 55(3): p. 303. 112. Berry, N., et al., Comparison of the detection of breast carcinoma metastases by routine histological diagnosis and by immunohistochemical staining. European Surgical Research, 1988. 20(4): p. 225-232. 113. Adachi, Y., et al., Prognostic significance of the number of positive lymph nodes in gastric carcinoma. British journal of surgery, 1994. 81(3): p. 414- 416. 114. Lee, H.S., et al., Prognostic implication of isolated tumor cells and micrometastases in regional lymph nodes of gastric cancer. World Journal of Gastroenterology: WJG, 2005. 11(38): p. 5920. 115. Pereira, M.A., et al., Detection of occult lymph node tumor cells in node- negative gastric cancer patients. ABCD. Arquivos Brasileiros de Cirurgia Digestiva (São Paulo), 2017. 30(1): p. 30-34. 116. Choudhury, M., et al., Immunohistochemical detection of axillary lymph node micrometastases in node negative breast cancer patients using cytokeratin and epithelial membrane antigen. South Asian journal of cancer, 2015. 4(1): p. 28. 117. Viale, G., et al., Predicting the status of axillary sentinel lymph nodes in 4351 patients with invasive breast carcinoma treated in a single institution. Cancer, 2005. 103(3): p. 492-500.

COLOR PLATES

FIGURE 1-INVASIVE LOBULAR CARCINOMA METASTATIC FOCI MISSED ON H&E

FIGURE 2 – INVASIVE LOBULAR CARCINOMA METASTATIC FOCI DETECTED ON IHC(CYTOKERATIN)

FIGURE 3 – INVASIVE DUCTO LOBULAR CARCINOMA METASTATIC FOCI DETECTED ON IHC(LOW POWER)

FIGURE 4 - INVASIVE DUCTO LOBULAR CARCINOMA METASTATIC FOCI DETECTED ON IHC(HIGH POWER)

FIGURE 5 –POORLY DIFFERENTIATED ADENOCARCINOMA (STOMACH) METASTATIC FOCI MISSED ON H&E

FIGURE 6 - POORLY DIFFERENTIATED ADENOCARCINOMA (STOMACH) METASTATIC FOCI DETECTED ON IHC

FIGURE 7 - POORLY DIFFERENTIATED ADENOCARCINOMA (STOMACH) METASTATIC FOCI MISSED ON H&E

FIGURE 8- POORLY DIFFERENTIATED ADENOCARCINOMA (STOMACH) METASTATIC FOCI DETECTED ON IHC

FIGURE 9- INVASIVE DUCTAL CARCINOMA METASTATIC FOCI MISSED ON H&E(LOW POWER)

FIGURE 10 - INVASIVE DUCTAL CARCINOMA METASTATIC FOCI MISSED ON H&E(HIGH POWER)

FIGURE 11 - -INVASIVE DUCTAL CARCINOMA METASTATIC FOCI DETECTED ON IHC(HIGH POWER)

FIGURE 12- MODERATELY DIFFERENTIATED ADENOCARCINOMA (STOMACH) METASTATIC FOCI DETECTED ON IHC(SCANNER)

FIGURE 13 - MODERATELY DIFFERENTIATED ADENOCARCINOMA(STOMACH) METASTATIC FOCI DETECTED ON IHC(LOW POWER)

S.N PATH AGE REGIONAL IP.NO PROCEDURE DONE HPE DIAGNOSIS O NO /SEX LYMPHNODES TNM (CK) TNM (H&E) STAGE (CK) STAGE STAGE(H&E) TOTAL NODES TOTAL H&E POSITIVE H&E NEGATIVE PANCK POSITIVE PANCK PANCK NEGATIVE PANCK HISTOLOGICAL GRADE HISTOLOGICAL

Invasive ductal carcinoma nos-Gr II - 1 1718/17 74/F 32288 Modified radical mastectomy Axillary lymphnodes Right breast G2 14 1 13 0 13 T2N1 IIB T2N1 IIB

2 1736/17 66/F 31674 Modified radical mastectomy Axillary lymphnodes Invasive lobular carcinoma-Right breast 12 3 9 0 9 T4aN1 IIIB T4aN1 IIIB Total hysterectomy with bilateral Well differentiated adenocarcinoma of 3 1765/17 75/F 30080 oopherectomy B/L Pelvic nodes endometrium G1 2 0 2 0 2 T1aN0 IA T1aN0 IA Ileal resection with mesenteric node Well differentiated adenocarcinoma of 4 1953/17 37/M 34482 dissection Mesenteric nodes ileum G1 6 2 4 0 4 T3N1 IIIA T3N1 IIIA Invasive ductal carcinoma nos-Gr II -Left 5 2067/17 68/F 40312 Modified radical mastectomy Axillary lymphnodes breast G2 13 0 13 0 13 T2N0 IIA T2N0 IIA Invasive ducto- lobular carcinoma nos-Gr 6 2068/17 45/F 40333 Modified radical mastectomy Axillary lymphnodes II -Left breast G2 11 8 3 2 1 T2N2 IIIA T2N3 IIIC Moderately differentiated squamous cell 7 2100/17 76/M 39163 Lip excision with MRND Cervical lymphnodes carcinoma of Lip G2 12 8 4 2 2 T2N2 IVA T2N2 IVA

8 2160/17 54/F 38019 Total gastrectomy with D2 Extended perigastric Well differentiated adenocarcinoma of lymphadenectomy lymphnodes Stomach G1 3 1 2 0 2 T3N1 III T3N1 III Invasive ductal carcinoma nos-Gr II -Left 9 2248/17 35/F 42043 Modified radical mastectomy Axillary lymphnodes breast G2 12 0 12 0 12 T3N0 IIB T3N0 IIB Invasive ductal carcinoma nos-Gr II - 10 2251/17 70/F 39491 Modified radical mastectomy Axillary lymphnodes Right breast G2 12 3 9 0 9 T4aN1 IIIB T4aN1 IIIB Total hysterectomy with bilateral B/L Pelvic nodes & Well differentiated adenocarcinoma of 11 2309/17 66/F 37996 oopherectomy paraaortic nodes endometrium G1 7 0 7 0 7 T1aN0 IA T1aN0 IA Total hysterectomy with bilateral B/L Pelvic nodes & Well differentiated adenocarcinoma of 12 2325/17 31/F 41331 oopherectomy paraaortic nodes endometrium G1 16 0 16 0 16 TIaN0 IA T1aN0 IA

13 2354/17 79/M 42942 Total gastrectomy with D2 Extended perigastric Poorly differentiated adenocarcinoma of lymphadenectomy lymphnodes Stomach G3 7 1 8 2 6 T3N1 III T3N2 III Invasive ductal carcinoma nos-Gr II - 14 2378/17 40/F 43536 Modified radical mastectomy Axillary lymphnodes Right breast G2 3 0 3 1 2 T3N0 IIB T3N1 IIIA Total hysterectomy with bilateral Serous papillary cystadenocarcinoma- LOW 15 2386/17 55/F 40698 oopherectomy B/L Pelvic nodes Ovary GRADE 7 0 7 0 7 T1aN0 IA T1aN0 IA Well differentiated squamous cell 16 2388/17 52/F 39451 Composite resection with MRND Cervical lymphnodes carcinoma -Right alveolus G1 19 0 19 0 19 T1N0 I T1N0 I Invasive ductal carcinoma nos-Gr II -Left 17 2441/17 75/F 44712 Modified radical mastectomy Axillary lymphnodes breast G2 13 0 13 0 13 T2N0 IIA T2N0 IIA S.N PATH AGE REGIONAL IP.NO PROCEDURE DONE HPE DIAGNOSIS O NO /SEX LYMPHNODES TNM (CK) TNM (H&E) STAGE (CK) STAGE STAGE(H&E) TOTAL NODES TOTAL H&E POSITIVE H&E NEGATIVE PANCK POSITIVE PANCK PANCK NEGATIVE PANCK HISTOLOGICAL GRADE HISTOLOGICAL

Moderately differentiated squamous cell 18 2513/17 80/F 42649 Composite resection with MRND Cervical lymphnodes carcinoma of Right Buccal mucosa G2 4 0 4 0 4 T1N0 I T1N0 I Invasive ductal carcinoma nos-Gr II - 19 2571/17 45/F 46605 Modified radical mastectomy Axillary lymphnodes Right breast G2 15 11 4 2 2 T1N3 IIIC T1N3 IIIC Invasive ductal carcinoma nos-Gr II -Left 20 2572/17 54/F 48309 Modified radical mastectomy Axillary lymphnodes breast G2 16 0 16 0 16 T1N0 IA T1N0 IA Well differentiated adenocarcinoma of 21 2595/17 67/F 41078 Wertheim's Radical Hysterectomy B/L Pelvic nodes endocervix G1 13 0 13 0 13 T1bN0 IB T1bN0 IB Partial gastrectomy with D1 22 2633/17 65/M 46226 lymphadenectomy Perigastric nodes Mucinous adenocarcinoma of Stomach 5 0 5 0 5 T3N0 IIB T3N0 IIB Partial gastrectomy with D1 Moderately differentiated adenocarcinoma 23 2644/17 55/M 44925 lymphadenectomy Perigastric nodes of Stomach G2 4 0 4 0 4 T4aN0 IIB T4aN0 IIB Invasive ductal carcinoma nos-Gr II - 24 2781/17 60/F 50814 Modified radical mastectomy Axillary lymphnodes Right breast G2 7 0 7 0 7 T3N0 IIB T3N0 IIB Moderately differentiated adenocarcinoma 25 2861/17 53/F 46978 Wertheim's Radical Hysterectomy B/L Pelvic nodes of endocervix G2 12 0 12 0 12 T1bN0 IB T1bN0 IB Partial gastrectomy with D1 Moderately differentiated adenocarcinoma 26 3108/17 70/M 55535 lymphadenectomy Perigastric nodes of Stomach G2 5 0 5 1 4 T3N0 IIB T3N1 III Partial gastrectomy with D1 Well differentiated adenocarcinoma of 27 3140/17 68/M 56072 lymphadenectomy Perigastric nodes Stomach G1 5 0 5 0 3 T4aN0 IIB T4aN0 IIB Amputation left footwith Left Ilio-inguinal Well differentiated keratinising squamous 28 3934/17 52/M 68296 block dissection Inguinal nodes cell carcinoma of left foot G1 4 0 4 0 4 T3N0 III T3N0 III Well differentiated adenocarcinoma of 29 4045/17 70/F 75980 Wertheim's Radical Hysterectomy B/L Pelvic nodes endometrium G1 3 0 3 0 3 TIbN0 IB T1bN0 IB Atypical medullary carcinoma-Gr-II of 30 4249/17 48/F 80583 Modified radical mastectomy Axillary lymphnodes Right breast G2 6 1 5 0 3 T2N1 IIB T2N1 IIB

31 4430/17 60/M 82884 Well differentiated keratinising squamous Composite resection with MRND Cervical lymphnodes cell carcinoma of Right buccal mucosa G1 4 0 4 0 4 T1N0 I T1N0 I Invasive ductal carcinoma nos-Gr II -Left 32 4470/17 45/F 82918 Modified radical mastectomy Axillary lymphnodes breast G2 16 9 6 2 4 T2N2 IIIA T2N3 IIIC Well differentiated adenocarcinoma of 33 307/18 60/F 358 Wertheim's Radical Hysterectomy B/L Pelvic nodes cervix G1 10 0 10 0 10 T2aN0 IIA T2aN0 IIA Invasive ductal carcinoma nos-Gr II -Left 34 429/18 47/F 6574 Modified radical mastectomy Axillary lymphnodes breast G2 9 0 9 0 9 T2N0 IIA T2N0 IIA 35 544/18 60/F 5089 Modified radical mastectomy Axillary lymphnodes Invasive lobular carcinoma-Left breast 12 0 12 1 11 T2N0 IIA T2N1 IIB S.N PATH AGE REGIONAL IP.NO PROCEDURE DONE HPE DIAGNOSIS O NO /SEX LYMPHNODES TNM (CK) TNM (H&E) STAGE (CK) STAGE STAGE(H&E) TOTAL NODES TOTAL H&E POSITIVE H&E NEGATIVE PANCK POSITIVE PANCK PANCK NEGATIVE PANCK HISTOLOGICAL GRADE HISTOLOGICAL

36 616/18 50/F 6613 Well differentiated keratinising squamous Composite resection with MRND Cervical lymphnodes cell carcinoma of Left buccal mucosa G1 11 1 10 0 10 T4aN1 IVA T4aN1 IVA Atypical medullary carcinoma-Gr-III of 37 700/18 52/F 13576 Modified radical mastectomy Axillary lymphnodes Left breast G3 10 0 10 0 10 T2N0 IIA T2N0 IIA Partial gastrectomy with D1 Poorly differentiated adenocarcinoma of 38 740/18 52/F 12605 lymphadenectomy Perigastric nodes Stomach G3 3 0 3 1 2 T4aN0 IIB T4aN1 III Moderately differentiated adenocarcinoma 39 789/18 72/F 16301 Hemicolectomy Mesenteric nodes of Colon G2 5 0 5 0 5 T3N0 IIA T3N0 IIA Invasive ductal carcinoma nos-Gr II -Left 40 1035/18 47/F 18252 Modified radical mastectomy Axillary lymphnodes breast G2 12 0 12 0 12 T2N0 IIA T2N0 IIA Total hysterectomy with bilateral Serous papillary cystadenocarcinoma- HIGH 41 1075/18 46/F 15279 oopherectomy B/L Pelvic nodes Ovary GRADE 20 0 20 0 20 T1aN0 IA T1aN0 IA Glossectomy with Supraomohyoid neck Moderately differentiated squamous cell 42 1184/18 45/M 17852 dissection Cervical lymphnodes carcinoma of tongue G2 15 2 13 0 13 T2N1 III T2N1 III B/L paratracheal 43 1202/18 52/F 17800 Total thyroidectomy dissection with FND Papillary carcinoma thyroid 29 0 29 0 29 T2N0 I T2N0 I

44 1669/18 49/M 30078 Radical cystoprostatectomy B/L Pelvic nodes Adenocarcinoma of urinary bladder G2 9 0 9 0 9 T4aN0 IIIA T4aN0 IIIA Partial gastrectomy with D1 Moderately differentiated adenocarcinoma 45 1941/18 64/F 36222 lymphadenectomy Perigastric nodes of Stomach G2 7 0 7 0 7 T4aN0 IIB T4aN0 IIB Total hysterectomy with bilateral Well differentiated endometrioid 46 1975/18 63/F 34724 oopherectomy B/L Pelvic nodes adenocarcinoma- ovary G1 8 0 8 0 8 T1aN0 IA T1aN0 IA Total hysterectomy with bilateral Endometrioid Endometrial 47 2041/18 65/F 30990 oopherectomy B/L Pelvic nodes adenocarcinoma-Gr II G2 20 2 18 0 18 T3bN1 IIIC1 T3bN1 IIIC1 Invasive ductal carcinoma nos-Gr II -Left 48 2046/18 70/F 30562 Modified radical mastectomy Axillary lymphnodes breast G2 5 0 5 0 5 T3N0 IIB T3N0 IIB Invasive ductal carcinoma nos-Gr II - 49 2135/18 29/F 39529 Modified radical mastectomy Axillary lymphnodes Right breast G2 21 0 21 0 21 T2N0 IIA T2N0 IIA Invasive ductal carcinoma nos-Gr II - 50 2185/18 40/F 73414 Modified radical mastectomy Axillary lymphnodes Right breast G2 5 1 4 0 4 T2N1 IIB T2N1 IIB