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Histopathological Pattern of Central Nervous System Infection: Experience of 61 cases at Referral Neuroscience Hospital in Bangladesh

 Huq N,1 Haque ME,2 Jahan N,3 Yusuf MA,4 Baqui MN,5 Rozhana S,6 Shirin S,7 Islam MN8

Abstract

Background: Central nervous system (CNS) infections presenting as space occupying lesions are not uncommon in a developing country like Bangladesh.
Objective: The purpose of the present study was to see the histomorphological pattern of CNS infections.
Methodology: This retrospective study was carried out in the Department of Neuropathology at National Institute of Neurosciences & Hospital (NINS&H), Dhaka, Bangladesh during the time period of June 2013 to March 2018 for a period of around five (05) years. All the patients biopsied in neurosurgical department and was reported as infectious lesions from the Department of Neuropathology of NINS&H were selected as study population.
Result: A total number of 2504 cases of surgical specimens were reported during the study period of which 61(2.4%) cases were infectious lesions. Among 61 infectious cases tubercular lesion, suppurative and fungal causes of infection were found in 33(54.1%) cases, 21(34.4%) cases and 7(11.5%) cases respectively. Majority of the study population was in less than 40 years of age group which was 48(80.0%) cases. Male was predominant than female which was 38(62.3%) cases and 23(37.7%) cases respectively.
Conclusion: In conclusion tubercular infection is the most common CNS infection followed by suppurative and fungal infection.

[Journal of Histopathology and Cytopathology, 2019 Jul; 3 (2):143-150]

Key words: CNS infection, Tuberculosis, Fungal infection

  1. *Dr. Naila Huq, Associate Professor, Department of Neuropathology, National Institute of Neurosciences & Hospital, Dhaka, Bangladesh. nailahuqpopy@gmail.com
  2. Mohammad Enamul Haque, Senior Consultant, Victoria Hospital, Narayanganj, Bangladesh. doctorenamk43@gmail.com
  3. Nasreen Jahan, Research assistant, National Institute of Neurosciences & Hospital, Dhaka, Bangladesh.dr.nasreen2014@gmail.com
  4. Md. Abdullah Yusuf, Assistant Professor, Department of Microbiology, National Institute of Neurosciences & Hospital, Dhaka, Bangladesh. ayusuf75@yahoo.com
  5. Nazmul Baqui, Senior Lecturer, Unit of Pathology, Faculty of Medicine, AIMST University, Kedah, Malaysia. doctornazmul@yahoo.com
  6. Sharmin Rozhana, Lecturer, Unit of Microbiology, Faculty of Medicine, AIMST University, Kedah, Malaysia. srozhana@gmail.com
  7. Sadia Shirin, Medical Officer, Department of Neuropathology, National Institute of Neurosciences & Hospital, Dhaka, Bangladesh. sadiashirin77@gmail.com
  8. Md. Nowfel Islam, Professor & Head, Department of Pathology, National Institute of Neurosciences & Hospital, Dhaka, Bangladesh. nowfel2008@gmail.com

 *For correspondence

 Introduction

Top of FormBottom of FormDiseases causing multiple ring enhancing lesions of the brain are infectious, neoplastic, inflammatory or vascular origin.1-3 Central nervous system (CNS) infections are important because of many emerging and reemerging pathogens. Now a day’s persons are at increased risk of CNS infection due to acquired immunodeficiency syndrome (AIDS). CNS becomes a watershed for Human Immunodeficiency Virus (HIV) infection.4 Infections are also common among the recipients of organ transplantation.2-5 Infections are caused by a wide variety of organisms including bacteria, fungi, parasite and virus. Environmental factors in tropical countries play a significant role in the pathogenesis of CNS fungal infections. Immunocompromised patients are commonly susceptible to fungal infection.5 But exposure to drug or contaminated devices can also cause CNS fungal infection in immunocompetent person. Even People of endemic region can get CNS infection following heavy exposure to fungi.4, 6

Radiology is not always helpful for the diagnosis of CNS infection. Some treatable infectious diseases can even mimic the MRI features of demyelinating disease like multiple sclerosis.3,7,5 Although neoplasms are the common considerations in the presence of enhancing lesions with perilesional edema and mass effect on neuroimaging; non-neoplastic conditions particularly, infectious lesions can have similar imaging characteristics.1

Histopathological diagnosis is essential in case of neoplasm for diagnosis, grading as well as treatment. But infectious lesions that could not be diagnosed by noninvasive procedure also need biopsy. Distinguishing non-neoplastic from neoplastic lesion is extremely important to relieve the patient from the potential side effect of chemotherapy and radiotherapy.3 On the other hand, though brain infections are rare but due to limited space and involvement of vital areas they are associated with high morbidity and mortality.2,8

As Bangladesh has conservative society; HIV infection is not so common. However, like other part of the world, Bangladesh deals with a considerable number of patients having organ transplantation. Moreover, it is one of the densely populated countries of the world. Communicable diseases impose heavy burden on health care system. Tuberculosis is an emerging global health problem. In a country with huge population burden, poor hygiene, malnutrition, less health awareness of people, tuberculosis as well as other infections with involvement of CNS is not rare. There are a very few study regarding the frequency or prevalence of CNS infection in Bangladesh. This study was focused on the frequency and distribution pattern of CNS infections in Bangladesh.

 Methods

The present retrospective study was based on the data collected from the Department of Neuropathology NINS&H, Dhaka, Bangladesh during the time period of June 2013 to March 2018 for a period of around five (05) years. In this study the data of the patients who were underwent CNS surgery and histopathologically diagnosed as infectious lesions were collected. Cases of meningitis were not included in this study as diagnosis of meningitis based only on CSF examination. All extra-cranial skin, soft tissue and bony infectious lesions were excluded from this study. Specimens were stained by routine Hematoxyline & Eosin (H&E) stain. Furthermore, Zeihl-Neelsen stain was performed in all cases of tuberculosis. Periodic Acid Schiff (PAS) stain was done in all cases of fungal infection. Statistical analyses were performed by Statistical Package for Social Science (SPSS) software, versions 22.0 (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp.). Continuous data that were normally distributed were summarized in terms of the mean, standard deviation and minimum, maximum. Categorical or discrete data were summarized in terms of frequency and percentages.

 Result

A total number of 2504 cases of surgical CNS specimens were reported during the study period of which 61(2.4%) cases were infectious lesions. Only cases of infectious lesions were further analyzed. The age range of patients was 9 months to 85 years. The patients were stratified into 4 groups which were less than 20 years, 20 to 40 years, 40 to 60 years and more than 60 years. Most of the patients belong to 20 to 40 years age group which was 26(42.6%) cases followed by less than 20 years which was 23(37.7%) cases. CNS infection was very rare in more than 60 years of age; only two cases were found in this age group. However, male predominance with a male to female ratio of 1.7:1 was seen. Male was 38(62.3%) cases and female 23(37.7%) cases respectively (Table I).

In 61.0% cases of histologically diagnosed CNS infections radiological diagnosis were tumors. However in 39.0% cases radiological diagnosis was infection which correlates with the histological diagnosis. In 19 cases radiological diagnosis correlates with the histopathological diagnosis and all of them were tubercular lesion. Radiological diagnosis did not correlate with histopathological diagnosis in 32 cases. And radiology was not available in 10 cases.

 

Of the total 61 cases 33(54.1%) cases were tubercular, 21(34.4%) cases were suppurative and 7(11.5%) cases were fungal infection. AFB stain done in 30 cases of tuberculosis and result was negative. All cases of fungal infection were further stained by PAS stain and found positive.

 Discussion

CNS includes a wide variety of diseases ranging from suppurative, tubercular, fungal, parasitic and viral infection. For the diagnosis of these cases combined approach of clinical, radiological, CSF and histopathological examination are necessary. There is a scarcity of research works related to the pattern of CNS infections in Bangladesh. As National Institute of Neurosciences & Hospital (NINS&H) is a tertiary level referral hospital in Bangladesh, it deals with different neurological diseases having a well-developed neurosurgery department. Therefore, a great number of patients attend in this hospital.

CNS infection producing space occupying lesions are not uncommon in Bangladesh. About 2.4% of surgical specimens of this study belong to infectious lesions. The age range of the study population was 9 months to 85 years with highest percentage 42.6% cases between 2nd to 4th decades, followed by 37.7% cases in less than 20 years. CNS infection was very rare in more than 60 years of age; only two cases found in this group. The age distribution is consistent with most of the previous studies conducted in different parts of India.9,10,11 In our study 62.3% cases were male and the male to female ratio was 1.7:1. A male predominance seen in most of the reported studies indicating that male are more prone to these infections.9,10,11 This may be due to more exposure of the male of this age group to the environmental agents, vectors due to their outward activities.9 Some zoonotic infections like Neurobrucellosis occur due to occupational exposure.12 Moreover, in the developing countries male got priority over the female in getting treatment facilities.9 This observation matches with Bangladesh having the similar socioeconomic pattern.

We analyzed the anatomical location of the total 61 cases. It has been observed that most of the cases 49(80.0%) were cranial and 12(20.0%) cases were in spinal location. The percentage could be different if lesion like Pott’s disease would include in the study. Rosenblum5 has reported that 20.0% of brain abscess are not associated with predisposing bacterial infection; however, remaining results are from established pyogenic infection in extraneural site. Contiguous spread of infection can result from sinusitis, otitis, dental sepsis and pyogenic infection of face and scalp which causes intracerebral abscess or subdural empyema.5 Contamination of the CNS can occur in three routes. Arterial route is the main route of contamination and the disposition of intracerebral arterial division causes development of diseases in this way at the junction of white matter and gray matter or in the territory of perforating artery. Therefore, frontal or temporal region lesion is most common. Spinal anomalies predispose to intramedullary abscess but such infections are extremely rare5. Herpes simplex, Rabies, Varicella Zoster or a few viruses disseminated through neural route. Direct contamination can result from cranial or vertebral wound or following intracranial surgery. All these explanations suggest that cranial location is more common site of CNS infection particularly in case of acute bacterial infection which is similar to our study.5,8 Another study has been mentioned that infectious lesions in the brain stem are very rare.13

Among 61 cases radiology is not available in 10 cases which are histologically diagnosed as CNS infection. In the remaining, radiological diagnosis correlates with the histopathological diagnosis in 39.0% cases. However, in 61.0% cases radiological diagnosis are tumors. From biostatistical point of view it is not the indicator of sensitivity of radiology. As cases of CNS tumors were not included in the study, it would be a biased statement.

In radiology, neoplasm is the common consideration in enhancing lesions with perilesional edema and mass effect. But infectious lesion, occasionally demyelinating disease can share the similar imaging characteristic1,5. There are some limitations of early imaging and overlapping imaging appearance of many of the infections. Pyogenic abscess often show amino acid at spectroscopy.  Neurotuberculosis, neurosyphilis shows nodular meningeal enhancement with or without vasculitis which can also be seen in fungal infection. Some diseases like, neuro-cysticercosis, echinococcosis may have highly characteristic imaging pattern. But many other infections share common imaging findings. So, a high degree of suspicion is required to assume the diagnosis of CNS infection in radiology.1,14

Of the total 61 cases 54.1% cases are tubercular; 34.4% cases are suppurative and 11.5% fungal. As primary tuberculosis has high prevalence in Bangladesh, CNS tuberculosis might be the commonest presentation in this study. Garg RK3 et al also mentioned intracranial tuberculoma as frequently encountered brain lesion in tropical country. Bacteria are responsible for majority of suppurative infections of CNS and its covering.5 According to working criteria, cases of meningitis are not included in this study. Pyogenic abscess is the second common presentation (34.4% ). Our study is limited by non-availability of relevant microbiological analysis. So type of organism producing pyogenic abscess could not be determined from this study.

We found 7 cases of fungal infection. One of them was suffering from rheumatoid arthritis and receiving treatment with methotrexate. And another was a baby of 9 monthsage. Rest of the patients has no known history of immunodeficiency. Incase of fungal infection we found highest incidence of aspergillosis(4) followed by zygomycosis(2). Only one case of phaeohyphomycosis was found. A hospital based study of India also shows increased incidence of hyphal form like aspergillosis and Zygomycosis in non immunocompromised host.15The cryptococcus usually causes meningitis and rarely presentas cryptococcoma.16,17 Therefore, the cryptococcus is not found in this study.

CNS parasitoses include a great variety of protozoa and helminth.5,17 We did not find any case of neurocysticercosis, toxoplasmosis, echinococcosis, malaria, schistosomiasis and also rare amoebic infection of CNS. A study in India by Bhalla et al9 showed that these infections produce intracranial mass lesion in HIV positive patients. An HIV infected patient having CD4 cell count less than 200 cells/µL is at high risk for opportunistic brain infection. HIV infection is not so common in Bangladesh in comparison with other South Asian country like India or Thailand. As patients of HIV get treatment only in Infectious Disease Hospital (IDH) of Bangladesh, the data of those patients could not be included in this study. These infections may be less frequent in our environment or poor access of our people to the tertiary level hospital like NINS&H may result such outcome. As most of the affected people belong to lower socioeconomic condition the people may die before reaching the health care system. Another study in India showed that some of these infections of nervous system diagnosed mostly in autopsy specimen.5,18 A study in Hill tracts of Bangladesh; endemic for malaria showed significant cases of cerebral malaria in autopsy specimens.19 As facilities of autopsy are not available in our institute the study might be missing these infections.


Conclusion

Tubercular infection is the most common CNS infection followed by suppurative and fungal infection in biopsied specimen. Less than 40 years age group is the most vulnerable age group. High clinical suspicion, awareness of imaging pattern and microbiological approach can minimize the need for brain biopsy. Further large scale multi-institutional study including autopsy should be carried out.

 References

  1. Santosh V, Mahadevan A, Chickabasaviah YT, Bharath RD, Krishna SS. Infectious lesions mimicking central nervous system neoplasm. Semin diagn pathol, 2010;27(2):122-35
  2. Sundaram C, Shankar SK, Thong WK, and Villamizar CAP. Pathology and diagnosis of Central nervous system infections. Pathology Research International, 2011, Article ID878263:1-4
  3. Garg R K, Singha M K, Multiple ring-enhancing lesion of the brain. Journal of Postgraduate Medicine 2010;56(4):307-316
  4. Wig N, Wali JP. Central nervous system and HIV/AIDS. Journal of Indian Academy of Clinical Medicine Vol. 2008;5(2):164-168
  5. Rosenblum MK. Central nervous system in Rosai and Ackermans Surgical Pathology.10th vol.2,2327-2338
  6. Rodriguez T. Fungal infection of the CNS: Diagnostic and Treatment Approaches. Neurology adviser, 2018 May, 1-8; Website: https://www.neurologyadvisor.com/topics/general-neurology/fungal-infections-of-the-cns-diagnostic-and-treatment-approaches/
  7. Rocha AJD, Littig IA, Nunes RH, Tilbery CP. Central nervous system infectious diseases mimicking multiple sclerosis: recognizing distinguishable features using MRI. Arq Neuropsiquiatr 2013;71(9-B):738-746
  8. Sarrazin J-L, Bonneville F, Blondel GM. Brain infections in Diagnostic and Interventional Imaging 2012;93:473-490
  9. Bhalla A, Kharbanda PS. Tropical CNS infections: one must not forget. Update on Tropical Fever 2011;75-86
  10. Joshi R, Clinical presentation, etiology and survival in adult acute encephalitis syndrome in rural Central India in Clin Neurol Neurosurg.2013;115(9):1753-1761
  11. Modi A, Atam V, Jain N, Guth M, Verma R. The etiological diagnosis and outcome in patients of acute febrile encephalopathy: a prospective observational study at tertiary care center. Neurol India 2012;60(2):168-173.
  12. Algahtani H, Shirah B, Abdulghani D, Farhan R, and Algahtani R,Occapational Neurobrucellosis Mimicking a Brain Tumor: A Case Report and Review of the Literature in Case Report in infectious Diseases ;Volume 2017, Article ID 1434051:1-5
  13. Hall WA, Infectious lesions of the brain stem in Neurosurg clin N Am. 1993;4(3): 543-51
  14. Robert Y. Shih LTC, Kelly K. Koellar, MD Bacterial, Fungal and Parasitic Infections of the Central Nervous System: Radiologic-Pathologic correlation and Historical perspectives. Radiographics 2015;35(4)4:1141-1169
  15. Shankar SK, Mahadevan A, Sundaram C, Sarkar C, Chako G, Lanjewar D N, Santosh V, Yasha TC, Radhakrishnan V. Pathobiology of fungal infections of the central nervous system with special reference to the Indian scenario. Neurology India 2007;55(3):198-215
  16. Gupta K, Radotra B. Non-neoplastic Lesions Mimicking CNS Tumors. Essentials of Diagnostic Surgical Neuropathology: 230-239
  17. Matthew P. Frosch, Douglas C. Anthony, Girolami UD, The central nervous system in Robbins and Cotran, Pathologic basis of disease, eighth ed. 1299-1309
  18. Shankar SK, Mahadevan A, Parmar S, Histological Atlas of Common Infections of the Nervous System With Teaching Slides:1-64
  19. Islam SMJ, Uddin MJ, Haque WS, autopsy findings in cerebral malaria, Journal of Armed Forces Medical College  Bangladesh, December 2006; vol 2(2).
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The Role of Special Stains in Trephine Biopsy of Lymphoma

 *Yesmin I,1 Begum F,2 Yunus A,3 Kabir AL,4 Baqui MN5

 *Dr. Israt Yesmin, Specialist Pathology, Pathology and Laboratory Medicine, Square Hospital, Dhaka. badhanydr29@gmail.com

  1. Ferdousy Begum, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka. ferdousy_begum2000@yahoo.com
  2. ABM Yunus, Professor & Chairman, Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Dhaka. abmyunus.bsmmu@gmail.com
  3. Amin Lutful Kabir, Associate Professor, DEPT of Haematology, BSMMU Email aminlutful@gmail.com
  4. Muhammad Nazmul Baqui, Senior Lecturer, Faculty of Medicine, AIMST University, Kedah, Malaysia. doctornazmul@yahoo.com

*For correspondence

 Abstract

Background: Accurate diagnosis of lymphoma is always challenging to histopathologists. Aside from routine H& E stain special stains play an important role inreaching the final diagnosing of  lymphomacases. Here we have studied the use of these special stains in trephine biopsy material of lymphoma cases.
Methods: This descriptive cross sectional study was carried out at the Department of Pathology Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, from July 2012 to June 2014. Total 17 cases, which underwent trephine biopsy were included in this study. Different special stains and immunostains were done to reach the final diagnosis. Statistical analyses were performed to find out the usefulness of these special stains.
Results: Fourteenpatients were male and rests were female. Mean age of the patients was 41.8±12.5 years. IHC of trephine biopsy material aids in diagnosis of lymphomatous infiltration with further sub classification. While conventional H&E and giemsa stain revealed only 6(35.3%) cases with lymphomatous infiltration of bone marrow. Among these 14 cases, 10 (58.8%) cases were B-NHL, 3 (17.6%) cases were T-NHL and rest 1(5.9%) was lymphocyte-rich classical Hodgkin lymphoma.
Conclusion: It was found that, use of special stains and IHC of trephine biopsy material of lymphoma cases improved the diagnostic accuracy of lymphoma.

[Journal of Histopathology and Cytopathology, 2019 Jul; 3 (2):134-142]

Key words: Trephine biopsy

Introduction

Trephine biopsy of the bone marrow has wide application in clinical medicine. Its important use is in the evaluation of patients with various haematological disorders like malignant lymphoma, acute leukaemias, myeloproliferative disorders, myelodysplastic syndromes, metastatic tumor, granulomatous disorder, myelofibrosis, aplastic anaemia and plasma cell dyscrasias. Examination of the bone marrow aspirate and trephine biopsy is essential for the diagnosis of bone marrow disorders. The aspirate and trephine biopsy provide complimentary and useful information. It is recommended that both bone marrow aspirate and biopsy be routinely performed for correlation of representative findings.

The bone marrow trephines are particularly useful in identifying focal lesion that may be less apparent on aspirate smear alone. The bone marrow biopsy is also a common and important staging procedure that is performed routinely on patients with hematologic and non-hematologic malignancy. Pathomorphological examination of trephine biopsies of the bone marrow represents a standard method for the diagnosis and staging of hematological neoplasms and other disorders involving the bone marrow.  The use of special stains in bone marrow pathology should be determined following examination of the routinely stained biopsy and the patient’s clinical history.1 Although conventional morphology remains the gold standard for paraffin embedded BM trephines, IHC staining has become an integral part of the diagnostic and prognostic workup.2 Distinction of the most common type of B-NHL in the trephine biopsy can be difficult on morphological ground alone due to overlapping cytological features and distribution pattern and therefore require IHC and occasionally molecular data for primary diagnosis.2

Non Hodgkin lymphoma (NHL) composed of small B lymphocytes comprises the majority of lymphoma infiltrates in the bone marrow. Bone marrow involvement is seen in virtually 100% of B cell chronic lymphocytic leukemia (B-CLL) / small lymphocytic lymphoma (SLL), in almost all cases of lymphoplasmacytic lymphoma (LPL) and hairy cell leukemia (HCL), in 55-93% of mantle cell lymphoma (MCL), in 50-60% of follicular lymphoma (FL) and invariably in splenic marginal zone B cell lymphoma (SMZL). In nodal and extra nodal marginal zone B cell lymphoma the marrow involvement is found in 10-30% cases.2

The pattern of bone marrow involvement is one of the most important hints for diagnosis. Different patterns of bone marrow infiltration by NHLs are recognized, namely paratrabecular, randomly focal / intertrabecular / non paratrabecular, interstitial, diffuse and sinusoidal / intravascular. In some cases, multiple or mixed pattern are present in the same specimen. Although most infiltrates are easily recognizable in standard stains, sparse interstitial and sinusoidal involvement can be practically undetectable by conventional morphology and requires IHC to highlight the neoplastic population.2

Lymphoma demonstrate significant increased incidence rates throughout the world and causes mortality.3 Though uses of special histochemical stain in trephine biopsy is an established practice in histopathology, the application of immunohistochemistry (IHC) to diagnostic bone marrow trephine biopsy specimen is a relatively new practice.4 IHC is a powerful auxiliary technique for the diagnosis of hematologic disorder in bone marrow trephine biopsies.2 IHC reveals clear architectural features and estimates tumor cellularity in paraffin sections. By using IHC an accurate characterization and primary classification of lymphoid neoplasm is possible in the majority of cases and expands the diagnostic scope of bone marrow histology.2 The ability to define such subgroups is increasingly important as treatment is tailored not only to specific types of lymphoma but also on the basis of their prognostic group.5 Therefore the aim of this study was undertaken to see the role of special stains in bone marrow trephine biopsy in the diagnosis of lymphoma and to detect lymphoma of bone marrow and their typing.

Methods

This descriptive cross sectional study was carried out at the Department of Pathology Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, from July 2012 to June 2014. A total of 17 patients of different age and sex were selected from the department of Hematology BSMMU, Dhaka and other different institutes of Dhaka by using non probability sampling method. Patients who underwent trephine biopsy and histologically and clinically suspected as cases of lymphoma and cases accompanied by clinical information were included in this study. Exclusion criteria include patients who were not suspected as cases of lymphoma in histological diagnosis, specimen having severe crush artifact and specimen less than 1 cm in length. According to standard protocol the biopsy samples were taken from the posterior superior iliac spines under local anesthesia. Formalin fixed trephine biopsy sample were allowed to fix over night at room temperature in 10% buffered formalin fixative. Trephine biopsy samples collected from haematology department of BSMMU and other different institutes of Dhaka were decalcified in 10% formic acid for four hours followed by wash in running tap water for an hour.  Routine processing and paraffin embedding were done according to BSMMU standard tissue processing protocol in automated machines.  Paraffin blocks were made.  Subsequent tissue sections were analyzed by using routine H&E stain, periodic acid Schiff (PAS) stain, Giemsa stain and a primary histological diagnosis were made.

Sections with at least five well preserved marrow spaces were studied for cellularity, normal hematological elements, presence of infiltration if any, the extent, histologic pattern and morphology of infiltration and other secondary changes. The morphology and histologic pattern of infiltration were categorized as diffuse, interstitial, focal (non-paratrabecular), paratrabecular and mixed patterns. Diffuse pattern was defined when there was extensive replacement of the marrow elements, both hematopoietic tissue and fat, obscuring the marrow architecture.  Interstitial pattern was indicated when individual neoplastic cells were interspersed between hematopoietic cells and fat, focal, where nodular aggregates were seen separated by normal hematopoietic marrow, paratrabecular, when infiltrate aggregates were seen immediately adjacent to bony trabeculae.

2–3 μm thick sections were cut from the paraffin blocks for IHC. EnVision method was followed. The immunohistochemical staining procedure of all antibodies except TdT was performed in the IHC lab of BSMMU by following its staining protocol. Table I shows the IHC panel done for diagnosis.

Results of immunostains were assessed by light microscopy using a 10× objective lens with subsequent use of 20 and 40 lenses for confirmation. Immunostains results were expressed as positive, weakly positive or negative. Cases where quantification of specific type of immunohistochemical markers positive cells are needed counted at x40 magnification, excluding cortical and trabecular bone, periosteal connective tissue, adipose tissue or areas of haemorrhage. The specific immunohistochemical marker positive cells were counted in five or more randomly selected fields and the number of positive cells was expressed as a percentage of total number of bone marrow nucleated cells.  Slides were reviewed by two senior histopathologists. Subsequently special histochemical stains and immunohistochemical finding was analyzed and final reports were issued. This was performed in the department of pathology of BSMMU, Dhaka.

 Results

Age, sex and clinical findings

It was observed that maximum case (35.3%) patients belonged to age grouped 31-40 years. The mean age was found 41.8±12.5 years with range from 20 to 70 years. Majority14 (82.4%) patients were male and 3(17.6%) were female indicating male predominance. Male and female ratio was 4.7:1. Out of 17 patients of the present study 13 patients had fever, 6 complained of weakness and 6 presented with hemorrhagic manifestation e.g. gum bleeding, cyanosis, bleeding spot etc. The other complains included weight loss, bone and joint pain, swelling of whole body, night sweat, pruritus, yellow discoloration of skin and sclera, neck swelling, oral ulcer etc. Anaemia was the most prevalent (88.2%) feature. Other signs included jaundice, oedema, cyanosis etc. Five (29.4%) patients had generalized lymphadenopathy, two (11.8%) had grouped palpable lymph node, one (5.9%) had bony tenderness and all had normal tonsil.

 Ultrasonographic findings

Among 17 cases USG reposts were not available in 5 cases. Two (11.8%) patients had splenomegaly, 2(11.8%) had hepatomegaly, 6(35.3%) had hepatosplenomegaly, 1(5.9%) had abdominal lymphadenopathy and USG findings were normal in 1(5.9%) cases.

Bone marrow examination

Comparison of Primary histological diagnosis with final diagnosis after IHC

Out of these 17 cases 14 cases as various types of lymphoma. In the present study, primary histological diagnosis on trephine biopsy was made as haematolymphoid malignancy unclassified in 11(57.1%) cases and NHL in 6(42.9%) cases. Fibrosis was not seen in any of the 17 cases.


Trephine biopsy samples of 17 cases were histologically analyzed with the help of H&E stain along with PAS and Giemsa stain. Five patients were finally diagnosed as NHL by primary histological diagnosis was made on the basis of different pattern of abnormal cellular infiltration in the bone marrow trephine biopsy.

Discussion

In this series it was observed that most of the patients with haematological disorder were in 4th decade and their mean age was 36.1±15.01 years. Male to female ratio was almost 3:1. Similarly study conducted by Chen et al. (2000) observed male to female ratio was 2.9:1.6 Similar observations regarding the male predominant were also made by Matsuo et al. (2003) and Lone & Naeem (2011).7 On the other hand Kumar et al. (2009) observed female predominant, where they found female to male ratio was almost 5:1.8

 The pattern of marrow involvement also differs to some extent in various NHL subtypes, for example paratrabecular infiltration is strongly associated with follicular lymphoma. A bone marrow biopsy performed in patients with low grade lymphoma sometime shows unexpected high grade transformation, which necessitates a different therapeutic approach.9

 Among initial 11 cases of haematolymphoid malignancy unclassified in trephine biopsy were finally seven of them were diagnosed as B-NHL and one case as Hodgkin lymphoma. The remaining three cases were finally excluded from haematolymphoid malignancy. This result indicates the importance of IHC in the diagnosis of haematolymphoid malignancy. The remaining six cases of NHL were further sub classified as B-NHL and T-NHL (Table IV).

It is very much clear that trephine biopsy reports are superior as it could specify the diagnosis in most of the cases with the exception of cases of B-NHL and T-NHL. The latter cases were diagnosed only on the basis of CD3, CD20 and CD79a immunomarker. The incidence of bone marrow involvement in extra nodal MZL is lower (~20%) than in other low grade B-NHL, albeit recent studies described BM infiltration in up to 44% of cases.10

West et al. (2002) studied 61 cases (26 follicular lymphoma and 35 benign or atypical aggregates). They found that no single stain is sufficient for identification of neoplastic lymphoid aggregates. However, this distinction was made possible by using a panel of antibodies. The most useful antibodies were CD10, bcl-2, CD5, and CD20. Most benign or atypical aggregates do not express CD10 and CD23.11

Bone marrow specimens from 317 patients with non-Hodgkin’s lymphoma (NHL) obtained at initial staging was evaluated for the presence of lymphoma or benign lymphoid aggregates were studied by Conlan et al. (1990). They have found thirty-two percent (102 patients) had lymphoma in their bone marrow, and 9% had benign lymphoid aggregates. Bone marrow lymphoma was present in 39% of low-grade, 36% of intermediate-grade, and 18% of high-grade lymphomas. The bone marrow was involved in 25% of patients with diffuse large-cell or immunoblastic NHL. Bone marrow involvement did not affect survival of patients with low-grade NHL, but survival was significantly shorter (P<0.05) for patients with intermediate- and high-grade NHL with bone marrow involvement. Bone marrow involvement was equally common in B-cell and T-cell NHL (31% v 32%).12

Bone marrow involvement by lymphoma is considered a systemic dissemination of the disease arising elsewhere, although some tumors may arise primarily in the bone marrow microenvironment 13. IHC is important for lymphoma in particular those with heterogeneous histology to establish accurate diagnosis and adequate therapy14.

Bone marrow aspiration and bone marrow biopsy are important diagnostic procedures for diagnosis of hematological, non-hematological malignancies and other diseases. These procedures are also valuable for follow up of patients undergoing chemotherapy, bone marrow transplantation and other forms of medical treatment. In the study conducted by Toi et al. (2010),  comparative evaluation of all such bone marrow aspiration (BMA) and bone marrow biopst (BMB), to see the complementary role of both the procedures, to study the advantages and disadvantages of both the procedures done simultaneously. There was 61.25% positive correlation between BMA and BMB in the study15.

Conclusion

It can be concluded that correct diagnosis can be made in majority of cases with the application of PAS, Giemsa and limited number of immunomarker. Correct and relevant clinical information along with appropriate special stain of bone marrow trephine biopsy improves the diagnostic accuracy of lymphoma cases.

 References

  1. Brunning RD, Arber DA, 2011. Bone marrow. In J, Rosai Rosai and Ackerman’s Surgical Pathology, Tenth edition, Mosby Elsevier Inc, pp. 1927-2012.
  2. Kremer M, Quintanilla-Martínez L, Nährig J, von Schilling C, Fend F. Immunohistochemistry in bone marrow pathology: a useful adjunct for morphologic diagnosis. Virchows Arch Int J Pathol. 2005 Dec;447(6):920–37.
  3. Tahmasby B, Marnani AB, Maleki M, Barouni M, Mousavi SH, Naseriyan B, et al. Blood malignancies in Mazandaran province of Iran. Asian Pac J Cancer Prev APJCP. 2013;14(2):1053–6.
  4. Olsen RJ, Chang C-C, Herrick JL, Zu Y, Ehsan A. Acute leukemia immunohistochemistry: a systematic diagnostic approach. Arch Pathol Lab Med. 2008 Mar;132(3):462–75.
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Study of Ovarian Tumours: Histomorphological Types

 *Ansari M,1  Khan AH,2 Hossain S,3 Khanom F4

  1. *Dr. MAS Ansari, Associate Professor of Pathology, Sylhet Women’s Medical College, Sylhet. drmasansari@gmail.com
  2. Professor Dr. Amjad Hossain Khan, Professor of Pathology, Sylhet Women’s Medical College, Sylhet.
  3. Sabbir Hossain, Associate Professor of Pathology, North East Medical College, North East Medical College, Sylhet.
  4. Farida Khanom, Medical Officer of Gynae & Obs, Jalalabad Ragib-Rabeya Medical Colleg, Sylhet.

 *For correspondence

 Abstract

Background: Ovarian tumour accounts for 3% of total cancer in females and is the 5th most common form of cancer related death in females with a varied clinical, morphological and histological features.

Objectives: The present  study was carreid out with an aim to find out  the frequency, age distribution and the histopathological  patterns of ovarian tumours.
Methods: Retrospective  study was done during the period  of 3 years comprised of 125 ovarian tumours diagnosed in the Department of Pathology, Sylhet Women’s Medical College, Sylhet. After thorough gross examination, representative bits were routinely processed and stained with H & E. Tumours were classified as per WHO classification.
Results: Out of 125 cases studied, majority were benign tumours (88%), followed by malignant (10.4%) and borderline tumour (1.6%). Age ranged from 11-70 years. Epithelial tumours were most common (76%), followed by germ cell tumours (16%), sex cord stromal tumours (7.2%) and metastatic ovarian tumours (0.8%). Serous cystadenoma was the commonest benign tumour and serous cyst adenocarcinoma was the commonest malignant ovarian tumour.
Conclusion: The prognosis and varying therapeutic strategies of ovarian tumours necessitate an accurate pathological evaluation. Although newer techniques like IHC and molecular analysis have made the diagnosis easier and more precise, in the institutes with provision of limited resources, histopathological study is still the gold standard in diagnosing most of these tumours.

 [Journal of Histopathology and Cytopathology, 2019 Jul; 3 (2):125-133]

Key words: Ovarian tumour, Histopathology

 Introduction

Ovarian tumours include complex, wide spectrum of neoplasms involving a variety of histological patterns ranging from epithelial tissues, connective tissues, specialized hormone secreting germinal and embryonal cells.1

The poor survival is due to the fact that they do not clinically manifest early and approximately 60-70% of the neoplasms present as either stage III or stage IV.1,3,4 Benign ovarian cysts are the commonest constituting about 80% of ovarian tumours and mostly occur in young women between the ages of 20-40 years. Borderline tumours occur at slightly older ages whereas the malignant tumours are common in older women between the ages of 40-65 years.1,4 Metastatic tumours subsequently involve the ovaries and mimic primary ovarian neoplasia. Approximately, 7% of lesions presenting clinically as primary ovarian tumours are of metastatic origin.5, 6 It is important to determine the histological pattern of ovarian tumour to achieve the optimum treatment response as prognosis depends on the degree of differentiation.2,3,7

Thus present study was undertaken to analyse the frequency of various histological subtypes, age distribution pattern and the  histopathological pattern of ovarian tumours.

 Methods

This retrospective  study was conducted for a period of 3 years (January2015 – December 2018) at Sylhet women’s Medical College, Sylhet.

Specimens sent in 10% formalin were routinely processed with paraffin embedding after adequate fixation. Paraffin sections and slides from fresh blocks and the retrieved blocks were stained with H & E. The slides were then reviewed microscopically in detail and tumours were classified according to the WHO classification of ovarian tumours.

Inclusion criteria

All histologically proven both primary and secondary ovarian tumours.

Exclusion criteria

Non- neoplastic ovarian lesions of study period.

Results

A total number of 125 cases were studied. Among these, majority were primary ovarian tumours (124; 99.2%), while one was metastatic ovarian tumours (1; 0.8 %). Of them 69 cases were benign (88%), 15 cases were malignant (10.4%) and rest 2 cases (1.6%) were borderline. Epithelial tumours (ET) were the most common histological type (95;76%), followed by Germ cell tumours (GCT) (20;16%) and sex cord stromal tumour (SST 9;7.2%; Table I).

Age Distribution

Majority of the tumours occurred in the reproductive age group (57; 45.6%) followed by 41-50 years of age group (26; 20.8%).Youngest patient was 13 years of age and older patient was about 68 years. Epithelial tumours and Sex cord stromal tumours had its peak between 31 to 40 years, whereas Germ cell tumours showed a peak in 21 to 30 years. Metastatic tumoursshowed  distribution between 61-70 years of age group.

 Gross Features

Most of the cases in this study were unilateral (117; 93.6%) and few were bilateral (8; 6.4%). Out of the total 95 Epithelial tumours, 76 were cystic in nature (80%), followed by those with cystic to solid in consistency (18; 18.9%) and solid (1; 1.05%), whereas most of Germ cell tumours were cystic in nature (16; 72.7%), followed by solid in consistency (5; 22.7%). Majority of sex cord stromal tumours (5; 71.4%) and all of the metastatic tumours were partly solid to cystic in consistency.

 Microscopy

The most common benign tumour was Serous cystadenoma (54; 43.2%), followed by Mucinous cystadenoma (14; 11.2%) and Mature cystic teratoma (16; 12.8%). Serous cyst adenocarcinoma was the most common malignant tumour (6; 4.8%), followed by Adult granulosa cell tumour (3; 2.4%). Borderline serous tumour were two in numbers. There was one case of metastatic ovarian tumours, was Krukenbergtumour. (Table II).

Discussion

Ovarian tumours are one of the major health problems and their diagnosis can be difficult due to variety of pathologic conditions affecting the ovaries. Thus knowledge of morphology and age-specific characteristics can help refine the diagnosis.8, 9

In the present study of 125 ovarian tumours, Primary ovarian neoplasms (99.2%) were the most common tumours of all ovarian tumours. Similar observations were made by Bhagyalakshmi A et al.10 (98.5%) and Vaddatti T et al.3 (98.9%). Most of the tumours belonged to Epithelial tumour category (76%) which was comparable to the results seen by Singh S et al.11 (69.17%), Krishna M & Maurya G12 (77.7%) and Badge S et al.13 (77%). GCT and SST accounted for 16% and 7.2%, respectively in our study compared to 42.2% and 3.1% reported in the others.14

In the present study, majority of the tumours were benign (88%) followed by malignant tumours (10.4%) and rest was borderline (1.6%). Findings of the present study correlated well with the studies of various authors.However, in this study the frequency of malignant tumours (10.4%) was little less than the study of Couto F et al.15 (40%).

In the present study, the patient’s age ranged from 11 years to 70 years and this was supported by the study done by Swamy GGEt al.16 where the youngest patient was 13 years old and the oldest was 68 years old. The majority of ovarian tumours (60.7%) were seen in the age group of 21-40 years, which was consistent with the study done by Kuladeepa A VK et al.17 (58.9%) and Pilli GS et al.18 (58.3%) .

In our study, majority of the tumours were unilateral (97.1%) and least were bilateral (2.9%) which were consistent with the findings of  VaddattiT et al.3, Jindal U8 and Prabhakar BR and Maingi K.19

In the present study, majority of the tumours were cystic (72%), followed by partly cystic & solid (18.4%) and few were solid (9.6%). These findings were comparable with the findings of Misra RK et al.20 where most of the tumours were cystic (78.2%) and few were solid (4.1%), whereas Couto F et al.15 have found 10.2% solid tumours which was close to our findings. Benign epithelial tumors were the commonest type (88.4%), followed by malignant epithelial tumors (9.47%) and the rest was borderline (2.10%). These findings were almost similar as observed in the study conducted by Kuladeepa A VK et al.17) Sharma I et al.21

In this study, maximum number of epithelial tumors (78%) were noted in 31-50 years age group. All malignant tumours were seen in the cases above 50 years of age. The results corroborated with the various studies done by Tushar K et al. (2005),22 Jha R & Karki S14 and Jindal U.8 Among the histomorphological types of epithelial tumors, Seroustumours (73.6%) were the most common, followed by Mucinous tumours (16.8%), Seromucinoustumours (8.42%) and least common were endometrioid tumours (1.05%). Similar results were seen by Krishna M & Maurya G12 andSarkar R.23 Serous cystadenoma was the commonest ET (53.3%) followed by Mucinous cystadenoma (21.7%) and serous cyst adenocarcinoma (8.4%). Similar results were reported by various authors in their studies.

In the present study, majority of the  germ cell tumours were benign (81.8%) and include mature cystic teratoma and Struma ovarii. These results were closer to the findings of Agrawal P et al.24 and Verma K & Bhatia A25 with 77.7% and 83.5% respectively. Malignant tumours include Dysgerminoma and Embryonal carcinoma. Germ cell tumours showed maximum cases below 35 years of age, with a gradual decline in 31-40 years age group and they were found to be uncommon after the age of 60 years. These findings were consistent with the studies of Jha R & Karki S14 and Agrawal P et al.24

In this study, majority of the sex cord stromal tumours were benign (57.1%) and the results were not similar to the findings of Rao KN et al.26 (55.6%) and were lower when compared to the findings of Jindal U8 (75%). On the contrary study of Badge SAEt al.13 (66.7%) showed mostly benign sex cord stromal tumours against malignant. The age range of sex cord stromal  tumours in our study was 41-70 years whereas Bhagyalakshmi A Et al.10 recorded age range of 21-70 years in their study. Adult granulosa cell tumour which is potentially malignant tumour occurred in 2.4% of all ovarian tumours. The frequency was consistent with the findings of Gupta SC et al.27 and Pilli GS et al.18 who recorded it as 4.4% and 3.54% respectively.

Our study showed one case of secondary tumours and constituted 0.8% of all ovarian tumours. This was lower when compared to the findings of Badge SA Et al.13  (10.9%) but was almost similar to the studies of Gupta N et al.28 (2%), Jha R & Karki S14 (2.4%) and Bhagyalakshmi A Et al.10 (1.5%). The former when compared with the studies of Misra RK et al.20 (1.07%), Prabhakar BR and Maingi K19 (1.57%) and Couto F et al.15 (1.46%) showed a good correlation.

 Conclusion

Ovarian tumour is a silent menace that presents as a tremendous clinical challenge to gynaecologist, medical oncologist and radiotherapists. Emergence of borderline tumours with prognostic difference from the benign and malignant counterparts, has added a wing to research in the field of ovarian tumours. Accurate diagnosis of ovarian tumours can be rendered in almost all of cases by correlating the clinical presentation, radiographic appearance and histomorphological features, which remains the gold standard. Even then, in the modern era by the application of specialised methods like special stains, IHC markers, ultrastructural studies and cytogenetics, there is a vast scope for reaching specific & reliable diagnosis of difficult dilemmatic cases of ovarian tumours, by which the therapeutic and prognostic implications could be modified.

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