pathology of b cell lymphoma and hodgkins

lymphoma

1. IC-712/23 ( S-997/23 ) DR ZAREEN 20-12-2023

2. CASE PRESENTATION HISTORY: A 7 year old boy presented with bilateral neck swelling since 1year.

3. HISTORY OF PRESENT ILLNESS : Patient was apparently alright 1year back . Then he developed bilateral cervical swelling , which was gradual in onset, progressive in nature. Associated with occasional cough. Not associated with fever, pain, loss of appetite and loss of weight. All the evaluation for Tuberculosis were Negative.

4. PAST HISTORY : No history of asthma, tuberculosis, diabetes and hypertension. FAMILY HISTORY : No similar history in the family. PERSONAL HISTORY : Diet- Vegetarian Appetite- Reduced Sleep- Sound Bowel and bladder- Regular Habits- Nil

5. GENERAL PHYSICAL EXAMINATION : Patient is conscious, oriented to time, place and person. Pallor, icterus, cyanosis, clubbing-not seen. L ymphadenopathy - seen. On Palpation: Cervical lymph node: Soft, matted , Left sided swelling was more prominent than right. Axillary Lymph node: Swelling noted on the left side. Inguinal Lymph Node: Bilateral swelling noted. Vitals: Temperature- Afebrile. Pulse rate: 130 beats/min Blood pressure: 110/60 mmHg Respiratory rate: 24 cycles/min

6. SYSTEMIC EXAMINATION: Central Nervous System- conscious and oriented Cardiovascular System- S1, S2 heard, no murmurs. Respiratory System- Normal vesicular breath sounds heard, no added sounds. Per abdomen- Soft, non-tender, no organomegaly .

7. Biopsy was done from the left cervical lymph node. (21/10/2022) HISTOPATHOLOGY REPORT: Specimen type: Lymph node. IMPRESSION: Features are of Reactive Non- Specific Lymphadenitis .

8. SPUTUM FOR AFB(17-05-2023) SPUTUM : Negative for Acid Fast Bacilli. SEROLOGY: (18-05-2023) MANTOUX TEST : Negative.( No induration seen).

9. RADIOLOGICAL INVESTIGATIONS ULTRASONOGRAPHIC OF NECK:( 02-11-2023) FINDINGS: Multiple enlarged round and oval hypoechoic cervical lymphnodes with loss of fatty hilum noted in bilateral level IB, II, III, IV and V and in left supraclavicular region largest measuring 4.1 x 1.5 cms in right level IB and measuring 2.8 x 1.6cms in left level IV. Few of them are showing the necrotic component. Matting of nodes noted on both sides. Few of them showing possible intranodal reticulations. Enlarged hypoechoic and necrotic lymphnodes noted in the left parotid gland largest measuring 21 x 11mm. IMPRESSION : Lateral cervical lymphadenopathy , possible lymphomatous nodes / Infective etiology. Hypoechoic lymphnodes noted in the left parotid gland.

10. ULTRSOUND OF WHOLE ABDOMEN:(02-11-2023) Liver is normal in size (11.8 cms ), shape and echotexture . No evidence of any focal solid or cystic lesions. No evidence of any intrahepatic biliary dilatation. Gall Bladder is contracted.BD and PV are normal.Pancreas is normal in size, shape and echotexture . No evidence of focal lesion or calcification. Spleen is normal in size (8.1 cms ), shape and echotexture . Few illdefined hypoechoic lesions noted in the parenchyma largest measuring 10 x 8mm. Right kidney measures 7.5 x 2.8 cms . Left kidney measures 7.1 x 3.0 cms . Both kidneys are normal in size, shape and echotexture . Cortical thickness and corticomedullary differentiation are normal. No evidence of any pelvicalyceal dilatation. No calculi seen. Urinary bladder is partially distended. No calculi seen. Pelvic organs are normal for age. No evidence of any free fluid in peritoneal cavity and pelvis. IMPRESSION : Few illdefined hypoechoic lesions in the splenic parenchyma infective/inflammatory etiology.

11. INVESTIGATIONS: COMPLETE BLOOD COUNT (04-12-2023) Haemoglobin - 10.9 g/dl (11.5-15 gm/dl) Total count- 5,510 cells / cumm (5,000-13,000 cells/ cumm ) Platelet count- 3.21 lakhs/ cumm ( 1.7-4.5 lakhs/ cumm ) Differential count:- Neutrophils - 70 % ( 40-62 %) Lymphocytes- 2 6 % ( 38 -45%) Monocytes- 03 % ( 4-8%) Eosinophils- 01 %( 2-8 %) Basophils- 0 % (<1%) ESR- 58mm/hour (10-20 mm/hr)

12. Lactate Dehydrogenase =352U/L Serum Creatinine =0.49mg/ dL . LIVER FUNCTION TEST: (04-12-2023) Total Bilrubin =0.48mg/ dL Conjugated Bilrubin =0.16mg/ dL Unconjugated Bilrubin =0.16mg/dl Total protiens =7.34g/dl Albumin=3.99g/dl Globulin=3.35mg/ dL A/G Ratio=1.19 SGPT(ALT)=40.0 U/L Alkaline Phosphatase =162U/L

13. RENAL FUNCTION TEST:(04-12-2023) Sodium=136.9mmol/l Potassium=3.93 mmol /L Chloride=106.4mmol/L Urea-15.0 mg/ Dl Creatinine =0.58mg/dl eGFR = 234.94

14. FDG PET – SCAN OF WHOLE BODY (5-12-2023) IMPRESSION: In pretherapy evaluation of Hodgkin's lymphoma, present PET CT scan findings revealed Multiple enlarged hypermetabolic supra and infra-diaphragmatic lymph nodes - lymphomatous involvement. Mild to moderate splenomegaly with low-grade metabolically active subtle hypodense lesions in spleen likely lymphomatous involvement. No other evidence of metabolically active disease in rest of whole-body survey .

15. CLINICAL IMAGE :

16. MICROSCOPIC EXAMINATION FNAC-GIEMSA,LOW POWER VIEW (10X)

17. FNAC-GIEMSA,HIGH POWER VIEW (40X)

18. GIEMSA – HIGH POWER VIEW(40X) GIEMSA – OIL IMMERSION VIEW(100X)

19. H AND E- SCANNER VIEW (4X )

20. H AND E- OIL IMMERSION VIEW (100X) H AND E- OIL IMMERSION VIEW (100X)

21. CD-15- HIGH POWER VIEW (40X) CD 30: HIGH POWER VIEW (40X)

22. CD 3 0:HIGH POWER VIEW (40X) CD 3 0:`OIL IMMERSION VIEW (100X )

23. PAX 5 : HIGH POWER VIEW (40X) PAX 5 : OIL IMMERSION VIEW ( 100 X )

24. DIAGNOSIS :- Hodgkins Lymphoma- Mixed Cellularity Subtype.

25. REVIEW OF LITERATURE DEFINITION : Hodgkin lymphomas (HLs) are lymphoid neoplasms usually affecting lymph nodes. They are composed of large dysplastic mononuclear and multinucleated cells surrounded by a variable mixture of mature non― neoplastic inflammatory cells. Abundant band―like and/or more diffuse collagen fibrosis may be present. On the basis of the immunophenotype and morphology of the neoplastic cells and the cellular background, two major types of HL are recognized: nodular lymphocyte predominant B –Cell Lymphoma ( NLPBL ) and classic HL (CHL).

26. EPIDEMIOLOGY: Approximately 90% of HLs are of classic type, and only 10% are nodular lymphocyte predominant B-cell Lymphoma ( NLPBL ). The age peak of NLPHL patients lies in the fourth and fifth decades of life, but NLPBL is also common in children. It is more common in males than in females. Mixed cellularity CHL (MCCHL) has a bimodal age distribution, with a peak in young patients and a second peak in older adults. Nodular sclerosis CHL (NSCHL) has a peak among individuals aged 15―35 years. There is a male predominance for most CHL subtypes, with the exception of NSCHL, in which the incidence is slightly higher in females

27. Individuals with a history of infectious mononucleosis have a higher incidence of CHL, in particular the mixed cellularity subtype. The wide geographic variation in the incidence of HL supports the belief that the disease is caused by an environmental, probably infectious, agent. In economically developed countries with a high standard of living, HL is rare in children and more frequent in young adults, displaying the histologic types that are associated with a favorable prognosis. In underdeveloped countries and populations with poor socio economic conditions, the incidence is highest in children, and the histologic types associated with a poor prognosis predominate.

28. ETIOPATHOGENESIS: The Hodgkin-Reed-Sternberg cells (HRSC) secrete cytokines and chemokines (reflected by yellow diamonds) that recruit an inflammatory cell infiltrate into the tumour and help sustain them. The microenvironment is richin lymphocytes (particularly Th2 and T- regs ). Granulocytes (GRAN), eosinophils (EO), mast cells (MC), tumour -associated macrophages (TAM)and fibroblasts are also recruited by the HRSC. Upregulation of NF- jB , JAK/STAT and PI3K pathways leads to altered signalling that is an essen-tial part of HRSC survival mechanisms. Common comparative copy number increases of a locus located at 9p24.1 can lead to overexpression ofPD-L1 and PD-L2 and increased JAK/STAT signalling . The presence of FOXP3+T-regs and CD20+B-cells incurs a favourable prognosis. Cyto -toxic lymphocytes (CTL) expressing granzyme -B or TIA1, and elevated TAM are associated with an inferior prognosis. S chematic representation of HRSC interaction with the microenvironment.

29. CLASSIFICATIONS OF HODGKIN LYMPHOMA

30. HODGKIN LYMPHOMA: WORLD HEALTH ORGANIZATION (WHO) CLASSIFICATION(2021) 1-Nodular Lymphocyte predominant B -cell Lymphoma . 2-Classical Hodgkin lymphoma Nodular sclerosis Lymphocyte-rich Mixed cellularity Lymphocyte depleted

31. ANN ARBOR STAGING CLASSIFICATION FOR HODGKIN DISEASE (AS MODIFIED AT COTSWOLDS):

32. Clinical features Patients with CHL usually present with peripheral lymphadenopathy , localized to one or two lymph node―bearing areas. Mediastinal involvement is most frequently seen in the nodular sclerosis subtype, whereas abdominal involvement and splenic involvement are more common in mixed cellularity cases. Most patients with NLPBL are asymptomatic and present with enlargement of peripheral lymph nodes; B symptoms are rare.

33. B SYMPTOMS About 33% present with B symptoms overall . Only 15-20% of stage I-III have B symptoms like 1 -Fever(>38^C) May first present as fever of unknown origin Fever persists for days to weeks followed by afebrile intervals and then recurrence. This pattern is called Pel Ebstein fever . 2-Drenching night sweats. 3- Weight loss (>10% in 6 months)

34. History. Complete physical examination. Confirmatory workup Excisional biopsy of the lymph node Staging workup Chest x ray(pa, lat) Usg neck, whole abdomen, CT scan thorax, abdomen and pelvis, FDG PET scan. Routine blood investigations. Complete blood count, Liver function, Renal function,Serum albumin,ESR,Lactate Dehydrogenase . OTHERS: Bone marrow biopsy. DIAGNOSTIC WORK-UP:

35. PET SCAN PET Scan has become an integral component of initial staging. Information provided by PET has been recently incorporated in the lymphoma guidelines for response evaluation after completion of treatment. Useful for follow up study to evaluate residual masses, dx of early recurrence and predicting outcome. It has a specificity of 90-95% Intense uptake of 18‐fluorodeoxyglucose of the bilateral cervical and mediastinal lymphadenopathy and spleen were observed at initial diagnosis (A) and disappeared after six cycles of ABVD therapy (B) in the maximum intensity projection image (MIP) of FDG‐PET study

36. Diagram showing overall approach in the diagnosis of fine needle aspiration cytology (FNAC) of lymph node

37. REACTIVE LYMPHOID HYPERPLASIA HODGKINS LYMPHOMA Polymorphic population. Lymphocytes,Centrocytes , Centroblasts,Immunoblasts,Plasma cells. Occasional neutrophils , eosinophils , mast cells , Tingible body macrophages. Polymorphic population. Mature lymphocytes, eosinophils , plasma cells and immunoblasts . Classical Reed-Sternberg cells: Large bi-nucleated cell with prominent nucleoli. Many mononuclear large atypical Hodgkin’s cells •Lymphocyte and histiocyte (L&H) cell/popcorn cell inlymphocyte predominance type. •Ill-formed epithelioid cell granulomas •Scattered elongated cells in nodular sclerosing variety

38. GROSS APPEARANCE: Here is a 5 cm lymph node (from a patient with lymphadenopathy ). The node should normally be soft and pink and less than 1 cm in size. This lymph node is involved with Hodgkin's disease. 

39. Diagnostic Approach to Hodgkins Lymphoma: NON-HODGKINS LYMPHOMA. HODGKINS LYMHPOMA

40. HODGKINS LYMHPOMA RS CELLS POPCORN CELLS VS FLOW CYTOMETRY RS Cells CD 15 + Cd30+ CLASSIC HODGKINS LYMPHOMA POPCORN CELLS CD 20 + CD45+ NON-CLASSIC HODGKINS LYMPHOMA

41. REED STERNBERG CELL Common feature of A Hodgkin Lymphomas. Large cells (>45um in diameter) with classically binucleate or bilobed central nucleus each with a large acidophilic central nucleoli surrounded by a clear halo. "owl's eye appearance“. Variants: mononuclear (Hodgkin's cell), mummified cell, lacunar cell, L/H cell. Classic Reed-Sternberg cell:CD15, CD30, CD20-, CD45-. Most current studies indicate the RS cells of HL are lymphocytic in nature and, in the great majority of cases, are of B-cell origin

42. NODULAR SCLEROSIS LYMPHOCYTIC RICH MIXED CELLULARITY LYMPHOCYTIC DEPLETED Nodular Lymphocyte predominant B -cell Lymphoma (NLPBL ) RS CELLS ; CD 15 +, CD30+ POPCORN CELLS: CD 20+, CD 45+ MOST COMMON 2 ND MOST COMMON BEST PROGNOSIS WORST PROGNOSIS

43. Classical Hodgkin lymphoma 1-NODULAR SCLEROSIS CHL (NSCHL): Most common type diagnosed. About 70%. Lacunar cells seen. CD15 , 30 positive. EBV negative. Only subtype without a male predominance. Seen in younger patients with stage I-II disease.

44. The classic histopathologic criteria for NSHL are: (a) Prominent nodularity . (b) Presence of lacunar RS cell variants , and Lymph node involved by nodular sclerosis CHL, with bands of dense fibrosis creating a nodular growth pattern (H&E, 20x)

45. Several lacunar cells (arrows) are present. LACUNAR VARIANT RS CELL : These variants possess large, multilobated , or irregular nuclei with finely dispersed chromatin; nucleoli are usually small. The cytoplasm of lacunar cells retracts when fixed in formalin, so the nuclei gives the appearance of cells that lie with empty spaces between them. (lacunae).

46. Nodular sclerosis classic Hodgkin lymphoma with aberrant CD3. The neoplastic cells are positive for CD3. Nodular sclerosis classic Hodgkin lymphoma. This case had aberrant expression of CD2, a finding usually seen in grade 2 disease. In this case, the CD2 antigen appears to be partially expressed on the outer surface of the cell membrane, suggestive of adsorption

47. Nodular sclerosis classic Hodgkin lymphoma, PAX5 is weakly positive in neoplastic cells.

48. 2. Mixed Cellularity Constitutes about 20%. More than 50% present as stage III or IV disease. Biphasic incidence, peaking in young adults and again in adults older than 55. CD15, 30, EBV positive. Presents in advanced stages. Tendency to involve spleen, bone marrow.

49. The mixed cellular infiltrate does not contain fibrotic bands. A typical binucleated Reed-Sternberg cell in a mixed cellular infiltrate with lymphocytes, macrophages, and eosinophils is visible.

50. Reed-Sternberg and Hodgkin cells selectively immunostained for CD15 marker. The staining is membranous and paranuclear . Anti-CD15 / peroxidase stain. Hodgkin lymphoma. Hodgkin and Reed-Sternberg cells with membrane and Golgi body staining by anti-CD30 antibody. Anti-CD30/ peroxidase stain.

51. DIFFERENTIAL DIAGNOSIS: 1-INFECTIOUS MONONUCLEOSIS LYMPHADENITIS: Infectious mononucleosis is induced byHHV-4, much better known as Epstein-Barr virus (EBV), a γ herpes virus . Viral spread is via direct contact with human secretions, most often by contact with human saliva. In conditions of inadequate standards of living, EBV infection occurs early in life, in infants and toddlers, and is almost universal. The peak incidence of EBV infection occurs between the ages of 10 and 19 years of age

52. Infectious mononucleosis involving lymph node.In this field, a sinus is distended by histiocytes , lymphocytes, and plasma cells. MICROSCOPY : Polymorphous inflammatory cells. Partial architectural effacement due to marked sinusoidal and capsular / extranodal infiltration by immunoblasts that often have Reed-Sternberg like features . Atypia Follicular hyperplasia with ragged or mottled edges, follicles have tingible body macrophages and marked mitotic activity. IHC: CD 20 positive.

53. 2 -EBV positive diffuse large B cell lymphoma : Mostly older patients (> 50 years), with peak in the eighth decade; second smaller peak in the third decade. M > F. Nodal and extranodal involvement is common.

54. MICROSCOPY: The lymph node architecture is completely effaced by a variably sized infiltrate, including very large cells with irregular nuclear membranes, vesicular nuclei, distinct nucleoli and moderate amount of cytoplasm. There is a mixed infiltrate composed of small lymphocytes and histiocytes . The background is hyalinized , with areas of necrosis and foreign body giant cells. There are areas with residual small lymphocytes. IHC: 1- Positive for CD20, PAX5, CD30,MUM1,BCL6 . 2- Negative for CD15 and CD10 . 3- BCL6 stains a subset of the large cells. 4-EBER and LMP1 are positive in the large cells. EBER is strongly positive

55. 3- Lymphomatoid granulomatosis Lymphomatoid granulomatosis is an angiocentric and angiodestructive lymphoproliferative disease involving extranodal sites composed of Epstein–Barr virus (EBV) positive B cell admixed with reactive T cells , which usually predominate. MICROSCOPY: Dense interstitial and diffuse infiltrates of small lymphoid cells with round nuclei, clumped chromatin and scattered large lymphoid cells with round nuclei, visible nucleoli and moderate amounts of cytoplasm. Admixed with these are a few aggregates of histiocytes and occasional eosinophils . IHC: Positive-   CD20,  EBV,  LMP1, EBER,  CD30 Negative- CD 15  lymphocytic vasculitis with infiltration of the vessel wall is seen

56. 3-Lymphocyte-depleted classic Hodgkin lymphoma Constitutes <5%. Worst prognosis of all subtypes. Older males, rare in children. Present as febrile illness with pancytopenia , hepatomegaly , and no peripheral lymphadenopathy . Advanced stage, Stage IV. The biologic hallmark of LDHL is a collapse of cell-mediated immunity, HIV infection. RS cells CD15+, CD30+; most EBV+. Differential Diagnosis: Large cell Non- Hodgkin's lymphoma. Nodular sclerosis HL

57. Lymphocyte-depleted classic Hodgkin lymphoma. Hodgkin/Reed-Sternberg cells are readily seen in a background rich in histiocytes and some small lymphocytes. Lymphocyte-depleted classic Hodgkin lymphoma. CD30 immunostaining .

58. Lymphocyte―rich classic Hodgkin lymphoma RS cells CD15+, CD30+; 40% EBV+ Uncommon. M>F. Tends to be seen in older adults. This is an uncommon form of classical HL. Reactive lymphocytes make up the vast majority of the cellular infiltrate: In most cases, involved lymph nodes are diffusely effaced, but vague nodularity due to the presence of residual B-cell follicles is sometimes seen. Differential Diagnosis: This entity is distinguished from the lymphocyte predominance type by the presence of frequent mononuclear variants and diagnostic Reed-Sternberg cells with a "classical" immunophenotypic profile. Very good to excellent prognosis.

59. Hodgkin lymphoma, lymphocyte-rich type. Few Reed-Sternberg cells or variants within a mass of lymphocytes. Hematoxylin,phloxine , and saffron stain. strong membrane variable paranuclear staining for CD30 in the neoplastic cells.

60. 1-Nodular Lymphocyte predominant B -cell Lymphoma ( NLPBL ) <5% of Hodgkin lymphoma. Mainly involves cervical, axillary or mediastinal . L&H cells or Popcorn ceils are seen. Positive for CD20, 45, CD79a, Bcl-6, J-chain, and PAX-5. EMA positive in 50% cases. Negative for CD15, 30 . Differential Diagnosis: Well differentiated lymphocytic lymphoma, mononucleosis, malignant melanoma,, progressive transformation of germinal centers

61. In, NLPBL L&H variants of RS cells are conspicuous with folded, multilobed nucleus and smaller nucleoli("popcorn nuclei"). In the nodular subtype of LPBL , there is almost total obliteration of the nodal architecture by a vaguely nodular process. NLPBL nodules are composed of small, round lymphocytes with varying numbers of epithelioid histiocytes which gives they a mottled appearance. L&H variants of RS cells may be numerous and are principally seen in the nodules. "Diagnostic" or "classic" RS cells are rare or nonexistent and are not required for the diagnosis of NLPBL.

62. H&E 40x; lymph node biopsy: vaguely nodular small lymphocytic proliferation. H&E 100x; lymph node biopsy: large neoplastic cells (arrows) with folded / irregularly contoured nuclei and small nucleoli in a background of small lymphocytes and histiocytes .

63. immunohistochemical stain: the small B cell nodules and LP cells express CD20.   immunohistochemical stain: T cells in the background express PD-1 and form rosettes around the neoplastic cells.

64. NLPBL

65. NLPBL In classic Hodgkin lymphoma, tumor cells are positive for CD30 and CD15. CD20 is negative and PAX5 is weak positive in large tumor cells. In nodular lymphocyte predominant B Cell lymphoma, tumor cells are positive for CD20 and Oct-2. CD30 is negative in tumor cells. CD3 stain shows peritumoral rosette-like T-cells.

66. REFERENCES 1 ) Kumar, V., Abbas , A. K., & Aster, J. C. (2017).  Robbins Basic Pathology  (10th ed.). Elsevier - Health Sciences Division.pg no.,611-616. 2 )WHO classification of Tumors of Hematopoietic and Lymphoid Tissues;5 TH Edition. pg no,423-442. 3 ) L. Jeffrey Medeiros,Ioachim's Lymph Node Pathology, 5th Ed. 2021.pg no.,306-333. 4) Dabbs DJ. Diagnostic Immunohistochemistry : Theranostic and Genomic Applications. 5th ed. Pg no,160-202. 5) Orell SR Sterrett GF.  Orell and Sterrett's Fine Needle Aspiration Cytology. 5th ed ; 2012.Pg no,77-117.

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