A LVEOLAR R HABDOMYOSARCOMA Katikaneni Sai Shreya Gr. 7

I NTRODUCTION Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The name is derived from the Greek words rhabdo, which means rod shape, and myo, which means muscle. the tumor is believed to arise from primitive muscle cells.

Skeletal muscles begin to form in embryos at about 7 weeks into early embryonic development. At that time, rhabdomyoblasts (cells that will eventually form muscles) begin to form. It is these cells that can develop into the cancer called rhabdomyosarcoma.

S TATISTICS Frequency- The incidence is 6 cases per 1,000,000 population per year (approximately 250 cases) in children and adolescents younger than 15 years. Because RMS is uncommon before 1 year of age and seldom seen in newborns, it is rarely detected by fetal screening. The Intergroup Rhabdomyosarcoma Study reported that 0.4% of infants presented with RMS in the first 30 days of life. Only 6 case reports on prenatal detection of RMS have been documented.

Mortality/Morbidity-In an analysis of data collected by the Surveillance, Epidemiology, and End Results (SEER) program, mortality was highly related to age, site, and histology. The 5-year survival was highest in children aged 1-4 years (77%) and was worst in infants and adolescents (47% and 48%, respectively). Orbital and GU sites were the most favorable (86% and 80%, respectively). Unfavorable sites included tumors of the extremities (50%), retroperitoneum (52%), and trunk (52%). Age- 87% of patients are younger than 15 years, and 13% of patients are aged years.

I NTRODUCTION Weber first described rhabdomyosarcoma in 1854, a clear histologic definition was not available until 1946, when Stout recognized the distinct morphology of rhabdomyoblasts. Stout described rhabdomyoblasts as appearing in round, strap, racquet, and spider forms. RMS histological sections have been found to resemble normal fetal skeletal muscle, and, as such, RMS is a member of the small blue round cell tumors of childhood. The Helman grouphas reported that the incidence of RMS between male groups is roughly equivalent, however the incidence of RMS in African American females is roughly half of that which is visible in Caucasian females. In contrast, a male preponderance in the generation of RMS with the disease being roughly 1.4 times more common in males than in females has been reported by the same group.

Common to all isoforms of RMS are certain morphological and biochemical characteristics which delineate it as being a muscle-derived cancer, such as morphological similarities to skeletal muscle in cross-striations, and positive immunohistochemical staining for muscle specific markers such as α-actin, myosin heavy chain, myoglobin, Z-band protein and the myogenic regulatory factor (MRF), MyoD. In addition to these immunohistochemical tests, RMS tumors stain positively for proteins not specific for muscle, such as desmin and the intermediate filament protein vimentin.

A NAMNESIS Rhabdomyosarcoma (RMS) usually manifests as an expanding mass; symptoms depend on the location of the tumor. Pain may be present. If metastatic disease is present, symptoms of bone pain, respiratory difficulty (secondary to lung nodules or to pleural effusion), anemia, thrombocytopenia, and neutropenia may be present. Disseminated rhabdomyoblasts in the bone marrow may mimic leukemia, both in symptoms and light microscopic findings. Typical presentations by the location of nonmetastatic disease are : Orbit - Proptosis or dysconjugate gaze Paratesticular - Painless scrotal mass; Prostate - Bladder or bowel difficulties Uterus, cervix, bladder - Menorrhagia or metrorrhagia Vagina - Protruding polypoid mass (botryoid, meaning a grapelike cluster) Extremity - Painless mass Parameningeal (ear, mastoid, nasal cavity, paranasal sinuses, infratemporal fossa, pterygopalatine fossa) - Upper respiratory symptoms or pain Physical findings depend on the location of the tumor. Tumors in superficial locations may be palpable and detected relatively early, but those in deep locations (eg, retroperitoneum) may grow large before causing symptoms.

R ISK F ACTORS The cause of rhabdomyosarcoma is unclear. Several genetic syndromes and environmental factors are associated with increased prevalence of rhabdomyosarcoma. Genetic syndromes include the following: Neurofibromatosis (4-5% risk of any one of numerous malignancies)- an autosomal dominant disorder that affects the bone, the nervous system, soft tissue, and the skin Li-Fraumeni syndrome (germline mutation of the tumor suppressor gene TP53 )- the autosomal dominantly inherited disorder characterized by the prevalence of soft tissue and bone sarcomas Rubinstein-Taybi syndrome- a well-delineated malformation syndrome characterized by facial abnormalities, broad thumbs, broad great toes, short stature, and mental retardation Gorlin basal cell nevus syndrome- an autosomal dominant cancer; develop multiple neoplasms, including basal cell carcinomas and medulloblastoma Beckwith-Wiedemann syndrome - combination of congenital exomphalos, macroglossia, and gigantism Costello syndrome- genetic disorder characterized by delayed development and mental retardation, distinctive facial features, loose folds of extra skin (especially on the hands and feet), and unusually flexible joints.

A higher prevalence of congenital anomalies are observed in patients who later develop rhabdomyosarcoma with locations as follows: Genitourinary (GU) tract CNS (ie, Arnold-Chiari malformation) GI tract Cardiovascular system Environmental factors appear to influence the development of rhabdomyosarcoma, as follows: Parental use of marijuana and cocaine Intrauterine exposure to X-rays Previous exposure to alkylating agents

L ABORATORY S TUDIES The following studies are indicated in rhabdomyosarcoma: CBC count : Anemia may be present because of inflammation, or pancytopenia may be present from bone marrow involvement. Liver function tests, including measurement of lactic acid dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and bilirubin levels: Metastatic disease of the liver may affect values of these proteins. Liver function must be assessed before chemotherapy. Renal function tests, including measurements of BUN and creatinine levels: Renal function must be assessed before chemotherapy. Urinalysis (UA): Hematuria may indicate involvement of the genitourinary (GU) tract. Blood electrolyte and biochemical analysis, including evaluation of sodium, potassium, chlorine, carbon dioxide, calcium, phosphorous, and albumin values: Assess for abnormalities before chemotherapy.

I MAGING S TUDIES Plain radiography : Radiography of the primary site and of the chest is helpful in determining the presence of calcifications and bone involvement of the primary tumor and to search for metastatic lung lesions. CT scanning Obtain a chest CT scan to evaluate for metastases to the lungs. Chest CT scanning is best performed before surgery to avoid atelectasis, which can be confused with metastasis. A CT scan of the primary site may also be helpful in evaluating for bone erosion, if present, and to follow up the patient's response to therapy. Obtain a CT scan of the liver in patients with abdominal or pelvic primary tumors to assess for metastatic spread. Ultrasonography is an alternative. MRI: MRI improves definition of the mass and its invasion of adjacent organs, especially in orbital, paraspinal, or parameningeal regions. Obtain an MRI of the head if the patient is symptomatic at diagnosis. Bone scanning : Search for metastases to the bones. Ultrasonography : Obtain sonograms of the liver in patients with abdominal or pelvic tumors. CT scanning is an alternative. Echocardiography : Assess cardiac function before chemotherapy.

P ROCEDURES Biopsy : Open biopsy best enables adequate tissue sampling for diagnosis and molecular studies. Core needle biopsy is an alternative. Depending on the location, definitive surgery can be postponed to allow for neo-adjuvant chemotherapy to shrink the tumor. Cytogenetics, Fluorescent in situ Hybridization (FisH) This study helps in determining if the translocations t(1;13) or t(2;13), which are associated with the alveolar subtype, are present. FISH also helps in the differential diagnosis if a translocation characteristic of another tumor type is present. FISH is most sensitive for these translocations and can be helpful in evaluating residual disease. Reverse transcriptase–polymerase chain reaction (RT-PCR) When cytogenetic testing is unavailable (eg, culture failure) or when its results are uninformative, order a RT-PCR reaction to assess for the characteristic translocations associated with alveolar rhabdomyosarcoma (ARMS) and other small, round blue-cell tumors of childhood. In many centers, the use of RT-PCR to screen for a panel of translocations associated with soft tissue sarcomas is becoming a routine adjunct to morphologic analysis to help ascertain the diagnosis. Bone marrow aspiration and biopsy : Assess for metastatic spread to bone marrow.

T HEORIES OF P ATHOGENESIS

I NTERRUPTED M YOGENESIS : Sarcomas are derived from mesenchymal, or loose connective tissue. Where exactly rhabdomyosarcomas are derived from is currently unknown, however, the morphology of transformed RMS cell lines appears to suggest that they are derived from an interruption in normal skeletal muscle development, and it has been proposed that RMS tumors are derived from satellite cells. The Shipley group has postulated that the cell from which RMS tumors originate could be either an uncommited mesenchymal stem cell, like a fibroblast, or a myogenic precursor. It is understood that skeletal muscle is derived from somites, mesodermal condensations which may differentiate into the dermomyotome and myotome. Migrating populations of cells from the dermomyotome develop into the skeletal muscle which populates the limbs, while a second nonmigrating population from the lateral dermomyotome develops into the axial skeletal muscles of the chest.

Studies in mice have shown at embryonic day 10, PAX proteins, such as PAX3 and PAX7 are detected in the somite, under the influence of proteins produced by the neural tube and notochord, such as sonic hedgehog (SHH) and WNT-1. Proliferation of dermomyotomal cells does not occur until PAX levels fall. Experimentally, homozygous deletion of PAX3, conducted in murine models produced a genetic model with no apparent limb musculature. This model is termed splotch, and arises as a result of a failure of the myogenic precursor cells to migrate from the lateral dermomyotome to the limbs. Similarly, during development, the process of myogenic determination is characterized by the sequential expression of members of the myogenic regulatory factor (MRF) family of proteins, such as MyoD, myogenin and MYF5. MYF5 null mice, as well as MyoD null mice, both develop skeletal muscle, however double mutants in PAX3 and MYF5 do not develop skeletal muscle. This may suggest a MYF5-driven pathway of myogenesis which is PAX3 and MyoD independent. Rhabdomyosarcomas display normal expression of PAX3 and PAX7 genes, as well as MRF family members. Mutation or deletion of signaling intermediates in the SHH-WNT-1-PAX3 pathway to myogenesis display an overinduction of IGF2, which appears to suggest a link between aberrant and disrupted myogenesis and the insulin-like growth factor 2 pathway.

H ISTOLOGICAL T YPES embryonal rhabdomyosarcoma (ERMS), which occurs in 55% of patients; the botryoid variant of ERMS, which occurs in 5% of patients; alveolar rhabdomyosarcoma (ARMS), which occurs in 20% of patients; and undifferentiated sarcoma (UDS), which occurs in 20% of patients.

Although the tumor is believed to arise from primitive muscle cells, tumors can occur anywhere in the body except bone. The most common sites are the head and neck (28%), extremities (24%), and genitourinary (GU) tract (18%). Other notable sites include the trunk (11%), orbit (7%), and retroperitoneum (6%). Rhabdomyosarcoma occurs at other sites in less than 3% of patients. The botryoid variant of ERMS arises in mucosal cavities, such as the bladder, vagina, nasopharynx, and middle ear. Lesions in the extremities are most likely to have an alveolar type of histology. Metastases are found predominantly in the lungs, bone marrow, bones, lymph nodes, breasts, and brain.

A LVEOLAR T YPE – CAUSE / PATHOGENESIS The alveolar variant is so named because of the thin criss- crossing fibrous bands that appear as spaces between cellular regions of the tumor (reminiscent of lung alveoli). This variant is usually associated with 1 of 2 chromosomal translocations, namely, t(2;13) or t(1;13). These result in the fusion of the DNA-binding domain of the neuromuscular developmental transcription factors, encoded by PAX3 on chromosome 2 or PAX7 on chromosome 1. The transcriptional activation domain of a relatively ubiquitous transcription factor, FKHR (or FOXO1a), is encoded on chromosome 13. The resulting hybrid molecule is a potent transcription activator. It is believed to contribute to the cancerous phenotype by abnormally activating or repressing other genes.

A LVEOLAR RHABDOMYOSARCOMA Alveolar rhabdomyosarcoma (ARMS)occurs more often in large muscles of the trunk, arms, and legs and typically affects older children or teenagers (10-25 yrs). ARMS cells look like the normal muscle cells seen in a 10-week-old fetus.

P ATHOLOGICAL FEATURES Alveolar pattern - The tumour consists of aggregates of undifferentiated tumour cells arranged in a alveolar pattern. Eosinophilic rhabdomyoblasts and giant cells with peripheral wreath- like nuclei are prominent. The cellular aggregates are separated by dense hyalinized fibrous septa. The tumour cells at the periphery are well preserved. The cells at the centre of the alveolar spaces are loosely arranged and poorly preserved. Cross striations are noted in only 10% cells. Solid pattern - Solid packed masses of tumour cells resembling round cell areas of embryonal rhabdomyosarcoma (may resemble carcinoma or malignant melanoma). A microalveolar pattern has also been described.

I MMUNOHISTOCHEMISTRY Demonstrate strong and widespread myogenin expression compared to embryonal type. The majority of the alveolar rhabdomyosarcomas are associated with specific chromosomal translocations (pARMS). Because alveolar variant is much more aggressive than embryonal variant, RMS subclassification has clinical relevance.

HISTO PATHOLOGY OF ALVEOLAR RHABDOMYOSARCOMA

Rearrangement in the chromosome material between chromosomes #2 and #13 is usually present. This rearrangement changes the position and function of genes, causing a fusion of genes referred to as a "fusion transcript." Patients have an abnormal fusion transcript involving two genes known as PAX3 and FKHR. This important discovery has led to improvements in diagnosing rhabdomyosarcoma.

S TAGING Staging of rhabdomyosarcoma is relatively complex. The process includes: Assigning a local tumor group (status postsurgical resection/biopsy). Assigning stage (consider site, size, group, presence/absence of metastases). Assigning a risk group (consider stage, group, and histology).

Favorable site Orbit; nonparameningeal head and neck; genitourinary excluding kidney, bladder, and prostate; biliary tract. Unfavorable siteAny site not considered favorable. T1Tumor confined to anatomic site of origin. T2 Tumor extension and/or fixative to surrounding tissue. aTumor 5 cm in maximum diameter. bTumor >5 cm in maximum diameter. N0 No clinical regional lymph node involvement. N1Clinical regional lymph node involvement. NX Regional lymph nodes not examined; no information. M0No metastatic disease. M1Metastatic disease.

GroupDefinition I Approximately 13% of all patients are in this group. A localized tumor that is completely removed with pathologically clear margins and no regional lymph node involvement. II Approximately 20% of all patients are in this group. A localized tumor that is grossly removed with: (A) microscopic disease at the margin, (B) involved, grossly removed regional lymph nodes, or (C) both A and B. III Approximately 48% of all patients are in this group. A localized tumor with gross residual disease after incomplete removal or biopsy only. IV Approximately 18% of all patients are in this group. Distant metastases are present at diagnosis. IRS Group Surgicopathologic Grouping System

Stage Sites of Primary Tumor Tumor Size Regional Lymph Nodes Distant Metastasis 1 Favorable sites Any size N0 or N1 or NX M0 2 Unfavorable sites T1a or T2aN0 or NXM0 3 Unfavorable sites T1a, T2aN1M0 T1b, T2b N0 or N1 or NX 4Any siteAny sizeN0 or N1M1 STS-COG Pretreatment Staging System

Risk GroupHistologyStageGroup Low RiskEmbryonal1I, II, III Embryonal2, 3 I, II Intermediate Risk Embryonal2, 3III Alveolar1, 2, 3 I, II, III High Risk Embryonal or Alveolar 4IV IRSG Rhabdomyosarcoma Risk Group Classification

A UTOPSY CASE A 24-yr old woman, G3P1L0-2 miscarriages, is 27 wk pregnant with breech presentation, malformation of fetus, tumor of left shoulder with fast growing tumor & vascularisation. H/o- chronic cystitis, nephroptosis, neuro-circulatory dystonia (hypotonic type) During the pregnancy, I trimester- she had complaints of hypercoagulation and was treated with Fraxiparin 0.3 SC hyperandrogeny and was given methyprednisolone 20mg/ml Inj. II trimester- in Ultrasonography diagnosed malformation of fetus, tumor on left shoulder with fast growing & vascularisation. At th week, performed termination of pregnancy. Biopsy done- abrasio cavi uterii

Macroscopic Findings tumor d=6.5 cm, soft consistency. section has fish meat appearance. Tumor has diversity of colors because of co- existence of light gray yellow & bluish focuses.

The tumour consists of aggregates of undifferentiated tumour cells arranged in a alveolar pattern.

Eosinophilic rhabdomyoblasts and giant cells with peripheral wreath- like nuclei are prominent.

M ETHODS OF T REATMENT surgery resection of tumor and/or metastases chemotherapy radiation therapy lumbar puncture (to administer medication and treat cancer cells if found in this site) supportive care (for the side effects of treatment) antibiotics (to prevent/treat infections) bone marrow transplant continuous follow-up care

Standard therapy for rhabdomyosarcoma (RMS) includes chemotherapy combined with surgical resection, radiotherapy, or both for local control, if necessary.

A NTI - NEOPLASTIC T HERAPY Vincristine (Oncovin) Inhibits tubulin polymerization, targeting dividing cells. Acts as vesicant. Adult- 2 mg IV slow push into central venous catheter or fresh IV line; acts as vesicant Pediatric- 1.5 mg/m 2 IV q1-3wk; not to exceed 2 mg/dose Dactinomycin (Cosmegen, actinomycin D) Antibiotic derived from Streptomyces bacteria. Adult mg/d IV push for 5 d Pediatric mg/kg/d IV push for 5 d or 1.5 mg IV push q3wk

Cyclophosphamide (Cytoxan) Alkylating agent believed to be cytotoxic to dividing cells by cross-linking cellular DNA. Processed in liver to active metabolites. Byproducts (eg, acrolein) accumulate in bladder and cause cystitis. Adult mg/m 2 /d PO for 5 d or g/m 2 IV q3- 4wk Pediatric g/m 2 /d IV for 1-3 d Etoposide (Toposar, VP16) Inhibits topoisomerase II and therefore toxic to cells undergoing DNA replication. Adult mg/m 2 /d IV for 5 d PO dose: 2 times IV dose rounded to nearest 50 mg Pediatric- 100 mg/m 2 /d IV for 5 d

Uroprotective antidote Mesna is a prophylactic detoxifying agent used to inhibit hemorrhagic cystitis caused by ifosfamide and cyclophosphamide Adult- 20% of ifosfamide dose IV (weight of solute per weight of solvent [w/w]); dosage depends on ifosfamide or cyclophosphamide; Pediatric mg/m 2 PO/IV q3h before and after cyclophosphamide or ifosfamide dose Colony-stimulating factors Filgrastim (Neupogen) Granulocyte colony-stimulating factor (G-CSF) that activates and stimulates production, maturation, migration, and cytotoxicity of neutrophils. Better tolerated than alternative GM-CSF. Adult mcg/kg/d IV/SC Pediatric- Administer as in adults

O UTLOOK Prognosis greatly depends on : i. the extent of the disease. ii. the size and location of the tumor. iii. presence or absence of metastasis. iv. the tumor's response to therapy. v. the age and overall health of your child. vi. your child's tolerance of specific medications, procedures, or therapies. vii. new developments in treatment.

R ESEARCH & C LINICAL T RIALS

G ENE EXPRESSION PROFILING OF ALVEOLAR RHABDOMYOSARCOMA WITH C DNA MICROARRAYS C ANCER G ENETICS B RANCH, N ATIONAL H UMAN G ENOME R ESEARCH I NSTITUTE, NIH, USA cDNA microarrays containing 1238 cDNAs were used to investigate the gene expression profile of a group of seven alveolar rhabdomyosarcoma (ARMS) cell lines characterized by the presence of the PAX3-FKHR fusion gene. Using the method of multidimensional scaling to represent the relationships among the cell lines in two-dimensional Euclidean space, it was determined that ARMS cells show a consistent pattern of gene expression, which allows the cells to be clustered together. By searching across the seven ARMS cell lines, it was found that 37 of 1238 genes were most consistently expressed in ARMS relative to a reference cell line. Only three of these genes have been previously reported to be expressed in ARMS. Among these 37 were genes related to both primary (PAX3-FKHR) and secondary (CDK4) genetic alterations in ARMS. These results in ARMS demonstrate the potential of cDNA microarray technology to elucidate tumor-specific gene expression profiles in human cancers.

G ENE FUSIONS INVOLVING PAX AND FOX FAMILY MEMBERS IN ALVEOLAR RHABDOMYOSARCOMA D EPARTMENT OF P ATHOLOGY AND L ABORATORY M EDICINE, U NIVERSITY OF P ENNSYLVANIA S CHOOL OF M EDICINE, USA. The chromosomal translocations t(2;13)(q35;q14) and t(1;13)(p36;q14) are characteristic of alveolar rhabdomyosarcoma, a pediatric soft tissue cancer related to the striated muscle lineage. These translocations rearrange PAX3 and PAX7, members of the paired box transcription factor family, and juxtapose these genes with FKHR, a member of the fork head transcription factor family. This juxtaposition generates PAX3-FKHR and PAX7-FKHR chimeric genes that are expressed as chimeric transcripts that encode chimeric proteins. The fusion proteins, which contain the PAX3/PAX7 DNA binding domain and the FKHR transcriptional activation domain, activate transcription from PAX-binding sites with higher potency than the corresponding wild-type PAX proteins. This increased function results from the insensitivity of the FKHR activation domain to inhibitory effects of N-terminal PAX3/PAX7 domains. In addition to altered function, the fusion products are expressed in ARMS tumors at higher levels than the corresponding wild-type PAX products due to two distinct mechanisms. The PAX7-FKHR fusion is overexpressed as a result of in vivo amplification while the PAX3-FKHR fusion is overexpressed due to a copy number-independent increase in transcriptional rate. Finally, though FKHR subcellular localization is regulated by an AKT-dependent pathway, the fusion proteins are resistant to these signals and show exclusively nuclear localization. Therefore, these translocations alter biological activity at the levels of protein function, gene expression, and subcellular localization with the cumulative outcome postulated to be aberrant regulation of PAX3/PAX7 target genes. This aberrant gene expression program is then hypothesized to contribute to tumorigenic behavior by impacting on the control of growth, apoptosis, differentiation and motility.

Gene silencing of the PAX gene & FKHR gene by altering the nuclear membrane reactivity and permeability, applying methodology of Differential Integration of Biological Systems (developed by Polisetty et.al.)

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