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AN ANALYSIS OF THE HISTOPATHOLOGY, IMMUNOHISTOCHEMISTRY AND CLINICAL FEATURES OF RHABDOMYOSARCOMA AND A PILOT STUDY TO

CORRELATE THE IMMUNOMORPHOLOGY OF ALVEOLAR AND UNCLASSIFIABLE RHABDOMYOSARCOMA WITH PAX3/7-FOXO1(FKHR)

TRANSLOCATION BY PCR.

A DISSERTATION SUBMITTED IN PART FULFILLMENT OF THE REGULATION FOR THE AWARD OF THE DEGREE OF

M.D. PATHOLOGY BRANCH III.

THE TAMIL NADU DR. M.G.R. UNIVERSITY CHENNAI, TAMIL NADU

APRIL-2017

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An Analysis of the Histopathology,

Immunohistochemistry and Clinical features of Rhabdomyosarcoma and a Pilot study to correlate the Immunomorphology of Alveolar

and Unclassifiable Rhabdomyosarcoma with PAX3/7-FOXO1(FKHR) translocation by

PCR.

A dissertation submitted in part fulfillment of the regulation for the award of the degree of

M.D. Pathology Branch III.

(3)

CERTIFICATE

This is to certify that this dissertation “An Analysis of the Histopathology, Immunohistochemistry and Clinical features of Rhabdomyosarcoma and a Pilot study to correlate the Immunomorphology of Alveolar and Unclassifiable Rhabdomyosarcoma with PAX3/7-FOXO1(FKHR) translocation by PCR “ is the bonafide work done by Dr. Radhika.S, in part fulfillment of the rules and regulations for the M.D. Branch III (Pathology) Degree Examination of Tamilnadu Dr.M.G.R.

Medical university, to be held in April 2017.

Dr.Vivi M Srivastava, MBBS, MD.

Professor and Head, Department of Pathology,

Christian Medical College, Vellore.

Dr. Anna Pulimood, MBBS, MD, Principal,

Christian Medical College, Vellore.

(4)

CERTIFICATE

This is to certify that this dissertation “An Analysis of the Histopathology, Immunohistochemistry and Clinical features of Rhabdomyosarcoma and a Pilot study to correlate the Immunomorphology of Alveolar and Unclassifiable Rhabdomyosarcoma with PAX3/7-FOXO1(FKHR) translocation by PCR “ is the bonafide work done by Dr.Radhika.S, in part fulfillment of the rules and regulations for the M.D. Branch III (Pathology) Degree Examination of Tamilnadu Dr.M.G.R. Medical university, to be held in April 2017. The candidate has independently reviewed the literature, standardized the data collection methodology and carried out the evaluation towards completion of the thesis.

Dr. Anne Jennifer Prabhu, MBBS, MD, Associate Professor of Pathology,

Department of General Pathology, Christian Medical College,

Vellore.

(5)

CERTIFICATE

This is to certify that this dissertation “An Analysis of the Histopathology, Immunohistochemistry and Clinical features of Rhabdomyosarcoma and a Pilot study to correlate the Immunomorphology of Alveolar and Unclassifiable Rhabdomyosarcoma with PAX3/7-FOXO1(FKHR) translocation by PCR “ is the bonafide work done by Dr.Radhika.S, in part fulfillment of the rules and regulations for the M.D. Branch III (Pathology) Degree Examination of Tamilnadu Dr.M.G.R. Medical university, to be held in April 2017. The candidate has independently reviewed the literature, standardized the data collection methodology and carried out the evaluation towards completion of the thesis.

Dr.Radhika.S,

PG Registrar,

Department of General Pathology, Christian Medical College,

Vellore.

(6)
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ACKNOWLEDGEMENT

I would like to thank the Almighty and my family and friends for their help in pursuing my thesis.

I express my warm thanks to Dr. Anne Jennifer Prabhu (General pathology) and Dr. Rekha Pai (Molecular lab, General pathology) for their guidance and persistent help for the completion of this dissertation.

I would also like extend my thanks to Dr. Leni Grace Mathew, Professor of Pediatrics, Pediatric Hematology-Oncology, Dr.Deepthi Boddu, Assistant Professor of Pediatrics, Pediatric Hematology-Oncology and Dr. Sam Prasad Mannam, Assistant Professor, Department of Medical Oncology for helping me in collecting the clinical and followup details.

I would extend my sincere thanks to Mr. Dhananjayan, Molecular Pathology in helping me with the PCR techniques and Mrs.Grace Rebekah, biostatistician for the statistical analysis.

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ABBREVIATIONS

RMS - Rhabdomyosarcoma

ARMS - Alveolar rhabdomyosarcoma PCR - Polymerase chain reaction

IRSG - Intergroup Rhabdomyosarcoma Study Group SEER - Surveillance, Epidemiology and End Results

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CONTENTS

1 Introduction 1

2 Aims and objectives 3

3 Review of literature 4

4 Materials and methods 34

5 Results 41

6 Discussion 68

7 Conclusions 95

8 Limitations 96

9 Bibliography 10 Appendix

Appendix 1- Procedure for immunohistochemical staining of Desmin and Myogenin.

Appendix 2 - Procedure for molecular analysis of PAX3/7- FOXO1 translocation.

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INTRODUCTION

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Rhabdomyosarcoma (RMS) is the most common soft tissue tumour of children and adolescents. RMS constitutes about 5% of pediatric tumours. It is the third most common extracranial tumour next to Wilms tumour and neuroblastoma(1).

The term rhabdomyosarcoma was described first in 1854 by Weber, however, distinct morphology of rhabdomyoblasts was described by Stout in 1946. The nomenclature belongs to the Greek word, “rhabdo” means rod like and „myo‟ means muscle(2). Rhabdomyosarcoma is a primitive malignant soft tissue tumour that recapitulates the phenotypic and biological features of embryonic skeletal muscle(3).

It is a malignant small round blue cell tumour solid tumour that arises from the mesenchymal tissues which normally differentiate to form striated muscle(1,4).

Rhabdomyosarcoma shows morphological, immunohistochemical, molecular and electron microscopic evidence of skeletal muscle differentiation(5).

The most common histological subtypes of Rhabdomyosarcoma are embryonal which predominantly occurs in infants and children, alveolar type which occurs mainly in adolescents and young adults and pleomorphic in adults with preponderance in males. The diagnosis is made by histomorphology and immunohistochemical features. A relatively new diagnostic modality for alveolar rhabdomyosarcoma is RT-PCR for PAX3/7-FOXO1 translocation which is the molecular signature for alveolar rhabdomyosarcoma. The prognostication depends on various clinicopathological and treatment related variables. Despite these prognostic factors, there is variability in the outcome of the tumours. The patients

(12)

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succumb to morbidity of chemotherapy and radiotherapy. To correctly gauge the prognosis, treatment protocols and a possibility of targeted therapy in the future, the molecular biology of rhabdomyosarcoma, particularly alveolar and the unclassifiable rhabdomyosarcoma becomes a significant diagnostic approach.

The treatment for rhabdomyosarcoma is multidisciplinary based on various prognostic factors and includes surgery, chemotherapy and radiotherapy. The treatment modality based on molecular translocation is under research.

(13)

AIMS AND OBJECTIVES

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a) To analyse the clinicopathological features of the different WHO subtypes of RMS in adult and pediatric population.

b) To identify PAX3/7-FOXO1(FKHR) translocation in alveolar and unclassifiable rhabdomyosarcoma.

c) To correlate the molecular characteristics of alveolar and unclassifiable rhabdomyosarcoma with the histological, immunohistochemical and clinical features.

(15)

REVIEW OF LITERATURE

(16)

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EPIDEMIOLOGY OF RMS:

The incidence of rhabdomyosarcoma among Indians is 2.3%(5). The incidence amongst Indians is 2-3 per million. The incidence of rhabdomyosarcoma is 4-6 per million among Europeans and Americans(5,6). The lower incidence amongst Asians is attributed to the low genetic susceptibility(7). With approximately 250 cases diagnosed yearly in the United States, it is the third most common extracranial solid tumor of childhood after Wilms tumor and neuroblastoma. Important epidemiologic, biologic, and therapeutic differences have been elucidated within the RMS family.

AGE:

About 250 cases are diagnosed per year in the United States. About 65% are less than 6 years of age, the remaining fall under 10-18 years of age(8). Alveolar rhabdomyosarcoma (ARMS) occurs mainly in adolescents and young adults, embryonal RMS predominantly occurs in infants and children. Pleomorphic RMS occurs exclusively in adults with a preponderance in males(9).

GENDER:

There is a slight male preponderance i.e. RMS is about 1.4 times more common in males compared to that of females(8).

RISK FACTORS FOR RMS:

There are no definite risk factors identified. However, few studies have found certain possible risk factors.

(17)

5

Genetic risk factors:

Rhabdomyosarcomas are sporadic but they can be associated with familial syndromes like Neurofibromatosis-I, Costello‟s syndrome, Beck-with Wiedemann syndrome and Li Fraumeni syndrome(2,10). Patients with neurofibromatosis- I have 20 fold increased risk of developing rhabdomyosarcoma.

Recent studies have found that patients diagnosed with embryonal rhabdomyosarcoma have a first degree relative with malignancy and most of their first degree relatives were diagnosed to have malignancy less than 30 years of age(11).

Congenital anomalies are associated with rhabdomyosarcomas in certain sites such as genitourinary tract, central nervous system (Arnold Chiari malformation) and gastrointestinal tract, etc.(12).

Environmental factors:

Radiation exposure in utero, maternal use of cocaine and children from low socioeconomic status are known to be associated with increased risk of rhabdomyosarcoma(13). Higher birth order and immune mediated diseases afford protective role against rhabdomyosarcoma(14).

EMBRYOLOGY:

RMS is a tumour of primitive mesenchymal cells. RMS develops as a consequence of regulatory disruption of growth and differentiation of myogenic precursor cells(15).

(18)

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Myogenesis is an orderly process. There are certain members of the myogenic bHLH family which help in orderly myogenic differentiation(16).

MyoD (Myogenic determination) gene helps in the conversion of nonmuscle cell into muscle cell. Three other related myogenic regulatory genes have been described which include myf 5, myogenin and MRF4. The proteins encoded by these genes are analogous to MyoD and help in inducing myogenic differentiation(17).

Myogenin is required for the maturation of skeletal muscle(8,18).

Rhabdomyosarcomas express markers according to the stage in which the maturation is arrested. MyoD is expressed in the early stages(19).

Embryonal RMS is believed to be due to early block in myogenesis before Myogenin expression, therefore embryonal RMS donot show strong positivity for Myogenin staining (16,20).

On the otherhand, alveolar RMS is believed to be due to late blockade during the myogenesis after the expression of Myogenin and so alveolar RMS shows a diffuse strong positivity for Myogenin. The other hypothesis as to why alveolar RMS strongly expresses Myogenin could be due to the fact that certain mechanisms involved in tumourigenesis tend to induce or upregulate the Myogenin(20).

SITE SPECIFIC CHARACTERS OF RMS:

Rhabdomyosarcoma (RMS) is a malignant tumor of mesenchymal origin thought to arise from cells committed to a skeletal muscle lineage. Common sites of primary

(19)

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disease include the head and neck region, genitourinary tract and extremities. Orbital and genitourinary tumours are mostly of embryonal histology. Those arising in the extremities are mainly of alveolar histology and those in the bladder and vagina are of botryoid type. The frequency of various sites involved are as follows:

1) Head and neck: 50%

Orbit- 20%

Oro/nasopharynx, palate- 15%

Sinuses, mastoid, middle ear- 15%

2) Genitourinary region: 25%

Paratesticular-20%

Bladder-5%

3) Extremities: 15%

4) Others:10%

Trunk and thorax-7%

Gastrointestinal tract-1%

Some of these sites correlate with better prognosis and are classified as favourable sites. The favourable sites include the orbit, head and neck (except parameningeal region), genitourinary region (except bladder and prostate) and bile ducts. The unfavourable sites include the parameningeal region, bladder, prostate, extremities and others (trunk and retroperitoneum)(21).

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Rhabdomyosarcomas of the head and neck region seem to occur at a lower age group compared to that of the other sites. Tumours of the parameningeal region show a predisposition to involve the CSF, thereby diagnosed by lumbar puncture(22).

A site-based tumor-nodes-metastasis staging system is being incorporated into use for assessing prognosis and assigning therapy in conjunction with the traditional surgicopathologic clinical grouping system(23).

CLINICAL FEATURES:

The clinical presentation depends to a certain extent on the site of involvement. The most common presentation is mass in the site of involvement. Tumours arising from the orbital region can produce proptosis and ophthalmoplegia(24). Tumours arising from the ear can present as aural polyp. Those arising in the parameningeal region can present as a small lesion or with mucopurulent discharge. Those in the extremities can present as a lump causing neuropathy. Retroperitoneal tumours can present with symptoms of renal failure due to obstructive uropathy and hydroureteronephrosis. Prostatic tumours can cause urinary obstruction. Embryonal rhabdomyosarcoma produces symptoms related to mass effects and obstruction(25).

Botryoid rhabdomyosarcoma usually arises beneath mucosal surface like bladder, conjunctiva, pharynx, biliary tract and auditory canal(25,26). Metastatic disease could be the initial presentation in some patients. Lymph node enlargement and bone pain can occur. Lung is the more common site of metastasis followed by bone and bone marrow (27).

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DIAGNOSTIC MODALITIES:

The diagnostic modalities undertaken should help in assessing the extent of primary disease and metastatic workup. Complete physical examination should be done so as to identify any lymph node enlargement. Laboratory investigations include complete blood count, liver function test, serum electrolytes and renal function test so as to help in deciding the treatment regimes. X-ray, CT or MRI of the primary site is to be done to exactly guage the size and extent of the tumour. Lumbar puncture has to be performed for tumours involving parameningeal region because they have higher propensity for spread to CSF. Patients with paratesticular RMS have to be subjected to abdominal CT to look for retroperitoneal lymph node involvement. X-Ray chest is recommended to identify any pulmonary metastasis as lung is the most common metastatic site to be involved. Bilateral iliac crest bone marrow biopsy is recommended as part of metastatic workup.

STAGING AND RISK STRATIFICATION:

To predict the outcome, RMS can be prognosticated using various clinical and histological parameters.

a) Surgicopathological grouping based on extent of disease after surgery but before chemotherapy and radiotherapy. (Table 1)

b) IRSG staging system which incorporates TNM staging, site, size, and presence or absence of lymphnode and distant metastasis. (Table 2)

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TNM staging depends on the following parameters a) Tumour size and location

b) Nodal status and c) Metastasis

TNM staging: T1- Confined to anatomical site T1a - <5cm, T1b->5cm

T2 - Extends beyond the site of origin T2a - <5cm, T2b->5cm

N0 - No nodal metastasis, N1- Nodal metastasis present M0 - No distant metastasis, M1- Distant metastasis present

(23)

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Table 1: IRSG surgical pathological grouping system (23) Group

I

Localised tumour, completely removed with pathologically clear margins and no regional lymph node involvement

Group IIa

Localised tumour, grossly removed with microscopically involved margins

Group IIb

Localised tumour grossly removed with involved grossly resected regional lymph node

Group IIc

Localised tumour grossly removed with both microscopically involved margins and involved lymph node

Group III

Localised tumour with gross residual disease after grossly incomplete removal or biopsy only

Group IV

Distant metastasis at diagnosis

(24)

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Table 2: IRSG staging system

Parameningeal sites includes the following regions

Infratemporal fossa, middle ear, mastoid, nasal cavity, nasopharynx, paranasal sinuses

Pterygopalatine fossa and the parapharyngeal region(23)

Risk stratification of RMS can be done with the histological subtype and the stage.

(Table 3)

Table 3: Risk stratification in RMS (27)

Low risk Embryonal RMS stage 1(Favourable site), Embryonal Stage 2-3 (Unfavourable site)

Intermediate

risk Embryonal stage 2-3 (Unfavourable site), Alveolar Stage 1-3

High risk Metastasis (Stage 4)

Stage 1

Favourable sites

TNM Any size,

N0/N1 M0

5 year overall survival 90%

Stage 2

Unfavourable sites

TNM Tumour size

≤5cm, N0 M0

5 year overall survival 85%

Stage

3 Unfavourable sites

TNM Tumour size

>5cm N1 M0

5 year overall survival 70%

Stage

4 Any site M1 5 year overall

survival 30%

(25)

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HISTOPATHOLOGY:

Rhabdomyosarcoma is a small round blue cell tumour(28). The hallmark for diagnosis of RMS is the demonstration of malignant skeletal muscle differentiation.

On light microscopy, cross-striations in tumor cells, characteristic of skeletal muscle or rhabdomyoblasts, can be noted(29).

HISTOLOGICAL SUBTYPES:

According to WHO 2013(3), RMS can be classified into the following types.

Embryonal Alveolar Pleomorphic Spindle cell and Sclerosing.

The relative percentage contributed by the individual subtypes is as follows.

Embryonal RMS-75%

Alveolar RMS- 20%

Pleomorphic- 5%

EMBRYONAL RMS:

Embryonal RMS has a predilection to affect children <15 years of age and most commonly involves the head and neck and genitourinary region(3). Embryonal type

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RMS constitutes 50-60% of RMS cases(30) and their 5 year disease free survival period is 81%(31).

GROSS APPEARANCE:

Embryonal RMS is usually a polypoidal, fleshy pale tan mass. Spindle cell variants can have a firm fibrous cut surface.

HISTOLOGY:

Embryonal RMS consists of primitive mesenchymal cells in different stages of maturation, predominantly small, round/spindle shaped undifferentiated cells admixed with variable number of strap, tadpole shaped eosinophilic rhabdomyoblasts in a myxoid stroma(20).

Embryonal rhabdomyosarcoma can histologically recapitulate different stages of myogenic differentiation. The most primitive cells are stellate with central oval nuclei and light amphophilic cytoplasm(32). As they differentiate, there is progressive eosinophilia of the cells and they become bipolar with tapered cytoplasmic processes or tadpole like cells with long cytoplasmic extensions(33).

The bright eosinophilia (bundles of actin and myosin) may be visible in the cytoplasm highlighted by Masson‟s trichrome or PTAH (phosphotungstic acid hemotoxylin)(34). Differentiation becomes more evident following chemotherapy(35).

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Botryoid type is a subtype of embryonal RMS with polypoid configuration. Botryoid subtype of embryonal RMS arises beneath a mucosal surface and the genitourinary tract. Histologically, there is a subepithelial cambial layer constituted by condensed relatively undifferentiated tumour cells(36) (Fig 1-6). Cartilagenous or osseous differentiation can be rarely seen(37).

Fig 1: Embryonal (botryoid) rhabdomyosarcoma - Hyper and hypocellular areas (H&E at 40x)

(28)

Fig 2: Embryonal (botryoid subtype) rhabdomyosarcoma (H&E at 40x)

Fig 3: Embryonal (botryoid subtype) rhabdomyosarcoma (H&E at 100x)

(29)

Fig 4: Anaplasia in embryonal RMS (H&E at 100x)

Fig 5: Embryonal rhabdomyosarcoma - IHC showing cytoplasmic positivity for Desmin (3+) at 100x

(30)

Fig 6: Embryonal rhabdomyosarcoma - IHC showing nuclear positivity for Myogenin (2+) at 100x

(31)

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ALVEOLAR RMS (ARMS):

ARMS occurs in adolescents and young adults and constitutes 20% of RMS cases. The

median age is between 6.8 and 9.0 years. ARMS most commonly arises in the extremities. Other sites include paraspinal, perineal region, paranasal sinuses and the female breast. The 5 year disease free survival period is 65%(38).

GROSS APPEARANCE:

Alveolar RMS presents as a rapidly growing grey tan mass.

HISTOLOGY:

ARMS is a cellular tumour and is composed of primitive cells with monomorphous round nuclei. It is a small round cell tumour. ARMS can be further subclassified into typical and solid variants.

TYPICAL ARMS produces fibrovascular septa that separate tumour cells into discrete nests with central clustering and discohesive periphery. Cells align along the septa in a

“picket fence” pattern (Fig. 7-10, 13-15).

The SOLID variant lacks the fibrovascular stroma and forms sheets of round cells with variable rhabdomyoblastic differentiation (Fig 11). Wreath like multinucleate giant cells can be seen in alveolar rhabdomyosarcoma (Fig. 12).

(32)

Fig 7: Alveolar rhabdomyosarcoma- typical subtype (H&E at 40x)

Fig 8: Alveolar rhabdomyosarcoma- typical subtype (H&E at 100x)

(33)

Fig 9: Alveolar rhabdomyosarcoma- typical subtype (H&E at 100x)

(34)

Fig 10: Alveolar rhabdomyosarcoma- solid subtype (H&E at 100x)

Fig 11: Alveolar rhabdomyosarcoma solid subtype (H&E at 200x)

(35)

Fig 12: Wreath like giant cell in alveolar rhabdomyosarcoma (H&E at 100x)

Fig 13: Alveolar RMS - IHC for Desmin (3+) at 200x

(36)

Fig 14: Alveolar RMS - IHC for Myogenin (3+) at 100x

Fig 15: Alveolar RMS- IHC for Myogenin (3+) at 200x

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Alveolar RMS has tendency to develop breast metastasis in female patients. They can also present like leukemia without a primary lesion. This pattern is due to extensive bone marrow involvement and is due to malignant transformation of the bone marrow mesenchymal stem cells. This is called rhabdomyoblastemia(39). Tumours with mixed embryonal and alveolar features were previously considered to be variants of ARMS, but most of these lack PAX3/7-FOXO1 fusions, thus biologically resembling ERMS.

Anaplasia can be seen in Rhabdomyosarcoma as seen in Wilm‟s tumour.

According to Sidhom et al(40), the presence of anaplasia is histologically defined by the marked nuclear enlargement (3 times that of the neighbouring nuclei), hyperchromasia and presence of multipolar mitotic figures.

The term „focal anaplasia‟ was restricted to tumour which showed anaplasia in localized regions and „diffuse anaplasia‟ if clusters of sheets of anaplastic cells were found.

Anaplasia was found more in patients more than 10 years of age, those who belonged to high risk group and displaying alveolar morphology(40).

PLEOMORPHIC RMS:

Pleomorphic RMS is a high grade pleomorphic sarcoma occurring in sixth and seventh decade. The most common site is the deep soft tissue of the extremity. The other sites include chest/abdominal wall, head & neck and retroperitoneum. Grossly, the tumour presents as well circumscribed large mass with firm white cut surface and areas of haemorrhage and necrosis. Microscopically, it is composed of undifferentiated round to

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spindle cells and an admixture of polygonal pleomorphic cells with densely eosinophilic cytoplasm (Fig.16-19). They are usually patternless but sometimes storiform pattern can be seen(41). Cross striations are rare in pleomorphic RMS (3).

(39)

Fig 16: Pleomorphic rhabdomyosarcoma (H&E at 200x)

Fig 17: Pleomorphic rhabdomyosarcoma - IHC for Desmin (3+) at 100x

(40)

Fig 18: Pleomorphic rhabdomyosarcoma - IHC for Myogenin (2+) at 100x

Fig 19: Rhabdoid cell in pleomorphic RMS (H&E at 400x)

(41)

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SPINDLE CELL RMS:

Spindle cell RMS is a rare type of RMS characterized histologically by the presence of pleomorphic spindle cells (Fig.20-22). They have a strong predilection for males. The paratesticular region is the most common site of spindle cells RMS (9).

Fig 20: Spindle cell rhabdomyosarcoma (H&E at 100x)

(42)

Fig 21: Spindle cell RMS - IHC for Desmin (3+) at 40x

Fig 22: Spindle cell RMS - IHC for Myogenin (1+) at 100x

(43)

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SCLEROSING RMS:

Sclerosing RMS is a rare variant which can occur both in pediatric age group and adults, the limbs being the most common location.

Histologically, there is evidence of stromal hyalinisation(42). (Fig 23-26)

(44)

Fig 23: Sclerosing rhabdomyosarcoma (H&E at 100x)

Fig 24: Sclerosing rhabdomyosarcoma (H&E at 200x)

(45)

Fig 25: Sclerosing rhabdomyosarcoma - IHC for Desmin (3+) at 100x

Fig 26: Sclerosing rhabdomyosarcoma - IHC for Myogenin (1+) at 100x

(46)

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UNCLASSIFIABLE RMS:

Those cases which could not be histologically classified under any of the above mentioned subtypes, were included under unclassifiable RMS (Fig. 27-29).

Fig 27: Unclassifiable RMS (H&E at 100x)

(47)

Fig 28: Unclassifiable RMS - IHC for Desmin (2+) at 100x

Fig 29: Unclassifiable RMS - IHC for Myogenin (2+) at 100x

(48)

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FEATURES OF MATURATION:

Higher degree of maturation in RMS is determined by three factors.

a) Presence or absence of necrosis b) Presence or absence of septa c) Increased amounts of cytoplasm

Increased amounts of cytoplasm (cytoplasm more than surface area of nuclei), absence of necrosis (necrosis less than 10% of the tumour surface), absence of septa (septa less than10% of the tumour surface) indicated higher degree of maturation. Those cases of RMS which showed a higher degree of differentiation were known to be associated with a better outcome(18). However, the current WHO does not recommend these parameters to prognosticate these tumours.

POSTTREATMENT INDUCED HISTOLOGICAL CHANGES:

Chemotherapy induces changes in the histology of the tumour cells and the stroma.

The various changes include cytodifferentiation, fibrosis, necrosis, myxoid areas, foamy macrophages, bizarre tumour cells and hyalinization (Fig 30-34).

The pattern of histological changes is as follows a) No histologic change

b) Tumour cell death and reparative changes- most common c) Tumour maturation- usually seen with primitive tumours

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d) Heterologous differentiation

e) Apoptosis of associated lymphocytes

Also seen are inflammation with macrophages, lymphocytes and plasma cells, fibrosis, and atrophic or regenerating non neoplastic skeletal muscle(43).

Cytodifferentiation i.e. maturation of tumour cells, is seen mostly in embryonal subtype and is less with alveolar rhabdomyosarcoma(44). Areas of cytodifferentiation can be intermingled with undifferentiated areas.

Such intermingling of undifferentiated cells with differentiated areas is common in alveolar rhabdomyosarcoma. So, careful search for these undifferentiated areas is required, especially in small biopsies.

(50)

Fig 30: Postchemotherapy change- hyalinisation and cytodifferentiation (H&E at 100x)

(51)

Fig 31: Postchemotherapy change - cytodifferentiation (H&E at 200x)

Fig 32: Postchemotherapy change - sheets of foamy macrophages (H&E at 100x)

(52)

Fig 33: Postchemotherapy change- fibrosis (H&E at 100x)

Fig 34: Postchemotherapy change - bizarre tumour cells (H&E at 100x)

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DIFFERENTIAL DIAGNOSIS:

Differential diagnosis of embryonal rhabdomyosarcoma include solid alveolar RMS, spindle cell RMS, infantile fibrosarcoma, Wilm‟s tumour, neuroblastoma, rhabdomyoma and pleuropulmonary blastoma(41).

The differential diagnosis of alveolar RMS include the whole spectrum of small round blue cell tumours including Ewing sarcoma, PNET, lymphoma, desmoplastic small round cell tumour and neuroblastoma(45).

Differential diagnosis of pleomorphic rhabdomyosarcomas includes all other pleomorphic sarcomas(41).

The differential diagnosis of spindle cell RMS include fibromatosis, solitary fibrous tumour, MPNST, synovial sarcoma and leiomyosarcoma(46).

IMMUNOHISTOCHEMICAL PROFILE:

Rhabdomyosarcomas express skeletal muscle markers like Myogenin, Desmin, myoD1 and myoglobin. The cytoplasmic nonfilamentous markers are Myoglobin and creatine kinase M. The cytoplasmic filamentous markers are Desmin, muscle specific actin and myosin. The nuclear markers are MyoD and Myogenin.

Myoglobin is seen in 60% of RMS. Occasional cases of leiomyosarcoma and undifferentiated sarcoma can also show Myoglobin positivity. So, Myoglobin lacks sensitivity as only differentiated cells express it(47).

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Desmin and Muscle Specific Actin are sensitive but they are also expressed by smooth muscles, myofibroblasts, myoepithelium and the pericytes. Also, certain tumours like leiomyosarcoma, malignant peripheral nerve sheath tumours, myofibrosarcomas and desmoplastic small round cell tumours can express Desmin and smooth muscle actin.

Muscle transcription factors MyoD and myogenin are sensitive and specific as they are expressed by cells with even little differentiation. Heat retrieval techniques employed during immunohistochemistry can produce non specific cytoplasmic staining. Only nuclear staining should be considered positive.

Embryonal RMS shows heterogenous staining pattern for myogenin whereas alveolar rhabdomyosarcoma shows a strong diffuse nuclear staining, which correlates with decreased survival(47).

MyoD (Myogenic determination) gene helps in the conversion of non muscle cell into muscle cell. Three other related myogenic regulatory genes have been described which includes myf 5, myogenin and MRF4. The proteins encoded by these genes are analogous to MyoD and helps in inducing myogenic differentiation(17).

Myogenin is required for the maturation of skeletal muscle(18). During myogenesis, MyoD expression represents the initial stage of commitment to myogenesis being present in undifferentiated cells. As the differentiation proceeds, MyoD is downregulated. But Myogenin expression correlates with the stage of onset of differentiation and maintenance of differentiated state. According to Morotti et al, the

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sensitivity and specificity for Myogenin immunohistochemical staining is 97% and 90%-91% respectively(34).

The staining for Myogenin and desmin is considered as 0 if all tumour cells are negative,

+ if 1-10% of the cells show moderate positivity, ++ if 10-50% of the cells show moderate positivity and

+++ if more than 50% of the cells show strong Myogenin positivity (16) .

Alveolar rhabdomyosarcomas do not always exhibit this classical alveolar histological pattern or the foci may be overlooked because of sampling errors. This heterogeneity in tumor histology makes it difficult to apply strict diagnostic criteria for subclassification.

As a consequence, alveolar rhabdomyosarcomas are often incorrectly diagnosed as embryonal rhabdomyosarcomas or conversely embryonal rhabdomyosarcomas may be incorrectly diagnosed as alveolar rhabdomyosarcomas. The currently applied histopathological criteria for diagnosing ARMS differ from the criteria used at the time of the IRS-III trial, which required alveolar pattern in at least 50% of the lesion for a diagnosis of ARMS. The International Classification of Rhabdomyosarcoma system, published in 1995, proposed that any focus with alveolar pattern was sufficient for a histopathological diagnosis of ARMS(48). Therefore the availability of protein markers that can be used to distinguish between alveolar and embryonal rhabdomyosarcomas by

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simple immunohistochemical techniques could significantly enhance a diagnosis of ARMS.

Myogenin is more sensitive and specific for diagnosis of rhadbomyosarcoma as compared to Desmin. Myogenin is not expressed in mature skeletal muscle fibres but it can be expressed in regenerated skeletal muscle fibres(49,50). Strong Myogenin expression is in favour of alveolar subtype. Moreover, strong staining for Myogenin was found to be correlating with PAX3/7- FOXO1 translocation status in other studies by Dias et al(16). In one study, 2 cases classified histologically as embryonal but which showed strong Myogenin staining, on molecular analysis turned out be alveolar subtype. So, strong Myogenin staining should alert to the possibility of alveolar subtype eventhough the histology doesnot seem so. Therefore, strong Myogenin staining can be used to triage patients for molecular analysis, thereby preventing unnecessary molecular analysis for all subtypes. Alveolar rhabdomyosarcomas expressed at least threefold more myogenin than embryonal rhabdomyosarcomas(16).

MOLECULAR PROFILE OF RMS:

The molecular analysis helps in understanding the tumourigenesis of RMS. Cytogenetic analysis show that recurrent translocation t(2;13)(q35;q14) occurs in most cases of ARMS while t(1;13)(p36;q14) occurs in a subset of cases(38). This corresponds to PAX3 or PAX7 on chromosome 2 and 1 respectively with FOXO1(FKHR) on chromosome 13, to generate chimeric genes that encode PAX3 and PAX7-FOXO1 fusion proteins. PAX3/7- FKHR fusion commits the mesenchymal stem cells to

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myogenic differentiation and inhibits terminal differentiation. PAX3/7-FKHR fusion induces skeletal myogenesis by transactivating MyoD and myogenin which transforms the mesenchymal progenitor cells to skeletal muscle lineage(10) .

These fusion proteins function as potent transcriptional activators with oncogenic effects. PAX3-FOXO1 translocation is identified in about 55-75% of alveolar RMS.

PAX7- FOXO1 translocation is identified in about 10-22% of alveolar RMS(51).

According to Sorenson et al(52), PAX3- FOXO1 gene fusion was present in 55% of the cases, PAX7- FOXO1 fusion gene in 22% of the cases and 23% were fusion negative. In few of these fusion negative cases, rearrangements such as PAX3 fused to FOXO4, NCOA1 or NCOA2 or FOXO1 fused to FGFR1 have been identified. Gene expression profiles of these fusion negative ARMS cases differ from those of fusion- positive ARMS, but are similar to those of ERMS. Fusion negative ARMS behave similar to ERMS, which has a favourable prognosis and is amenable for cure(53).

ERMS is also associated with loss or gain of chromosomes. There is loss of heterozygosity of chromosome 11(54). Thus, Rhabdomyosarcomas may appear similar morphologically but they are biologically diverse.

Identification of this genetic translocation helps in deciding the treatment strategy. RT- PCR and FISH are the gold standards for analyzing fusion status(55). Presence of PAX3-FOXO1 translocation has a worse outcome as compared with other translocations(56).

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There is an association of fusion status with site and nodal status. The association with site is characterized by a low incidence of fusion-negative tumors in the extremities and high incidence in nonbladder/prostate genitourinary sites compared to the two fusion- positive categories(57). PAX7-FKHR-positive tumors occur in younger patients. Nodal status is characterized by a very low frequency of nodal involvement in the fusion- negative tumors(57).

SENSITIVITY AND SPECIFICITY OF THE TEST:

According to Thway et al, the sensitivity and specificity of detecting the PAX3/7- FOXO1 translocation by RT-PCR is 85.7% and 100% respectively. However FISH is considered to be a more sensitive test(58).

ELECTRON MICROSCOPIC APPEARANCE:

Electron microscopically, rhabdomyoblasts are characterized by the presence of myofilaments, thick and thin filaments, intermediate filaments and the Z- band (59).

PROGNOSIS OF RHABDOMYOSARCOMA :

Prognosis of rhabdomyosarcoma is based on various clinicopathological and treatment related variables(60). Various clinical and the biological factors influence the prognosis of RMS. This includes site, size, nodal status and the histology.

Patients with non-alveolar histology, primary site other than parameningeal region, local recurrence and recurrence after completion of therapy, have better prognosis(61).

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Prognosis also depends on the stage of the tumour(8). Presence of PAX3-FOXO1 translocation has a worse outcome as compared with the other translocation(62).

Head and neck region involvement, other than parameningeal region has a favourable prognosis.

Size less than 5cm has a favourable prognosis.

Nodal involvement implies a poor prognosis.

Extent of surgery and margin status also affect the outcome(63).

Metastasis and poor response to chemotherapy are also poor prognostic indicators(64).

TREATMENT:

SURGERY:

Treatment of Rhabdomyosarcoma involves a multimodality approach. For superficial non orbital lesions- wide local excision is the treatment of choice with 2cm adequate margins. If the tumour is near the orbit and a clearance of 2cm is not feasible, the clearance can be reduced. For recurrent or locally persisting orbital lesions- exenteration is recommended. For microscopic residual disease- reexcision with wide local margins is recommended. Surgery is not recommened for metastatic disease.

CHEMOTHERAPY:

The treatment of ARMS is multimodal with a combination of surgery, radiation and intensive chemotherapy(65). Alveolar rhabdomyosarcoma is usually removed by surgery. Chemotherapy can be either neoadjuvant or adjuvant chemotherapy with

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Vincristine, Actinomycin and cyclophosphamide (VAC) regimen. Radiotherapy is advised to reduce the chance of recurrence of tumour.

For embryonal RMS with favourable features, i.e. low risk ERMS, favourable site, ERMS which is completely resected or ERMS with microscopic residual disease- Vincristine, Actinomycin& Cyclophosphamide (VAC) regimen is recommended(66,67). The overall survival is >90% for this group of patients.

For patients with ERMS with unfavourable features i.e. ERMS with gross residual disease, metastatic ERMS and nonmetastatic alveolar RMS - VAC regimen with radiotherapy is the treatment of choice. The International Rhabdomyosarcoma group V- recommends addition of Topotecan. Irinotecan is recommended for metastatic disease.

The overall survival is 55-70%. Chemotherapy is ideally given 2-3 months before starting radiotherapy except for rhabdomyosarcoma involving the parameningeal region for which radiotherapy is started at the time of diagnosis because of risk of meningeal spread. Radiotherapy is given for 5-6 weeks. During radiotherapy, radiosensitising drugs like Actinomycin should be avoided. Intracranial meningeal extension of parameningeal rhabdomyosarcoma requires whole brain irradiation and radiotherapy to the primary tumour(66–68).

RADIOTHERAPY:

Radiotherapy is indicated for all alveolar RMS cases with positive margins.

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THERAPEUTIC IMPLICATIONS:

Currently, chemotherapy or surgery with or without radiotherapy is the treatment of choice. Drug resistance and metastatic disease pose an important problem(66). The development of intensive multimodality treatment protocols tested in large-scale international trials has resulted in significant improvements in outcome, especially for patients with local or locally extensive disease for whom a 60%-70% disease-free survival can be expected. Despite aggressive approaches incorporating surgery, dose- intensive combination chemotherapy and radiation therapy, the outcome for patients with metastatic disease remains poor. Future challenges include the development of less toxic therapy for patients with localized disease and new approaches for patients with metastatic disease.

TARGETED THERAPY:

Proapoptotic gene, NOXA is found to be upregulated by PAX3-FOXO1 gene fusion in a p53 independent mechanism. Cells expressing this translocation have increased apoptosis in response to Bortezumib(69). Screening libraries of kinome SiRNA defined PLK1 as an upstream acting regulator, interacting and phosphorylating PAX3- FOXO1 at a novel site S503, leading to protein stabilisation. PLK1 inhibitors (I 2536) led to degradation of the chimeric PAX3- FOXO1oncoprotein, leading to tumour regression (70). Post transcriptional modifications includes phosphorylation of PAX3/7-FOXO fusion protein. So, by inhibiting this phosphorylation by certain agents like Lithium and Tideglusib, there is an expected benefit in the high risk cases(72).

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COMPLICATIONS OF TREATMENT:

Chemotherapy can cause various side effects. The side effects may vary according to the age. Patients more than 10 years of age had less myelosupression as compared with those less than 10 years of age. The most common side effects observed were the following: neutropenia, thrombocytopenia, anaemia, infection, peripheral nervous system (PNS) toxicity and mucositis.

(63)

MATERIALS AND

METHODS

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This study, partly prospective and partly retrospective, was carried out in the Departments of General Pathology and Molecular Pathology, Christian Medical College and Hospital during the period 2012-2016. All cases diagnosed as Rhabdomyosarcoma during the period January 2012-March 2016 were included in this study.

Inclusion criteria:

All adult and pediatric cases diagnosed as rhabdomyosarcoma between January 2012 to March 2016 in the Norman Institute of Pathology, Christian Medical College, Vellore were included in the study. Those cases referred from elsewhere were also included, provided the slides/ blocks were available. Those cases which were diagnosed as alveolar and unclassifiable subtypes were analysed for molecular translocation, provided the blocks were adequate and the area covered by the tumour was atleast 50%.

Exclusion criteria:

1) Blocks handed over to the patients.

2) Biopsy sample inadequate to proceed with immunohistochemistry and molecular analysis (blocks with less than 50% tumour area).

3) Referred cases with only slides submitted for review.

4) Tumours with rhabdomyoblastic differentiation as a minor component.

Clinical details of the cases:

The clinical details of these patients were obtained from the charts retrieved from the Medical Records Department. The clinical features that were analysed include age,

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gender, size, location, radiology, surgical treatment given, bone marrow biopsy, lymph node metastasis, distant metastasis, chemotherapy status, recurrence, complications, followup and the stage.

Staging:

Staging was done based on the following details, size, location and the presence or absence of lymph node and distant metastasis (As in Table 2).

Follow-up:

The period of followup was noted and divided into three groups.

Group1:Followup duration of less than 6 months from diagnosis in CMC.

Group2:Followup duration of 6 months-1 year from diagnosis in CMC.

Group3:Followup duration of 1 year to 3 years from diagnosis in CMC.

Complications as a part of disease course or due to treatment were noted, including mucositis, diarrhoea, neuropathy, infections, bladder obstruction and its consequences.

Histopathological assessment of Rhabdomyosarcoma:

Gross features:

The gross features were assessed only for the those cases in which excision specimen was available. Circumscription, cut surface, colour, consistency, necrosis, myxoid areas and margins were studied.

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Microscopic features:

The slides were reviewed by 2 pathologists and the following features were noted.

Pattern:

Sheets, fascicles, trabeculae and cords.

Shape of the tumour cell:

Round, spindle, polygonal and others . Cytoplasm:

Scant, moderate and abundant cytoplasm.

Rhabdomyoblasts:

The presence or absence of rhabdomyoblasts.

Anaplasia:

The tumour was examined thoroughly in all the available sections for anaplasia . Anaplasia was categorized into focal and diffuse anaplasia.

According to Sidhom et al, the presence of anaplasia is histologically defined by the marked nuclear enlargement (3 times that of the neighbouring nuclei), hyperchromacity and presence of multipolar mitotic figures. The term „focal anaplasia‟ was restricted to tumour which showed anaplasia in localized regions and „diffuse anaplasia‟ if clusters of sheets of anaplastic cells were found (40).

Pleomorphism:

Mild, moderate and marked pleomorphism.

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Mitosis:

Mitosis in 10 hpf was counted as follows.

<5/10hpf, 6-9/10hpf and >10/10hpf

The presence or absence of atypical mitotic figures was also noted.

Wreath like giant cells:

Presence of wreath like giant cells, a feature of alveolar rhabdomyosarcoma was noted.

Necrosis:

Present or absent.

Heterologous differentiation:

Heterologous differentiation including bone and cartilage, etc. was noted.

Inflammation:

Mild, moderate and severe.

Subtype:

The tumour was subclassified according to the WHO 2013 as alveolar, embryonal, pleomorphic, spindle cell, unclassifiable. Alveolar was further subclassified into two categories- typical and solid.

Immunohistochemistry:

Technical procedure as described in Appendix-1.

Evaluation of IHC:

The following parameters were assessed.

Dias et al(16) have scored the immunostaining for Desmin and Myogenin as follows:

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0 if all tumour cells are negative,

+ if 1-10% of the cells show moderate positivity, ++ if 10-50% of the cells show moderate positivity and

+++ if more than 50% of the cells show strong Myogenin positivity(16)

In our study we used this scoring system, with a slight modification and considered weak staining for Myogenin and Desmin also as 0.

IHC for Desmin and Myogenin was performed on 65 small biopsies.

Of the 21 excision specimens, IHC was done for Desmin (15 cases) and Myogenin (13 cases) and compared with their prechemotherapy scoring.

Molecular assessment of translocation:

Procedure as described in Appendix- 2.

The presence or absence of PAX3/7-FOXO1 translocation was identified using PCR for all those cases classified as alveolar and unclassifiable subtypes.

PCR was done for both PAX 3/7-FOXO1 translocation in all 27 cases of alveolar and unclassifiable RMS. PAX3/7-FOXO1 gene translocations was amplified using primer as in Appendix 2. The control used was human cell line ATCC-CRL-2061 SJCRH 30 (RMS 30) homo sapiens.

EXCISION SPECIMENS:

For those cases in which excision specimen was available the following histological parameters were analysed.

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Margin status:

Involved or negative and distance of the tumour from the nearest resection margin was noted.

Post chemotherapy changes:

The following postchemotherapeutic changes were looked for.

Foamy macrophages, hyalinization, myxoid change, bizarre tumour cells, fibrosis, necrosis and inflammation.

Chemotherapy in excision specimen was classified as follows.

a) No histologic changes - 2

b) Tumour cell death and reparative changes -16 c) Tumour maturation-6

d) Induction of heterologous differentiation-1 e) Apoptosis -4

Lymphovascular and perineural invasion:

Present or absent.

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Statistical analysis:

The study data were summarized using descriptive statistics. Pearson‟s Chi square test was used to evaluate associations between categorical variables using SPSS software.

In all the statistical analysis a P value of <0.05 was considered to be statistically significant. Survival analysis was done using Kaplan Meier test.

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RESULTS

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A total of 75 cases were reported as rhabdomyosarcoma between January 2012 to March 2016. The archived slides and blocks were retrieved from the pathology records, Department of General Pathology, CMC, Vellore. Of these cases, 71 cases were included in the study, 70 of which were small biopsies, 21 with followup surgical excision also. Two of the 75 cases were eliminated as the excisions were reported as Malignant peripheral nerve sheath tumour with rhabdomyoblastic differentiation (Malignant Triton tumour). Two more cases were eliminated because both slides and blocks were not available. One case had only excision specimen without small biopsy.

This case was still included. The clinical and morphological features were analysed for all 71 cases and immunohistochemical features were analysed only for 65 cases because the immunohistochemistry slides/blocks could not be retrieved. 26 biopsies which were subclassified as alveolar and 9 cases which fell into the unclassified category were planned for translocation analysis. Of these 35 cases, molecular study was feasible only on 27 cases, as in 2 cases, blocks could not be retrieved/ handed over to the patient and 1 case was slide for review. Blocks with scanty tumour(5) were also not subjected to molecular study. (Fig 35)

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75 cases of RMS

71 cases

70 small biopsies 1 excision (Including 20 excision)

Study group (71)

Embryonal Alveolar Unclassifiable Pleomorphic Sclerosing Spindle (26) (26) (9) (6) (2) (2)

Translocation analysis (35)

Translocation analysis (27) Fig 35: Selection of cases included under study.

2- Blocks not available.

1-Slide for review only 5- Too scanty

2- Malignant Triton tumour 2- Slide and block not available

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AGE:

The median age at diagnosis for all cases of rhabdomyosarcoma was 18 years (2months-65 years) with a standard deviation of 17.15. The youngest patient was 2 months old and the oldest patient was 65 years old.

0-10 years: 34 (48.57%) 11-19 years: 15 (21.43%) 20-44 years: 12 (17.14%) 45-64: 8(11.43%)

65-84: 1(1.43%). (Fig 36)

Details of age was not provided for one case.

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Figure 36: Number of cases in each age group

The most common age group involved was children less than 10 years of age.

Embryonal was most common in <10 years (21/34 cases) Alveolar was most common in 11-19 years (11/15 cases)

Pleomorphic rhabdomyosarcoma was common in 45-64 years (6/6 cases). (Fig 37)

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Fig 37: Different histological subtypes in accordance with the age.

GENDER:

Of the 71 cases, 48 cases were males, 23 were females.

The distribution of cases showed a male preponderance. (Fig.38)

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Figure 38: Sex distribution of rhabdomyosarcoma Alveolar subtype was common in females.

Embryonal subtype was common in males.

CLINICAL FEATURES:

Most of the patients presented with a palpable mass. A minority of patients presented with abdominal pain, aural/nasal polyp, diplopia, proptosis, lower urinary tract symptoms (LUTS), lymphnode enlargement, etc. (Table 4)

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Table 4: Clinical presentation of rhabdomyosarcoma

CLINICAL PRESENTATION NUMBER OF PATIENTS (n=70)

Mass 47

LUTS*, urinary retention 8

Lymphnode enlargement 5

Pain 3

Polyp 2

Others 6

*LUTS (Lower urinary tract symptoms) SITE:

The most common site involved was the head and neck (20) including the parameningeal region (Table 5). Of the 20 cases involving the head and neck, 10 cases involved the parameningeal region.(Fig 39)

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Table 5: Site predilection of rhabdomyosarcoma

SITE INVOLVED NUMBER OF CASES(n=70)

Head&neck* 20

Pelvis 15

Extremities 13

Abdomen including retroperitoneum 9

Metastatic sites 7

Others* 6

Head and neck* includes the parameningeal region.

Parameningeal region includes infratemporal fossa, middle ear, mastoid, nasal cavity, nasopharynx, paranasal sinuses, pterygopalatine fossa and the parapharyngeal region.

Others* includes endometrium, chestwall and paratesticular region.

Figure 39: Site distribution of rhabdomyosarcoma

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Alveolar subtype most commonly involved the extremities.

Embryonal subtype most commonly involved the pelvis, followed by the parameningeal region.

The most common parameningeal region to be involved was nasal cavity and the nasopharyngeal region.

SIZE:

The tumour size could not be assessed in 17 of 71 cases, since 8 cases were slides and/or blocks sent to our institution for review; in 8 cases the size of the tumour was not mentioned (only radiological extent was mentioned) and 1 case was outside tissue with no details of size.

Of the remaining 54 cases, 16 cases were <5cm, 19cases were 5-9cm, 14 cases were 10- 14cm and 5 cases were 15-25cm. ( Fig 40)

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Figure 40: Size distribution of rhabdomyosarcoma HISTOLOGY ON SMALL BIOPSIES:

The 70 small biopsies cases were classified into one of the following histological types according to the WHO criteria - Alveolar, embryonal, pleomorphic, spindle cell, sclerosing and unclassifiable. The alveolar type was further subtyped as the typical and the solid variant.

Embryonal - 26 Alveolar, typical - 17

Alveolar, solid - 8 Unclassifiable - 9

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Pleomorphic - 6 Spindle cell - 2

Sclerosing - 2 (Fig 41)

Fig 41: Different subtypes of rhabdomyosarcoma

In pediatric age group, the most common subtype was embryonal (48.92%) followed by alveolar (40.82%) and unclassifiable (8.16%) whereas in adults 28.57% were alveolar subtype, 9.52% were embryonal, 28.57% were pleomorphic and 23.81% were unclassifiable. (Fig 42)

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Figure 42: Histological subtypes of RMS in pediatric age group and adults.

PATTERN:

Patterns of tumour cell arrangement seen were sheets, fascicles, alveolar pattern, trabeculae, cords and nests. (Fig 43)

Sheets - 43 Alveolar -1 Fascicles - 1 Trabeculae – 1 Cords - 5

Sheets and alveolar pattern – 5

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Nests – 1 Mixed – 13

The tumour cells were mostly arranged in sheets.

Figure 43: Distribution of different patterns in rhabdomyosarcoma SHAPE OF THE CELLS:

The cells were round in 44 of 70 cases, spindle in 18 of 70 cases and polygonal in 3 of 70 cases and 5 cases had more than one shape of the cell.

Most of the cells in rhabdomyosarcoma were round in shape. (Fig 44)

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Figure 44: Distribution of different shapes in rhabdomyosarcoma CYTOPLASM:

The cytoplasm was scant in 32 of 70 cases, moderate in 35 of 70 cases and abundant in 3 of 70cases.

CAMBIUM LAYER:

Cambium layer was present in 2 of 26 embryonal rhabdomyosarcoma. This feature was not noted in other histological types.

DEGREE OF PLEOMORPHISM:

The degree of pleomorphism was classified as mild, moderate and severe.

Mild pleomorphism – 5

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Moderate pleomorphism– 58 Severe pleomorphism - 7 ANAPLASIA:

Anaplasia was seen only in the embryonal subtype.

Focal – 4 Diffuse – 1

MITOTIC ACTIVITY:

The mitotic activity was

<5/10hpf - 52cases 6-9/10hpf - 13cases

>10/10hpf - 5 cases

Majority of the cases had mitotic activity less than 5/10 hpf.

ATYPICAL MITOSIS:

Atypical mitosis was present in 11 cases.

Of the 11 cases which showed atypical mitosis, 3 cases belonged to alveolar subtype, 4 cases were embryonal, 3 were pleomorphic and 1 was spindle cell subtype (Fig 45).

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Fig 45: Tripolar mitosis in pleomorphic RMS (H&E at 200x) APOPTOSIS:

Apoptosis was noted in only 41 cases, mostly in the alveolar subtype.

WREATH LIKE GIANT CELLS:

Tumour giant cells were seen in 5 cases. One alveolar subtype had wreath like giant cells.

NECROSIS:

Necrosis was present in 20 of 71 cases.

HETEROLOGOUS DIFFERENTIATION:

Heterologous osseous differentiation was not identified in any case.

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INFLAMMATION:

Most cases did not show inflammation(48). Mild inflammation was seen in 16 cases and inflammation was moderate in 6 cases.

STROMAL FEATURES IN SMALL BIOPSIES:

Stromal features observed in the small biopsies were hyalinisation, sclerosis, myxoid change and fibrosis. 10 cases had history of prior chemotherapy and showed hyalinization in 4 cases, fibrosis in 4 cases and sclerosis in 2 cases.

IMMUNOHISTOCHEMISTRY:

Immunohistochemistry for Desmin and myogenin were analysed in 65 cases., with variable expression as described below. IHC was not available in the remaining 5 cases as the blocks were not available.

Table 6: IHC expression of Desmin in rhabdomyosarcoma

IHC expression Number of cases

0 1

1+ 7

2+ 15

3+ 42

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There was no difference in Desmin expression amongst the different histological subtypes.

Table 7: IHC expression of Myogenin in rhabdomyosarcoma

IHC expression Number of cases

0 2

1+ 14

2+ 14

3+ 35

Of the 14 cases which expressed 2+ for myogenin 2 were of the alveolar subtype. Of the 35 cases which expressed 3+ for myogenin, 19 were alveolar. Thus, Myogenin was strongly expressed in alveolar subtype compared to the other subtypes.

IHC expression in postchemotherapy excision specimen:

IHC for Desmin:

Out of 15 postchemo excision specimen cases, 7 cases showed (0,1+,2+) positivity and 8 cases shows (3+) positivity.

IHC for Myogenin:

Out of 13 postchemo excision specimen cases, 11 cases showed (0,1+,2+) positivity and 2 cases shows (3+) positivity.

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BONE MARROW BIOPSY:

Bone marrow biopsy was done for 29 cases, 23 of which were bilateral. Only 4 cases showed bone marrow involvement, all of which had undergone bilateral biopsies. Two of the cases with bone marrow metastasis were of the embryonal subtype and two were alveolar subtype.

TNM LAWRENCE/GEHAN PRETREATMENT STAGING CLASSIFICATION FOR THE INTERGROUP RHABDOMYOSARCOMA STUDY (IRS) IV:

Staging for 70 small biopsy cases is as follows.

Stage 1 - 8 (11.27%) Stage 2 - 9 (12.68%) Stage 3 - 12 (16.90%) Stage 4 - 25 (35.21%)

In 17 cases, staging couldnot be done because the tumour size, nodal status or the metastatic details were not available. (Fig 46)

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Figure 46: Distribution of staging in rhabdomyosarcoma Most of the cases belonged to Stage 4.

Of the 25 cases who belonged to Stage 4, 4 cases were Stage 4 at the time of initial presentation . Remaining 21 cases developed metastasis during the course of the disease.

METASTASIS:

Metastasis was present in 28 of 71 cases, of which 9 had multiple metastatic sites. The most common site of metastasis was lymphnode followed by lung. (Table 8)

Locoregional metastasis is defined as metastasis to the regional lymphnodes. Metastasis to any other site other than regional lymphnodes is called distant metastasis. The frequency of locoregional and distant metastasis in our study is depicted in Fig 47.

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Alveolar rhabdomyosarcoma was the most common histological subtype to metastasize and mostly to the lymphnode.

Metastasis was proved by FNAC in 2 cases, biopsy in 10 cases, radiology in 11 cases and more than one of these modalities in 5cases.

Table 8: Distribution of metastatic sites in rhabdomyosarcoma

Site Number of cases

Lymphnode 16

Lung 7

Osseous metastasis 4

Miscellaneous sites 5

Bone marrow 4

Breast 1

Liver 2

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Figure 47: Type of metastasis in rhabdomyosarcoma RECURRENCE:

Recurrence at the same site was present in 12 of the 71 cases, 5 of which had also metastasized to other sites.

Recurrence was most common in alveolar subtype.

COMPLICATIONS:

The most common complication was infection and obstructive uropathy.

Obstructive uropathy - 5 Infection - 5

Febrile neutropenia - 4

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Neuropathy - 4 Miscellaneous - 3

More than one complication was seen in 6 cases.

FOLLOWUP:

The patients in the study were divided into 4 groups, depending on duration of follow up.

Group I - <6 months =17

Group II - 6months to 1 year= 13 Group III- >1 yr =9 (4=1-2yr, 5=>2yr)

Group IV- Less than 1 month/ left after diagnosis = 32 TREATMENT STATUS:

Treatment status was assessed for 39 patients who were followed up for a period ranging from 1month to 2 years. The group IV patients with a followup of <1 month were not included in assessing the status.

A total of 15 of these 39 patients whose treatment status was assessed, had already undergone chemotherapy elsewhere at the time of initial diagnosis in our institution.

The treatment status of the 39 cases is as follows

References

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