AN EPIDEMIOLOGICAL , CLINICAL AND PATHOLOGICAL STUDY OF LYMPHOMAS OF THE BONE AND MUSCULOSKELETAL SYSTEM AMONG POPULATION FROM THE SOUTH OF IRAQ

Lymphomas of the bone and musculoskeletal tissue are uncommon. Yet, they constitute a certain frequency that they merit certain consideration. A study was conducted to clarify the behavior of such type of lymphomas in the South of Iraq, epidemiologically, clinically, pathologically, biochemically and immunologically. Results show that they form minority among total lymphoma cases, but they were the third common type among extranodal lymphomas with male gender, adult age and urban residency predominance. There was a predominance of low performance status, absence of B symptoms, neurological and musculo-skeletal manifestations, secondary bone, especially the spine, involvement. NonHodgkin lymphoma (NHL) was the most predominant, mainly the intermediate grade, with diffuse large cell lymphoma (DLCL) and Burkitt lymphoma (BL) as the main histological types encountered. Hematologically, there was mild anemia, rapid ESR (> 50 mm), normal platelets count in the majority, leucocytosis in < half and monocytopenia predominance. Biochemically they were characterized by normal liver function tests with the exception of high serum alkaline phosphatase, normal total serum proteins, hypoalbuminemia, normal serum urea, creatinine & uric acid, hypercalcemia, with serum LDH and B2MG elevation in the majority. Serum anti EB virus antibodies were seen in 1/4th of cases and the majority were of the higher IPI scores (both high intermediate & high). Those results were comparable to some and contradicting to other studies elsewhere. Introduction ymphomas constitute a heterogeneous group of clonal lymphoid system neoplasms arising as a result of a one or more of somatic mutations in a lymphocyte progenitor 1 ). They include distinct disorders defined by their clinical, histological, immunological, molecular, and genetic characteristics. Each one of these represents a clonal expansion of a normal precursor cell 2 . They rank as the 6th in incidence and mortality among malignancies at global level 3 . However, in Basra City, they occupied the 3rd rank among malignancies in males and the 2nd in females and represent 8.2% of all new malignant cases registered over the years 2005-2008 4 . Primary bone lymphomas (PBL), (defined as lymphoma localized to the bone without evidence of lymph nodes or other tissues at presentation: a modification of the initial Coley criteria in 1950 of ‘Lymphoma presenting in an osseous site with no evidence of disease elsewhere for at least six months after diagnosis’) 5,6 are uncommon among extranodal lymphomas. They accounts for <5% of all bone malignancies and are mostly are NHL. Secondary bone involvement in nodal NHL is uncommon, too, and typically is L Study of lymphomas of the bone and musculoskeletal system Z Al-Barazanchi, J Al-Ali & A Abbadi Bas J Surg, June, 20, 2014 27 restricted to aggressive types where the lesions are usually lytic 7 . For staging purposes, involvement of bone only is regarded as stage I, while involvement of bone along with other sites, or secondary to nodal disease is considered as stage IV, which differs entirely in prognosis. Diffuse large B-cell lymphoma is the most common type in both primary and secondary lymphomas. Primary bone NHL lymphomas usually arise from the appendicular skeleton or from flat bones of the axial skeleton, while secondary bone lymphomas usually involve the axial skeleton (skull, spine, ribs, and pelvis). Primary HL of the bone is very rare, whereas secondary involvement is found in 10%–25% of patients 8 . The imaging findings of bone lymphoma at conventional radiography, CT, and MR imaging are nonspecific, and usually reflect an aggressive pattern of bone destruction. It had been proved that FDG PET is more specific and sensitive than conventional bone radiology in identifying bone involvement by malignant lymphoma 8,9 . Aims of the study The study aims to clarify the incidence, epidemiology, clinical presentations, staging, classification, pathological types and hematological, biochemical, immunological alterations of lymphomas of the bone and musculoskeletal tissue (both primary and secondary) among patients from the south of Iraq. Materials and methods During the period of June 2008-February 2012, 669 newly diagnosed lymphoma patients, from Basra, Missan, Thi Qar, Muthanna, Babylon and Wasit Governorates, were studied. Of them, 347 cases had extranodal lymphomas, out of which, bone and musculoskeletal lymphomas (both primary and secondary) were 43 cases. Most of the workup had been done in the Laboratory Departments of both Basra General Hospital and Basra Children Specialty Hospital. Some of the work had been accomplished in the researcher’s Private Laboratory. All cases were subjected to a thorough clinical study including: age, gender, residency of origin (the site they were living for the last 20 years). Those from Basra, were categorized into 5 regions: City Center, North, South, and East and West (Table I). Table I: Geographical divisions of Basra Governorate adopted Territory Included Areas Center All districts and avenues within the official boundaries of City Centre. North Garma, Hartha, Daer, Qurna, Midaena, Talha. East Shatt-Al-Arab, Nashwa. west Shiaeba, Safwan. South Zubair, Um Qasir, Fao, Abul-Khaseeb. Patients were segregated into rural or urban in origin depending on whether they at live, for the last 5 years, in rural or urban areas. The smoking habit, the duration of smoking and the number of cigarettes consumed per day were asked for. The drinking habit was asked for, too. A history of a possible exposure to radiation (a direct exposure to a blast or war explosion, the presence of suspected war remnants in the vicinity of residence for > 5 years, or, the direct exposure to radiation in the form of therapy for another disease) was asked for. A positive family history of any malignant disease (affecting the 1stdegree relatives) and the type of the tumor was ascertained too. The chief complaint and duration of illness (in months) were inquired for. The presence of "B" symptoms: an increase in body temperature >37.3 °C for >10 days Study of lymphomas of the bone and musculoskeletal system Z Al-Barazanchi, J Al-Ali & A Abbadi Bas J Surg, June, 20, 2014 28 (matched with measuring body temperature by examination), any drenching sweating especially at night, and loss of >10 % of body weight over a period of 3 months, were asked for 10 . The same was done for the presence of intense itching, any change in bowel habit and any history of a bleeding tendency. The general status of the patient was assessed, according to the Eastern Cooperative Oncology Group Performance Status (Table II) 11 . Table II: The Eastern Cooperative Oncology Group Performance Status (11). Grade Description 0 Fully active, able to carry on all pre-disease performance without restriction. 1 Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature (eg, light housework, office work). 2 Ambulatory and capable of all self-care but unable to carry out any work activities, up and about more than 50% of waking hours. 3 Capable of only limited self-care, confined to bed or chair more than 50% of waking hours. 4 Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair. 5 Dead. Patients were examined thoroughly for the presence of fever (by measuring the core temperature of the patient at the time of initial presentation), pallor, jaundice, hepatomegaly, splenomegaly (the last two were matched with the findings on abdominal ultrasound examination), neurological and cutaneous manifestations, ecchymotic patches anywhere or any evidence of a bleeding tendency, the presence of palpable lymphadenopathy and their sites. An enlargement of any two or more groups of the non-contiguous groups above was considered to be "generalized" 12 . All the patients were subjected to chest radiographs and abdominal ultrasound examinations and then were staged using the Ann Arbor Classification System 13 into 4 stages: I, II, III and IV. All patients were subjected to hematological investigations, including full blood count, using the BeckmanCoulter 5 differential hematology analyzer which was properly calibrated according to the manufacturer's instructions with quality controls included between batches 14 , erythrocyte sedimentation rate (ESR) (Westergren method) 15 , peripheral blood smear examination to find the presence of abnormal lymphoid/leukemic peripheral blood cells, bone marrow aspiration, touch imprint and trephine biopsy (Figure I). Both direct and indirect antigolobulin tests (DAT & IAT) were done, too 16 . Normal results used were those adopted by the International Council of the Standardization in Hematology (ICSH) and the British Committee for Standardization in Haematology (BCSH) 17,18 . For those with palpable, accessible lymph nodes, fine needle aspiration was performed, stained with haematoxylin stain 19 and examined. Hodgkin lymphomas were classified according to the original Rye classification since it is popular among both pathologists and clinicians for its simplicity, ease of use, the association of individual types with prognostic differences when treated with current therapy and because of the lack of immunophenotypic studies 20 , while the non-Hodgkin's lymphomas were classified using the International Working Formulation because of the lack of cytogenetic, immunological and molecular studies 21,22 . Patients were subjected to a battery of biochemical tests including: liver function Study of lymphomas of the bone and musculoskeletal system Z Al-Barazanchi, J Al-Ali & A Abbadi Bas J Surg, June, 20, 2014 29 and renal function tests, serum uric acid, calcium, lactate dehydrogenase (LDH), beta-2-microglobulin 23-33 and serological tests including the anti gliadin antibodies (AGA), anti tissue transglutaminase (ATTA), anti Epstien-Barr virus (EBV) antibodies and anti Helicobacter pylori a


Introduction
ymphomas constitute a heterogeneous group of clonal lymphoid system neoplasms arising as a result of a one or more of somatic mutations in a lymphocyte progenitor 1 ).They include distinct disorders defined by their clinical, histological, immunological, molecular, and genetic characteristics.Each one of these represents a clonal expansion of a normal precursor cell 2 .They rank as the 6th in incidence and mortality among malignancies at global level 3 .However, in Basra City, they occupied the 3rd rank among malignancies in males and the 2nd in females and represent 8.2% of all new malignant cases registered over the years 2005-2008 4 .Primary bone lymphomas (PBL), (defined as lymphoma localized to the bone without evidence of lymph nodes or other tissues at presentation: a modification of the initial Coley criteria in 1950 of 'Lymphoma presenting in an osseous site with no evidence of disease elsewhere for at least six months after diagnosis') 5,6 are uncommon among extranodal lymphomas.They accounts for <5% of all bone malignancies and are mostly are NHL.Secondary bone involvement in nodal NHL is uncommon, too, and typically is L restricted to aggressive types where the lesions are usually lytic 7 .For staging purposes, involvement of bone only is regarded as stage I, while involvement of bone along with other sites, or secondary to nodal disease is considered as stage IV, which differs entirely in prognosis.Diffuse large B-cell lymphoma is the most common type in both primary and secondary lymphomas.Primary bone NHL lymphomas usually arise from the appendicular skeleton or from flat bones of the axial skeleton, while secondary bone lymphomas usually involve the axial skeleton (skull, spine, ribs, and pelvis).Primary HL of the bone is very rare, whereas secondary involvement is found in 10%-25% of patients 8 .The imaging findings of bone lymphoma at conventional radiography, CT, and MR imaging are nonspecific, and usually reflect an aggressive pattern of bone destruction.It had been proved that FDG PET is more specific and sensitive than conventional bone radiology in identifying bone involvement by malignant lymphoma 8,9 .

Aims of the study
The study aims to clarify the incidence, epidemiology, clinical presentations, staging, classification, pathological types and hematological, biochemical, immunological alterations of lymphomas of the bone and musculoskeletal tissue (both primary and secondary) among patients from the south of Iraq.Patients were segregated into rural or urban in origin depending on whether they at live, for the last 5 years, in rural or urban areas.The smoking habit, the duration of smoking and the number of cigarettes consumed per day were asked for.The drinking habit was asked for, too.

Materials and methods
A history of a possible exposure to radiation (a direct exposure to a blast or war explosion, the presence of suspected war remnants in the vicinity of residence for > 5 years, or, the direct exposure to radiation in the form of therapy for another disease) was asked for.
A positive family history of any malignant disease (affecting the 1stdegree relatives) and the type of the tumor was ascertained too.
The chief complaint and duration of illness (in months) were inquired for.The presence of "B" symptoms: an increase in body temperature >37.3 °C for >10 days (matched with measuring body temperature by examination), any drenching sweating especially at night, and loss of >10 % of body weight over a period of 3 months, were asked for 10 .The same was done for the presence of intense itching, any change in bowel habit and any history of a bleeding tendency.The general status of the patient was assessed, according to the Eastern Cooperative Oncology Group Performance Status (Table II) 11 .Patients were examined thoroughly for the presence of fever (by measuring the core temperature of the patient at the time of initial presentation), pallor, jaundice, hepatomegaly, splenomegaly (the last two were matched with the findings on abdominal ultrasound examination), neurological and cutaneous manifestations, ecchymotic patches anywhere or any evidence of a bleeding tendency, the presence of palpable lymphadenopathy and their sites.An enlargement of any two or more groups of the non-contiguous groups above was considered to be "generalized" 12 .All the patients were subjected to chest radiographs and abdominal ultrasound examinations and then were staged using the Ann Arbor Classification System 13 into 4 stages: I, II, III and IV.All patients were subjected to hematological investigations, including full blood count, using the Beckman-Coulter 5 differential hematology analyzer which was properly calibrated according to the manufacturer's instructions with quality controls included between batches 14 , erythrocyte sedimentation rate (ESR) (Westergren method) 15 , peripheral blood smear examination to find the presence of abnormal lymphoid/leukemic peripheral blood cells, bone marrow aspiration, touch imprint and trephine biopsy (Figure I).Both direct and indirect antigolobulin tests (DAT & IAT) were done, too 16 .Normal results used were those adopted by the International Council of the Standardization in Hematology (ICSH) and the British Committee for Standardization in Haematology (BCSH) 17,18 .For those with palpable, accessible lymph nodes, fine needle aspiration was performed, stained with haematoxylin stain 19 and examined.Hodgkin lymphomas were classified according to the original Rye classification since it is popular among both pathologists and clinicians for its simplicity, ease of use, the association of individual types with prognostic differences when treated with current therapy and because of the lack of immunophenotypic studies 20 , while the non-Hodgkin's lymphomas were classified using the International Working Formulation because of the lack of cytogenetic, immunological and molecular studies 21,22 .Patients were subjected to a battery of biochemical tests including: liver function and renal function tests, serum uric acid, calcium, lactate dehydrogenase (LDH), beta-2-microglobulin [23][24][25][26][27][28][29][30][31][32][33] and serological tests including the anti gliadin antibodies (AGA), anti tissue transglutaminase (ATTA), anti Epstien-Barr virus (EBV) antibodies and anti Helicobacter pylori antibodies [34][35][36][37] .
Cases were, after that, categorized according to the International Prognostic Index scoring system (Table-3) 38 .Data were fed and analyzed using the SPSS (Statistical Package for Social Sciences) version-17 39 .

Table III:
The International Prognostic Index for Non-Hodgkin Lymphoma 39 Risk

Results
Lymphomas of the bone and musculoskeletal system represented a minority among total lymphoma cases (6.4 %) and the third most common group (12.4%) of extranodal lymphomas (ENL).Patients ranged between 1.0-79.0years of age, with a mean of 39.98 years (Table-IV).Adults were much more affected than children (79.1 vs 20.9%) and the most frequent age interval was between 40.1-50.0years (16.2%).Males formed 62.8% of them.More than half of patients were from Basra Governorate (55.8%) and in those from Basra, City Center was the greater source of cases (58.3%) and 65.1% of cases were urban in origin.Smoking habit was seen among 34.9% of patients, most of them (73.3%)smoked for >20 years and 53.3% were smoking <20 cigarettes /day.Only 2.3% of them were alcohol-consumers.Those who gave a positive history of exposure to chemicals and radiation hazards constituted 9.3% and 18.6%, respectively.No case with a positive family history of a malignant disease was encountered (Table-V).Histopathologically, positive fine needle aspiration (FNA) was seen in 90.0% of cases to whom the test was done (10 cases).Non-Hodgkin lymphoma constituted the majority of cases compared to HL (86.0%vs 14.0%) and 66.6% of HL cases were of lymphocytic depletion (LD) and 16.7% were both mixed cellularity (MC) and nodular sclerosis (NS), equally.The most frequent histological type of NHL was the diffuse large cell lymphoma (DLCL) (37.9%) followed by Burkitt lymphoma (BL) (13.5%) and follicular mixed cell lymphoma (FMCL) ( *More than one bone may be involved in one patient.Hematologically, the hemoglobin concentration ranged between 4.70-13.40g/dl with a mean of 10.72 g/dl.The majority were anemic (82.4%), with mildmoderate anemia predominating on the severe (60.5%, 18.6% and 2.3%, respectively).Hematocrit ranged between 16.00-43.20%with a mean of 33.44% and the majority, (88.4%), was below 40%.Red cell sedimentation (ESR) ranged between 10-130 mm/hr with a mean of 82.74mm and the majority (90.7%) had >50 mm, of them, 20.9% had >100 mm sedimentation.The total WBCs count ranged between 2.80-107.00x 109/L, with a mean of 12.48 x 109/L.Less than half (43.5%) of cases had leucocytosis, mostly (44.2%), below 50.00 x 109/L.Neutrophil differential count ranged between 2-87%, with a mean of 60.79% and the majority (93.0%) of them had normal counts.Differential lymphocytes count ranged between 10-57% with a mean of 30.00% and 20.9%, 72.1% and 7.0% of cases had below normal, normal and above normal counts, respectively.Differential monocytes counts ranged between 1-10%, with a mean of 2.74% and 60.5% of them had below, and 39.5% had normal counts.Differential eosinophils count ranged between 0-25%, with a mean of 3.05% and 51.2% of them had below normal, 44.1% had normal and 4.7% of them had high counts.Platelets counts ranged between 21.0-618.0x 109/L, with a mean of 259.35 x 109/L and the majority, (93.0%), had normal counts while only 7.0% had thrombocytopenia.Malignant lymphoid cells were seen in the peripheral blood of 7.0% of cases and 4.7% presented with leukemic phase.Prolonged PT and PTT were seen in 2.3% of cases while positive Coombs' tests (both DAT and IAT) were seen 7.0 % and 9.3% of cases, respectively (Table-VIII).Biochemically, total serum bilirubin ranged between 0.4-5.4mg/dl, with a mean of 1.01 mg/dl and 69.8% of them had normal and 30.2% presented with above normal levels.Serum AST and ALT ranged between 13-213 and 14-234 IU/L, with their means 33.02 and 37.56 IU/L, respectively and the majority of patients, (81.4% and 79.1%), had normal levels of both .Serum alkaline phosphatase ranged between 34-287 IU/L, with a mean of 138.79 IU/L and 74.4% of cases had elevated, while 25.6% of them had normal levels.Total serum proteins ranged between 54-89 g/L with a mean of 70.33 g/L and 76.7% presented with normal level, while 14.0% of them had hypoproteinemia.Serum albumin, ranged between 20-52 g/L, with a mean of 32.51 g/L> and 81.4%, of cases presented with hypoalbuminemia.Serum globulins ranged between 22-55 g/L with a mean of 37.84 g/L and 46.5% and 51.2% of them had normal and elevated serum levels, respectively.Serum urea ranged between 20.4-66.0 mg/dl with a mean of 37.85 mg/dl and serum creatinine ranged between 0.70-1.30mg/dl with a mean of 1.02 mg/dl and the majority (81.4% and 97.7%), had normal levels of both.Serum uric acid ranged between 2.3-8.9 mg/dl, with a mean of 5.01mg/dl and 76.7% of cases had normal levels.Serum calcium ranged between 7.9-13.9mg/dl with a mean of 11.21 mg/dl and 65.1% of cases had hypercalcemia.Serum LDH ranged between 187.00-657.00IU/L with a mean of 396.95 IU/L and the majority (93.0%) of cases had elevated and 48.8% of them had more than double the normal levels of this enzyme.Serum B2 microglobulin ranged between 3.11-10.40u/ml with a mean of 4.31 U/ ml and all cases (100.0%) had elevated level (Table-IX).

Discussion
The frequency of lymphomas of the bone and musculoskeletal tissues in this study (6.4% of the total lymphoma cases, 12.4% of extranodal lymphomas (ENL) with age ranging between 1.0-79.0years and a mean of 39.98 years), seems higher than that reported by Ludwig, 2002 and Aboulafia et al 1998, who stated that primary lymphomas of bone (PBL), are one of the rarest primary bone malignancies, accounting for less than 5% of all primary bone tumors, 3-5% of all bone tumors and 5% of all primary extranodal NHL 40,41 , while secondary involvement of bone and marrow, as stated by Baar et al 1999, is seen in 5-15% of patients with HL and 30-53% of patients with NHL 42 .It is still higher than that reported by Fletcher, et al 2002 43 who reported that bone lymphoma (both primary and secondary) comprises 8.8% of ENL, while Maruyama et al 2006 stated that the incidence of PBL is 7% of all malignant bone tumors, 4-5% of all extranodal NHL and less than 1% of all malign.lymphomas 44 .However, Suresh et al stated that primary lymphomatous involvement of skeletal muscle is seen in only 0.3% of HL and 1.5% of NHL 45 .The higher incidence of bone lymphomas among adults & male sex is comparable to the findings of Mathur and Gellman 2011 who reported a male predominance (male: female ratio 1.8:1), and main affection in the fifth to seventh decade of life 46 .It is also comparable to Jawad et al, 2010 who reported a male predominance (53.9%) with the main age affected was > 60 years (55.7%) 47.The predominance of low performance status (status 4) and B symptoms is higher than that reported by Mathur and Gellman, 2011 46 , where systemic (B) symptoms, were seen in fewer than 10% of patients in true stage IE lymphoma of bone., who stated that in adults, diffuse large B-cell lymphoma is the most common subtype, accounting for (60-90% of cases).It is also comparable to that reported by Jawad et al 2010 47 , where DLBCL was the most predominant, accounting for 66.3% of cases and the high grade was markedly predominating (97%).However, Krishnan et al 2003 and Ueda, et al 1989, stated that 75% of all classifiable PBL were intermediate grade (diffuse mixed small and large cell or large cell lesions) in the Working formulation.On the basis of the current WHO classification, the majority of cases were classified as diffuse large Bcell lymphomas 52,53 .The majority of cases in this study, (90.7%), had > 50 mm ESR, of them, 20.9% had > 100 mm sedimentation, is compatible to Mathur and Gellman, 2011 who stated that the ESR is frequently elevated, especially when there is systemic disease involvement 46 .Hypercalcemia in about 2/3 rd , (65.1%) of cases is contradicting to Mathur and Gellman 2011 46 , who stated that hypercalcemia is seen in some pediatric patients and has been associated with a poorer prognosis, and to Bhagavathi and Fu 2009 and Desai et al, 1991, too, who reported that rarely, symptoms of hypercalcemia (eg, lethargy, constipation, and somnolence) are present 54,55 .The predominance of serum LDH elevation in the majority (93.0%) of cases (48.8% of them show > double the normal level), is also compatible with that reported by Mathur and Gellman 2011 46 who stated that most patients have elevated (LDH) levels, which is directly proportionate to the disease load and also to, but higher than, Ahmad et al 2009 where LDH was elevated among 71.4% of cases of PBL 56 .

Conclusions and recommendations
We conclude, from this study, that bone and musculoskeletal lymphomas, though are uncommon among population from the Sothern Part of Iraq, they seem more common than reported elsewhere with more severe and aggressive features at presentation.Thus a detailed study of them and the co-operation between orthpeditians, hematologists and histopathologists is necessary to clarify the detailed behavior of such tumors among them.This necessitates the need of the use of the revised WHO classification which needs the introduction of immunophenotyping and molecular and genetic study of cases.

Fig. 1 :
Fig. 1: Bone marrow aspiration and trephine biopsy procedures to one of the study cases, showing the way how to spread marrow smears and how to make marrow touch imprint from the biopsy piece.ResultsLymphomas of the bone and musculoskeletal system represented a minority among total lymphoma cases (6.4 %) and the third most common group (12.4%) of extranodal lymphomas (ENL).Patients ranged between 1.0-79.0years of age,

Fig. 2 :
Fig. 2: Bone involvement by lymphoma: a and b: Destruction of lumbar spine by lymphomatous lesions, c and d: involvement of right humeral head, e and f: involvement of dorsal spine and paravertebral soft tissue.

Table VI : Clinical study of bone & musculoskeletal lymphomas in south of Iraq.
10.8%).Most of NHL cases were of the intermediate and high grades (59.5% and 21.6%, respectively) (Table-VII).

Table X : Serological study and IPI distribution among bone and musculoskeletal lymphoma cases in the South of Iraq.
Histological subtype distribution of NHL in this study is comparable to that of Mathur and Gellman, 2011 46 , Kitsoulis et al 2006 50 and Chan et al 1991