Why are ancillary techniques essential in round cell tumors (RCTs)?
l RCTs are a group of highly undifferentiated neoplasm’s composed of monotonous population of round cells with high N:C ratio. The histological similarity and lack of differentiating features on hematoxylin and eosin sections in most of them makes definite diagnosis on H&E difficult, and requires the additional support of immunohistochemical and molecular studies.

l Advent of newer relatively specific treatment protocols in pediatric oncology means that Pathologist must accurately classify a RCT and he /she can no longer give a diagnosis of “Small blue /round cell tumor” on histology.

l These tumors have propensity for odd presentations and disseminated metastases and hence require additional techniques for diagnosis.

E.g. the diagnosis of an abdominal desmoplastic round cell tumor (DSRCT) can be made on histology but a similar tumor at an odd site like bone requires molecular confirmation of translocation.

l Accurate diagnosis is also important to understand biology and natural history

What is the gold standard?
Light microscopic histology is still the main stay in diagnosis
l Some tumors that can be recognized on H&E without ancillary techniques. Thus histology in right clinical setting may be used to make a light microscopy diagnosis e.g. of tumors that can be recognized on H&E
a) Lymphoblastic lymphoma and Burkitt’s lymphoma have classic cellular features that make identification on histology and FNAC easy.

b) Embryonal rhabdomyosarcoma with strap cells confirming skeletal muscle differentiation

c) Differentiated neuroblastoma

l Preliminary histology also guides on application of ancillary techniques hence without familiarity with usual histology, diagnosis will be hampered.

l Around 10% RCTs cannot be classified inspite of ancillary techniques and in this setting one has to resort to a likely diagnosis on H&E

Type of ancillary technique
The need for accurate diagnosis of RCTs has ushered an new era of newer & expensive technology for accurate diagnosis. In 80-90% of cases diagnosis can be made on H&E coupled with IHC. However in 15-20% there is a need for molecular or electron microscopic diagnosis.

a) Immunohistochemistry (IHC)
Amongst all the ancillary techniques IHC has gained maximum popularity.
Advantages of IHC are
l Application to paraffin fixed tissue.

l Wide range of detection and is applicable to most tumors.

l Relatively easy to establish and hence most laboratories can use this method.

l Most cost effective amongst the ancillary techniques.

Disadvantages of IHC
l Infidelity of markers – a good example is the MIC2 antigen which was initially thought to be specific for EWS-PNET but now is recorded in many tumors like osteosarcoma, mesenchymal chondrosarcoma with widely different treatment or chemotherapy options. This is where sound histology diagnosis, molecular diagnosis, clinical and X-ray picture will help.

l Aberrant expression of markers – Sometimes tumors express unusual markers e.g. RMS expresses LCA, desmin and cytokeratins are seen in EWS-PNET group of tumors.
l Undifferentiated tumors – These tumors often have no markers and even lack histological clues to the diagnosis.

l Forming a differential diagnosis on histopathology is an important prerequisite so that a suitable IHC panel can be designed and requested.

l Need for strict quality control

b) Electron microscopy
The wider application of immunocytochemistry for tumour diagnosis is endorsed, but electron microscopy should be retained for selected cases in which the results of immunocytochemistry might be predictably ambiguous or otherwise unhelpful.

Advantages of EM
l Rhabdomyosarcomas – In tumors with aberrant muscle markers or when only Desmin is positive on IHC, the typical skeletal muscle differentiation on EM helps in diagnosis.

l Unsuspected diagnosis - Ensuring that the sample is representative, it is possible to find pointers towards an unsuspected diagnosis on ultrastructure (in contrast to IHC).

l Specific examples - to document neuroendocrine or melanocytic differentiation in poorly differentiated tumors (better than IHC).

Disadvantages - Not in common use, because;
l Need for expertise in interpretation: EM reporting requires relatively greater interpretational skills, training and experience of pathologists.
l Cost factor
l Sampling – the tissue studied is limited and may be non-representative.

c) Molecular techniques
Advantages and proven uses -

1. Undifferentiated or difficult cases -Molecular diagnostic studies most definitely demonstrate their merit when they clarify problematic cases. Histology alone may be diagnostically inconclusive in some RCTs leading to conflicting interpretations. In 45-50% of undifferentiated RCT which can not be resolved by IHC or histology, identification of the translocation helps in accurate treatment. See TMH experience below.

2. Improved & refined diagnostics – PCR based testing of MRCT Why are ancillary techniques essential in round cell tumors (RCTs)?

l RCTs are a group of highly undifferentiated neoplasm’s composed of monotonous population of round cells with high N:C ratio. The histological similarity and lack of differentiating features on hematoxylin and eosin sections in most of them makes definite diagnosis on H&E difficult, and requires the additional support of immunohistochemical and molecular studies.
l Advent of newer relatively specific treatment protocols in pediatric oncology means that Pathologist must accurately classify a RCT and he /she can no longer give a diagnosis of “Small blue /round cell tumor” on histology.
l These tumors have propensity for odd presentations and disseminated metastases and hence require additional techniques for diagnosis.
E.g. the diagnosis of an abdominal desmoplastic round cell tumor (DSRCT) can be made on histology but a similar tumor at an odd site like bone requires molecular confirmation of translocation.
l Accurate diagnosis is also important to understand biology and natural history

What is the gold standard?
Light microscopic histology is still the main stay in diagnosis
l Some tumors that can be recognized on H&E without ancillary techniques. Thus histology in right clinical setting may be used to make a light microscopy diagnosis e.g. of tumors that can be recognized on H&E
a) Lymphoblastic lymphoma and Burkitt’s lymphoma have classic cellular features that make identification on histology and FNAC easy.
b) Embryonal rhabdomyosarcoma with strap cells confirming skeletal muscle differentiation
c) Differentiated neuroblastoma

l Preliminary histology also guides on application of ancillary techniques hence without familiarity with usual histology, diagnosis will be hampered.
l Around 10% RCTs cannot be classified inspite of ancillary techniques and in this setting one has to resort to a likely diagnosis on H&E

Type of ancillary technique
The need for accurate diagnosis of RCTs has ushered an new era of newer & expensive technology for accurate diagnosis. In 80-90% of cases diagnosis can be made on H&E coupled with IHC. However in 15-20% there is a need for molecular or electron microscopic diagnosis.

a) Immunohistochemistry (IHC)

Amongst all the ancillary techniques IHC has gained maximum popularity.
Advantages of IHC are
l Application to paraffin fixed tissue.
l Wide range of detection and is applicable to most tumors.
l Relatively easy to establish and hence most laboratories can use this method.
l Most cost effective amongst the ancillary techniques.

Disadvantages of IHC
l Infidelity of markers – a good example is the MIC2 antigen which was initially thought to be specific for EWS-PNET but now is recorded in many tumors like osteosarcoma, mesenchymal chondrosarcoma with widely different treatment or chemotherapy options. This is where sound histology diagnosis, molecular diagnosis, clinical and X-ray picture will help.
l Aberrant expression of markers – Sometimes tumors express unusual markers e.g. RMS expresses LCA, desmin and cytokeratins are seen in EWS-PNET group of tumors.
l Undifferentiated tumors – These tumors often have no markers and even lack histological clues to the diagnosis.
l Forming a differential diagnosis on histopathology is an important prerequisite so that a suitable IHC panel can be designed and requested.
l Need for strict quality control

b) Electron microscopy
The wider application of immunocytochemistry for tumour diagnosis is endorsed, but electron microscopy should be retained for selected cases in which the results of immunocytochemistry might be predictably ambiguous or otherwise unhelpful.

Advantages of EM
l Rhabdomyosarcomas – In tumors with aberrant muscle markers or when only Desmin is positive on IHC, the typical skeletal muscle differentiation on EM helps in diagnosis.

l Unsuspected diagnosis - Ensuring that the sample is representative, it is possible to find pointers towards an unsuspected diagnosis on ultrastructure (in contrast to IHC).

l Specific examples - to document neuroendocrine or melanocytic differentiation in poorly differentiated tumors (better than IHC).

Disadvantages - Not in common use, because;
l Need for expertise in interpretation: EM reporting requires relatively greater interpretational skills, training and experience of pathologists.

l Cost factor

l Sampling – the tissue studied is limited and may be non-representative.

c) Molecular techniques
Advantages and proven uses -

1. Undifferentiated or difficult cases -Molecular diagnostic studies most definitely demonstrate their merit when they clarify problematic cases. Histology alone may be diagnostically inconclusive in some RCTs leading to conflicting interpretations. In 45-50% of undifferentiated RCT which can not be resolved by IHC or histology, identification of the translocation helps in accurate treatment. See TMH experience below.

2. Improved & refined diagnostics – PCR based testing of MRCT provided diagnostically relevant information in 78% of the cases.

3. Scanty material – RTPCR comes in handy when biopsy specimen is small and may not sample the entire histological spectrum e.g. In ARMS the alveolar pattern may not be clear in a small specimen but presence of PAX-FKHR group of translocation helps in recognition.

4. Polyphenotypic tumors – In tumors showing positivity for multiple markers on IHC, such as DSRCT, identification of the translocation can help in accurate classification.

5. Help in understanding tumor biology – Discovery of the EWS-FLI translocation has allowed for EWS/PNET renaissance and for EWS to be lumped together with PNET.

6. Prognostication – Favorable outcome has been observed with detection of EWS-FLI fusion transcript in EWS-PNET group of tumors. Patients with ARMS fusion transcript PAX3-FKHR have increased chances of metastases and failure of treatment, whereas PAX7FKHR imparts relatively favorable prognosis. Like wise Stage IVS neuroblastoma behaves better because it lacks the Chr1p deletion & N-myc amplification.

Disadvantages :
Being widely available studies, emphasizing the RTPCR related diagnostic issues are on record.
1. Type of tissue – Fresh tissue provides the highest frequency of positive cases in comparison to paraffin processed tissue. A difference of 20-40% in positivity is seen between the two.

2. False Negative result – Not all tumors of a particular group of tumors will harbor the translocation e.g. only 90% EWS-PNET have EWS-FLI1 translocation.

3. Product has atypical size – In this set there is a translocation but the product is atypical. DNA sequencing will pick up cases that have fusions transcripts of atypical size but are translocation positive

4. Unexpected results :
– Classical tumor but unusual translocation : Sporadic reports of t(11;22) in OGS, mesenchymal chondrosarcoma & RMS. Some tumors may harbor additional translocations as part of a nonspecific epiphenomenon. Hence sound base of histologic identification is essential to avoid errors.

– False positive : In 6% of cases there will be a typical band but it will be due to nonspecific artifacts (false positive). DNA sequencing may also help resolve the issue.

In case of discrepant diagnosis;
- It is essential to have a reappraisal of all clinical and pathological information.

- Around 10% RCTs cannot be classified inspite of ancillary techniques and in this setting one has to resort to a likely diagnosis on H&E.

- Given a case with discordant diagnosis oncologists often manage patient as per genetic result (if suggestive) and their clinical impression.

Methods available for molecular diagnosis of RCTs
a) Cytogenetics : In this tumor is grown in a tissue culture and analyzed for the chromosomal abnormality. The problem with this technique is that it requires a larger sample than RTPCR and the success rates for tumor growth in culture are low. It is however an excellent screening technique in tumors with unknown chromosomal abnormality, as it does not require any probes etc.

b) RTPCR : In this technique the tumor RNA is extracted amplified and probed with primers which are specific for a particular translocation. This is the most popular technique as it can be applied to small samples and FNA material and also on paraffin processed tissue. Fusion transcript extracted amplified & detected, report may be available in 2-3 days. The only disadvantage is that it can only identify a known translocation.

c) FISH (Fluorescent in situ hybridization): Fluorescent probe is used to highlight the translocations in RCTs. The advantage of this technique is that it can also be performed on paraffin block and the translocation in the tumor cells can be seen on microscopy. But it is definitely more time consuming and success rates are low.

TMH experience
Of a 100 RCTs that we have subjected to RTPCR for translocations in last one year we found molecular methods helped us in

– classifying 3/ 8 undifferentiated round cell tumors which were EWS-FLI positive
– Accurately differentiating two cases where the dilemma was between small cell osteosarcoma and EWS-PNET
– Of 12 cases of Embryonal Rhabdomyosarcoma(ERMS) PAX3FKHR was positive and were reviewed and labeled as ARMS

References :
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2. Dar AUH, Hird PM, Wagner BE, Underwood JCE. Relative usefulness of electron microscopy and immunocytochemistry in tumour diagnosis: 10 years of retrospective analysis. J Clin Pathol 1992;45:693-696

3. de Alava E, Kawai A, Healey JH, Fligman I, Meyers PA, Huvos AG, Gerald WL, Jhanwar SC, Argani P, Antonescu CR, Pardo-Mindan FJ, Ginsberg J, Womer R, Lawlor ER, Wunder J, Andrulis I, Sorensen PH, Barr FG, Ladanyi M.EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing’s sarcoma. J Clin Oncol. 1998 Apr;16(4):1248-55

4. Fletcher CD, Fletcher JA, Cin PD, Ladanyi M, Woodruff JM.
Diagnostic gold standard for soft tissue tumours: morphology or molecular genetics?. Histopathology. 2001 Jul;39(1):100-3.

5. Hill DA, O’Sullivan MJ, Zhu X, Vollmer RT, Humphrey PA, Dehner LP, Pfeifer JD.Practical application of molecular genetic testing as an aid to the surgical pathologic diagnosis of sarcomas: a prospective study. Am J Surg Pathol. 2002 Aug;26(8):965-77.

6. Mierau GW, Weeks DA, Hicks MJ Role of electron microscopy and other special techniques in the diagnosis of childhood round cell tumors. Hum Pathol. 1998 Dec;29(12):1347-55.

7. Peydro-Olaya A, Llombart-Bosch A, Carda-Batalla C, Lopez-Guerrero JA Electron microscopy and other ancillary techniques in the diagnosis of small round cell tumors. Semin Diagn Pathol. 2003 Feb;20(1):25-45

8. Pfeifer JD, Hill DA, O’Sullivan MJ, Dehner LP. Diagnostic gold standard for soft tissue tumours: morphology or molecular genetics? Histopathology 2000;37:485-50

9. Sorensen PH, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, Maurer HM, Bridge JA, Crist WM, Triche TJ, Barr FG. PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children’s oncology group. J Clin Oncol. 2002 Jun 1;20(11):2672-9.