Spectrum of Nodal and Extra Nodal Lymphoma in a Regional Cancer Centre, Odisha, India

Lymphoma encompasses a large group of cancers, ranking 7th among the common malignancies. Commonly, lymphoma originates in the lymph nodes. Infiltration of malignant lymphomatous cells in organs other than the lymph node is termed as Extra Nodal Lymphoma. In this retrospective study of 253 cases of Lymphoma, the spectrum of histopathological features of Extra Nodal Lymphoma is illustrated to ascertain the histological and anatomical distribution of ENL & NL. ENL constituted about 16% of all lymphomas studied during this period. Gastro-intestinal tract is the most common anatomical site in ENL. The peak incidence is found among middle aged population. Cervical Lymph Node is the most prevalent node involved in case of Nodal Lymphoma. This study was carried out at the Acharya Harihar Regional Cancer Centre (AHRCC), Cuttack, Odisha, India. This activity outlined the epidemiological and morphological evaluation of ENL & NL. future research will look to focus and highlight the survival analysis and genetic profile to understand the complex biology of primary Extranodal Lymphoma.


Introduction
Lymphoma is an umbrella term that encompasses a large group of cancers, ranking 7th among the common malignancies in both the sexes together [1].This neoplastic proliferation of lymphoid cells often affects the lymph nodes; with infiltration into the bone marrow, spleen and thymus.These are the organs constituting the primary lymphoid organs [2].In nodes, uncontrollable proliferation and growth of cells due to lymphatic mutation results in tumorigenesis [3].In extra nodal lymphoma, the sites involved include organs other than lymph nodes, spleen, thymus and pharyngeal lymphatic rings [4].However, spleen being involved is considered as a nodal disease in Hodking's disease, where as an extra nodal case in Non Hodking's lymphoma [5].The cause of lymphoma still remains unclear; however certain individuals show more susceptibility towards this disease.These individuals may include those who are infected with virus and bacteria like HIV, Helicobacter pylori, Epstein-Barr virus or Human T-Lymphotrophic virus [3].Genetic link of an individual also increases his susceptibility [6].In spite of complexity in classification of lymphoma, it can be broadly classified into Non-Hodking's Lymphoma and Hodking's Lymphoma, with their sub classes ranging to 30 in number [7][8][9][10].This neoplastic disease is highly treatable and often curable when caught early.However extra nodal lymphomas pose a challenge during their diagnosis due to varied clinical presentations, morphological mimicry and molecular alterations [11,12].Although the incidence of ENL has seen a hike in the last 20 years, the literature on ENL is limited.Hence the study was undertaken to ascertain the incidence, anatomical distribution and other factors.

Aims and Objectives
To ascertain the incidence, distribution and histological subtypes of nodal and extra nodal lymphoma in a Regional Cancer Centre, Odisha.

Materials and Method
This hospital based retrospective study was conducted on patients who came to the department of Acharya Harihar Regional Research Centre (AHRCC), 0disha and diagnosed to have lymphoma.The study was carried out over a period from January, 2018 -January, 2019 and included prospective data collected during the period from February, 2019 -April,2019.During this period, 350 patients are diagnosed with lymphoma.Among which 253 cases were included for the study who were confirmed of lymphoma based on histopathological test and the rest 93 cases were excluded due to incomplete evaluation and lack of data in the records.The tissues from the patients who underwent biopsy of the lesion was processed and stained with haematoxylin and eosin.The patients with nodal lymphoma underwent excisional biopsy and the patients with extranodal lymphoma were diagnosed based on operated histopathological specimen.IHC studies were performed using a panel of antibodies depending on morphology of tissue section of the cases resected during biopsy.The protocol followed for H&E stain & IHC test have been mention below.HISTOPATHOLOGICAL TECHNIQUES Tissue collection: Tissue resected from biopsy was fixed in normal buffer formalin (NBF) 10% formalin immediately [100ml formalin (37.4% stock solution) + 900 ml of distiiled water + 9mg NaCl + 12gm Na2HPO4] for 6-72 hours.

Figure 1: Automatic Tissue Processor
Grossing and processing: The tissue was grossed and the sections were processed in automatic tissue processor (histokinetics).The processing of tissue included dehydration in increasing gradients of alcohols followed by clearing in xylene & then embedding in paraffin wax.
Section-cutting: After impregnation with paraffin, thin sections of 5 micrometer thickness were cut using microtomy and then stained with H&E staining.H&E staining: The tissue section of slide first undergoes deparafinization by xylene 1 & 2 for 10-20 mins each.Then follows hydration with decreased concentration of alcohol (100%, 85%, 70%, 50%).Then it is counter stained with haematoxylene for 5-10 minutes and washes and then stained with eosin.This is followed by dehydration with increasing concentration of alcohol (50-100%).It was then cleaned with acetone and xylene and observed with DPX mount.

Figure 2: Experimental set up for H & E Staining
The data pertaining to patients, demography, clinical signs and symptoms, routine CBC, imagery finding, IHC reports were received from the medical records.

Statistical Analysis
Measurement data were expressed as mean +/-SD.Count Data were expressed as percentage.The age group distribution, organ involvement were compared for nodal and extra nodal cases separately using histograms and pie-charts.

Results
Extranodal lymphoma constituted 16.06% (35/218) of all lymphoma studied during this period.The mean age at presentation for ENL and nodal lymphoma was 52 years and 48 years respectively (range 4-95 years).There were 20 male and 15 female in ENL group compared to 154 male and 64 female in nodal lymphoma group [Table 2].The peak incidence was in 5th decade in both ENL and NL.Studies from Pakistan, Korea and China have also reported incidence ranging from 45% to 62%.This present study showed the incidence to be 16.06% which is lower than the studies followed in and around India.In

Conclusion
This study showed that the overall incidence of ENL in our centre is less common in comparison to the data from other parts of India and Asia.The GIT was the most common site of involvement among the ENL as seen in other studies.Compared to previously published studies of extra-nodal lymphoma series, no major specific differences were noted with respect to the gender preponderance and the histopathology.Majority of the patients presented at younger age, with early stage of lymphoma and were immunecompetent.This study being mostly an epidemiological and morphological study, failed to address the outcomes as the long-term follow-up data pertaining to the survival analysis are lacking.However, we do believe at this point of time that more studies of similar kind with focus on outcomes and also highlighting the genetic profile, must be carried out to understand the complex biology of the primary extra-nodal lymphomas.

Figure 3 :
Figure 3: Stained Cancer Cells Under MicroscopePatients diagnosed with lymphoma in organs other than lymph nodes and primary lymphoid organs were considered for extra nodal cases.Nodal lymphoma involvement of primary lymphoid organ or otherwise not specified were considered nodal by default.Classification of lymphoma classes were considered according to the WHO classification.

Table 1 .
World Health Organization (WHO) Classification of Lymphoid Neoplasms Non

Figure 7 :
Figure 7: Gender Distribution Among Different Age Groups this study, in comparison to the patients with extra nodal lymphoma, patients diagnosed to have NL were younger [mean 52.29 years vs 48.15 years] which differed from the study done by Pai et al in South India that stated the ENL where younger than the nodal.Follicular lymphocytic, small cell lymphoma and mantle cell lymphoma seen in nodal lymphoma, were not seen at extra nodal sites and thin may be possibly due to the geographic variations in molecular expression profiling of the lymphoma as reported by Biagi and Seymour et al.A similar result was obtained from this study of mine.The incidence of GIT lymphomas has been increasing throughout the world has been reported to be the most common site and involvement among extra nodal cases [18].After GIT, the most common involved extra nodal site in head and neck DLBCL being the commonest histological type.The ENL have been known to have complex biological behaviour as shown in various studies.In spite of significant diagnostic and treatment development, the therapeutic outcome has been worse for patients with ENL involving rare sites as shown by Yun et al.Studies have also shown that the age, stage of lymphoma, performance status and serum LDH level have 71 been independent prognostic variables, where as the site involvement did not bear any prognostic significance as shown by Lal et al In this present study 117 at initial presentation, patients with NL were younger than extra nodal lymphoma.