Significance of Immunohistochemistry in Accurate Characterisation of Hodgkin Lymphoma

Background: Hodgkin lymphomas are malignant disorders of cells residing in lymphoid tissue and containing Reed-Sternberg (RS) cells and its variants and account for about 0.7% of all new cancers. Hodgkin lymphomas are two types: Classical Hodgkin lymphoma (CHL) that is CD30 positive and CD45 negative in specific pattern; Lymphocyte-predominant Hodgkin lymphoma (LPHL) that is CD30 negative and CD45 positive in specific pattern. Objective: To evaluate the value of immunohistochemistry in the diagnosis and accurate characterisation of Hodgkin lymphoma. Materials and Methods: This cross-sectional study was carried out in the department of Pathology of Sir Salimullah Medical College & Mitford Hospital, Dhaka from January 2010 to June 2012. Histopathologically diagnosed 45 cases of Hodgkin lymphoma and 5 cases of other than Hodgkin lymphoma were selected and then immunomarkers CD30 and CD45 were applied. Results: Among 50 cases 37 were classical Hodgkin lymphoma and 8 cases were lymphocytepredominant Hodgkin lymphoma histopathologically. When immunomarkers were applied in 50 cases then 33 cases were classical Hodgkin lymphoma and 9 cases were lymphocyte-predominant Hodgkin lymphoma. Conclusion: Immunohistochemistry helped in accurate characterisation of Hodgkin lymphoma.

Lymphomas are malignant disorders of cells residing in lymphoid tissue and are classified into two main types: Hodgkin lymphoma and non-Hodgkin lymphoma.About 1,700 cases are diagnosed as Hodgkin lymphoma in United Kingdom each year.About 1 in every 200 cancers diagnosed is a Hodgkin lymphoma. 1Hodgkin lymphomas account for 0.7% of all new cancers in the United States.Male female ratio in Hodgkin lymphoma is 1.5:1 (except nodular sclerosis). 2 Hodgkin lymphomas are of two types: a) Classical Hodgkin lymphoma (CHL), b) Lymphocytepredominant Hodgkin lymphoma (LPHL).These two entities of Hodgkin lymphoma differ in their clinical features and behaviour, and in their morphology, immunophenotype and immunoglobulin transcription of the neoplastic cells as well as in the composition of their cells and their cellular background. 3LPHL have distinct pathobiology: an indolent natural history, unimodal age distribution, marked male predominance, frequent axillary presentation, and association with low stage disease.Long survival of patient with LPHL without any treatment could favour a 'watch and wait' strategy. 4ntroduction 23

Significance of Immunohistochemistry in Accurate Characterisation of Hodgkin Lymphoma
Hodgkin lymphoma are actually proved to be large cell non-Hodgkin lymphoma.In 3-5% cases of lymphocytepredominant type Hodgkin lymphoma resembling diffuse large B cell lymphoma, marker study can help to resolve the problem. 3e marker CD30 is positive and marker CD45 is negative in RS cells in classical Hodgkin lymphoma.
On the other hand study with the same markers on RS variant L&H cells (popcorn cell) present in nonclassical lymphocyte-predominant variety resulted in opposite finding, that is, the cell is negative for CD30 but positive for CD45 in specific pattern. 5munohistochemistry (IHC) is a new technique in our country.As IHC has provided a feasible approach to performing immunostaining on routinely processed tissues, and this method is now becoming a routine practice in surgical pathology laboratories in some hospitals, we designed to conduct this study in our population for accurate diagnosis and characterisation of Hodgkin lymphoma using the two immunohistochemical markers CD30 and CD45.

Study procedure
In this study comparison between two diagnostic procedures, histopathology and immunochemistry, were done for characterisation of Hodgkin lymphoma.After selection of specimens immunomarkers CD30 and CD45 were applied on each of these.By seeing specific staining pattern of immunomarkers, characterisation of Hodgkin lymphoma were done and compared with histopathology using diagnostic performance tests (sensitivity, specificity, positive predictive value [PPV], negative predictive value [NPV] and accuracy).

Immunohistochemical staining procedure
Immunohistochemistry was done on the tissue specimens histopathologically diagnosed as Hodgkin lymphoma.Paraffin embedded specimens were sectioned at a thickness of 4-5 microns.Sections were deparaffinised and rehydrated through graded alcohol to water prior to staining proper.Antibodies for CD30 and CD45 were used in all cases for diagnosing and characterisation of subtypes of Hodgkin lymphoma.

Routine staining for light microscopy
Tissues were fixed in 10% formalin and embedded in paraffin block and processed routinely.The paraffin blocks were sectioned at 4-5 micrometer thickness and were stained with H&E stain.

Ethical clearance
Prior to the commencement of the study, the research protocol was approved by the Ethical Committee of Sir Salimuallah Medical College & Mitford Hospital, Dhaka.There was no threat or risk for study population.Patients' private information was not disclosed.

Data processing and analysis
All the data were checked and edited after collection and computerised.Statistical analysis of the results was obtained by using Windows based computer software devised with Statistical Packages for Social Sciences (SPSS 13.0) (SPSS Inc, Chicago, IL, USA).The results were presented in tables and figures.The statistical terms included in this study are mean, standard deviation and percentage.Statistical significance was set at p<0.05 and confidence interval set at 95% level.

Results
The age of the subjects in this study was 7-79 years with mean ± SD 33.90 ± 17.70 years.Regarding immunohistochemistry study in 45 cases of Hodgkin lymphoma, 32 were CD30 positive and CD45 negative in specific staining pattern and 9 cases were CD45 positive in specific staining pattern and CD30 negative.Out of five cases other than Hodgkin lymphoma, four cases were both CD30 and CD45 negative in specific staining pattern; only one case was CD30 positive and CD45 negative in specific pattern.Lymphocyte-predominant Hodgkin lymmphoma may differentially be diagnosed as lymphocyte rich classical Hodgkin lymphoma. 12Histopathologically classical RS cells have either bilobed nuclei or two nuclei, each containing distinct nucleoli and L&H cells have a single, large, folded or multilobated nucleus (popcorn cell).L&H cell nuclei typically contain multiple small basophilic nucleoli that are smaller than those of classical RS cells. 3Curiously, the European Task Force on Lymphoma (ETFL) study reported the coexistence of RS cells in addition to L&H cells in substantial percentage of LPHL cases. 8The current consensus, however, is that although cells that appear to be RS cells by morphologic criteria can occasionally be seen in LPHL, such cells are negative for CD15 and CD30 and positive for CD45 and therefore are not classical RS cells. 13mphocyte-predominant Hodgkin lymphoma occurs in 5-10% cases of Hodgkin lymphoma. 13But number of cases of lymphocyte-predominant Hodgkin lymphoma was higher (17.78%) in this study.Lymphocyte-predominant Hodgkin lymphoma occurs mainly in cervical and axillary regions. 13In the present study, maximum number of lymphadenopathy occurred in cervical (n=28) and axillary (n=5) regions.Submental, inguinal and mesenteric lymph node swellings were absent.
Lymphocyte-predominant Hodgkin lymphoma occurs mainly in 30-40 years with a male predominance. 13In this study, overall mean age was 33.90 years and number of males was 28 (62.22%) and that of females was 17 (37.78%).
In this study CD30 stained RS cell membranes and showed dot like cytoplasmic staining in cases of classical Hodgkin lymphoma and no specific staining pattern in cases of lymphocyte-predominant Hodgkin lymphoma.In few cases CD30 stained centroblast and centrocytic cells of lymphoma.Jaffe 14 also got similar staining pattern.
In this study CD45 stained popcorn cell membranes of lymphocyte-predominant Hodgkin lymphoma whereas there was no specific staining pattern in classical Hodgkin lymphoma; but it showed diffuse staining pattern by staining all haemopoetic cells of classical Hodgkin lymphoma.Lee 15 also got the same findings.
In 2 reactive changes and 2 non-Hodgkin lymphoma cases of 5 cases other than Hodgkin lymphoma, CD30 showed no staining and CD45 showed diffuse staining of haemopoietic cells like T cells and B cells.Similar staining patterns were also found by other researchers. 16,17 one case of non-Hodgkin lymphoma, CD30 showed staining that was like RS cells.CD45 showed diffuse staining pattern of haemopoietic cells in non-Hodgkin lymphoma.Immunohistochemistry (IHC) is built on the foundations of histopathology.It does not replace histopathology but rather serves as a valuable adjunct that greatly extends the variety of tissue components that can be demonstrated specifically within tissue section or other cell preparations.The basic critical principle of IHC, as with any other special staining method, is a sharp localisation of target components in the cell and tissue based on a satisfactory signal.Amplifying the signal while reducing non-specific background staining is a major challenge to achieve a satisfactory and practically useful result. 9om the findings of the present study it can be concluded that immunohistochemistry has high sensitivity, specificity and diagnostic accuracy in case of Hodgkin lymphoma and it can safely be used in the diagnosis of Hodgkin lymphoma along with histopathology.

Limitations of the study
The major limitation of this study is small sample size; only 50 cases were included in the study.Secondly, there are many markers for Hodgkin lymphoma, but due to difficulty in procurement and high cost, only two markers (CD30 and CD45) were used in this study.
There are also many potential problems affecting the outcomes of the immunostaining procedure.There were reasons to believe that the findings and conclusion of the study may not be completely representative though it may give general idea of certain clinicopathological aspect of Hodgkin lymphoma amongst the population of Bangladesh.

Fig 1 .Fig 2 .Fig 3 .Fig 4 .
Fig 1. Photomicrograph of classical Hodgkin lymphoma showing CD30 +ve cells (H&E stain) Table I shows the sex distribution of Hodgkin lymphoma patients.Male to female ratio was 1.64:1.Table II shows the sites of lymphadenopathy of Hodgkin lymphoma.All these lymphadenopathies were superficial.Histopathlogical types of Hodgkin lymphoma are shown in Table III.
Table V: Immunohistochemisty results in five cases other than Hodgkin lymphoma

Table VII :
Table VII shows the findings of validity test.The sensitivity, specificity, PPV, NPV and accuracy are 94.11%, 87.5%, 96.96%, 77.78% and 92.85% respectively.Diagnostic performance test of PPV, Positive predictive value; NPV, Negative predictive value

Table VI :
8iagnostic performance test results (n=50=45+5)Out of 45 cases of Hodgkin lymphoma, 28 (62.22%) were male patients and 17 (37.77%)werefemale.Male to female ratio was 1.64:1.Desforges et al8stated that in their study younger group showed equal male to female ratio in contrast to higher male to female ratio in older age group in Hodgkin lymphoma.Bhatia 9 found male to female ratio 1.47:1.Rosillo 10 found male to female ratio 1.8:1 in 127 patients with Hodgkin lymphoma.
Gutenson & Cole 17 and Hall 18 also found similar findings.