Cytomorphology and Morphometry of Small Round-Cell Tumors in the Region of the Kidney
Savithri Ravindra, D.C.P., D.N.B., and Usha Kini, M.D., D.C.P., D.N.B .*
Small round-cell tumors (SRCTs), with malignant cell components measuring 10 m or less in diameter with scanty cytoplasm in alcohol-fixed smears, pose a diagnostic challenge at fine-needle aspiration cytology (FNAC), especially when they are situated in and around the kidney and need facilities such as electron micros- copy, immunohistochemistry, tissue culture, and cytogenetics for their subtyping. A precise cytodiagnosis of SRCTs is important because a definite diagnosis is mandatory in preoperative diag- nostic workup for presurgical chemotherapy in these cases. With this view in mind, an attempt has been made to diagnose SRCTs in the region of the kidney based on cytomorphology and morphom- etry alone so as to facilitate its diagnosis in a simple cytology laboratory of a developing country where facilities for auxiliary techniques are not easily available.
Of 2,028 abdominal aspirates in a 12-yr period, 36 SRCTs were diagnosed in the region of the kidney by correlating with histology, radiology, and clinical features. The smears were studied for cellularity, morphology, pattern of cell arrangement, and smear background and morphometrically analyzed using an ocular mi- crometer. An aspirate with preponderant malignant round cells that were larger or double the size of red blood cells in air-dried smears or measured less than 10 u in diameter in alcohol-fixed smears was considered as a small blue-cell tumor.
Twenty-one were diagnosed as Wilms’ tumor (WT), 10 were diagnosed as neuroblastoma (NB), 3 were ganglioneuroblastoma (GNB), 1 was a cellular congenital mesoblastic nephroma (CMN), and 1 was an adrenocortical carcinoma (ACC). Cell clusters with neuropil and cytoplasmic processes were diagnostic of NB, gan- glion cells of GNB, and blastema with tubular differentiation in WT. Aspirates from CMN and ACC were considered as simulators/ mimickers of SRCT because they had superficial resemblance to SRCT and their differentiating cytomorphological features ob- served at histology were too subtle to be noted at cytology. The latter were appreciated only on retrospective analysis after histo- logical confirmation.
Department of Pathology, St. John’s Medical College, Bangalore, India *Correspondence to: Usha Kini, M.D., D.C.P., D.N.B., St. John’s Med- ical College, Bangalore 560034, India. E-mail : zipcb028@bgl.vsnl.net.in Received 11 June 2004; Accepted 3 November 2004 DOI 10.1002/dc.20225
Published online in Wiley InterScience (www.interscience.wiley.com).
Thus, morphometry in correlation with cytology, clinical his- tory, physical findings, and radiological data is helpful in guided FNA for a definite diagnosis of SRCT in the region of the kidney. One needs to keep in mind the mimickers of small round-cell lesions at this anatomic site. Diagn. Cytopathol. 2005;32: 211-216. @ 2005 Wiley-Liss, Inc.
Key Words: small round-cell tumor; Wilms’ tumor; neuroblas- toma; ganglioneuroblastoma; cytology; morphometry; FNAC
Introduction
Small round-cell tumors (SRCTs) are a group of malignant neoplasms characterized predominantly by round to oval cells.1 They do pose diagnostic problems in fine-needle aspiration cytology (FNAC) because the differentiating fea- tures are subtle. Electron microscopy, immunohistochemis- try, tissue culture, and cytogenetic studies are useful in enhancing the diagnostic accuracy,2-4 but these hi-tech fa- cilities are not available in most of the centers in developing and underdeveloped countries. We have attempted to eval- uate the combined role of cytomorphology and morphom- etry in the diagnosis and differential diagnosis of SRCTs in the region of the kidney.
Materials and Methods
The FNA material for this study, done under guidance, was taken from both retrospective and prospective studies per- formed on patients with SRCTs of the abdomen who visited St. John’s Medical College Hospital, Bangalore, India for diagnosis and treatment from November 1989 to December 2001. All relevant demographic data, clinical summaries, laboratory findings, radiological data, and relevant cytology and histopathology slides were retrieved from the Medical Records section and Archives. Bleeding parameters such as bleeding, clotting, and prothrombin times were performed routinely to rule out bleeding diathesis. The prothrombin
| Features | WT (n=21) | NB/GNB (n = 10 and 3) | CMN (n = 1) | ADR.CA (n = 1) |
|---|---|---|---|---|
| Age/sex (M:F) Cellularity | 2-6 yrs 1:3 Abundant, cohesive, and dyshesive | 8 mo-3 yr 0:4 Abundant, cohesive, and dyshesive | 3 days 1:0 Poor and dyshesive | 42 yr 0:1 Poor and dyshesive |
| Rosette | 0 | 2 | 0 | 0 |
| Neuropil and cytoplasmic processes | 0 | 10 | 0 | 0 |
| Tubule formation | 18 | 0 | 0 | 0 |
| Nucleoli | 0 | 1 | 0 | 1 |
| Nuclear chromatin | Hyper chromatic 8.2 µ (blastemal) | Stippled | Vesicular | Hyper chromatic |
| Mean cell diameter | 9.2 µ (epithelial) | 7.2 µ | 7.3 µ | 8.2 μ. |
time with a value of 2 sec more than the control was considered abnormal and the case was deferred from the FNAC procedure. FNAs were performed by the pathologist through posterolateral approach with either ultrasound or CT guidance using a 22-gauge needle attached to a 10-ml disposable plastic syringe. Because of a “hit-or-miss” chance in unidirectional aspirates in intra-abdominal masses, aspirations were repeated at more than one site depending on the patient’s condition, his cooperation, and the size of the neoplasm. Smears were made from each aspirate by direct smear technique and fixed in 95% ethyl alcohol for 20-30 min and later stained by Papanicolaou (Pap) and hematoxylin and eosin (H&E) stains. A few smears were air-dried and stained with Leishman stain. Any hemorrhagic material or excess material that was collected was processed for cell block preparation by centrifuging it with egg albumin/agar. The patients were kept on watch for a period of 24 hr for any intra-abdominal bleeding.
All of the smears were examined for
1. Cellularity. A smear was said to be cellular when the material was sufficient for cytological assessment leading to a definite diagnosis.
2. Morphology. A “small blue cell” was considered when a malignant cell measured less than 10 µ in diameter with round/oval large hyperchromatic nuclei and scanty cytoplasm in alcohol-fixed smears. Details of the nucleus, nucleolus, and cytoplasm were noted along with details of a smear background.
3. Patterns of cell arrangement.
4. Morphometric analysis was performed on 50 uni- formly dispersed cells in alcohol-fixed smears stained by Pap or H&E stain using a Leitz (Wetzlar, Ger- many) calibrated ocular micrometer. The cell diameter was considered for all purposes as being equivalent to the nuclear diameter because the amount of cytoplasm was scanty. These cells were compared with adjacent red blood cells, small lymphocytes, and endothelial cells in the smears.
A final cytological diagnosis was made after correlation with clinical history, physical findings, radiological data, and clinical outcome. All of the cases chosen for the study were further confirmed and analyzed with corresponding histopathology from Tru-Cut biopsies taken in the same sitting as that of the aspirate wherever possible. The patients were followed-up for a period of 2 yr.
Unguided and inadequate aspirates and those that were abdominal/retroperitoneal from the lesions not in the region of the kidney were excluded from the study.
Results
Of the 2,028 guided intra-abdominal aspirates, 36 cases of SRCTs localized to the kidney and perirenal region were chosen for the study. All patients had a palpable abdominal mass in the region of the kidney, which was confirmed radiologically. The cases were seen in all age groups rang- ing from a 3-day old child to a 42-yr-old woman but most of the patients were in the paediatric age group with a mean age of 3.5 yr and female predominance (male/female ratio [M:F] = 1:4; Table I). Twenty-two aspirates were done under ultrasound guidance and the remaining 14 aspirates were CT guided.
For morphometrical reference, red blood cells, small lym- phocytes, and nuclei of endothelial cells were considered and they measured an average of 4 µ in diameter in alcohol- fixed smears stained with H&E or Pap and, in contrast, their mean measurement was 7 µ in air-dried smears stained with Leishman stain. In comparison, the cells of the SRCT mea- sured 8-10 µ in diameter in alcohol-fixed smears, i.e., they measured almost double the size of the reference cells. A definite cytological diagnosis was made with confidence in 21 cases of Wilms’ tumor (WT), 3 cases of neuroblastoma (NB), and 1 case of ganglioneuroblastoma (GNB). Two cases labelled as SRCT cytologically could not be catego- rized further. They were later proved at histology to be one each of congenital mesoblastic nephroma (CMN) and adre- nocortical carcinoma (ACC).
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Fig. C-1
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Twenty-two cases chosen for the study were clinically WT (Fig. 1); 16 cases were in stage 2 of National Wilms’ Tumour Study Group (NWTS) and 6 cases were in stage 3. Aspirates from 21 cases of WT were cellular and were sampled through the posterolateral approach. Eighteen cases showed a bimodal cell population comprising both blastemal and epithelial cell components. The blastemal components were characteristically small cells with round nuclei having a mean diameter of about 8.2 p. The nuclei showed fine granular chromatin without nucleoli and scanty cytoplasm. The epithelial component comprised of larger cells of an average diameter of 9.2 u with a larger amount of cytoplasm were seen in small groups with definite tubule formation in all 18 cases. The remaining three cases showed a chiefly blastemal component with no obvious epithelial
differentiation. The cell block preparation was very useful in identifying the epithelial-cell component in those three aspirates that showed only a blastemal component in the smears. Stromal components were identified as small groups of spindle cells in an hemorrhagic background in about five cases. There was no evidence of any heterologous element. Thus, all 21 cases were diagnosed as biphasic WT by combining both smear and cell block findings. No tripha- sic WTs were identified. The Tru-Cut biopsy specimens taken simultaneously ratified the cytological features in 21 of the 22 cases.
One of the aspirates from a 3-day-old child with a clinical diagnosis of WT stage 1 showed predominantly epithelial- like components. They were round to oval cells with a mean diameter of 8 µ with scanty cytoplasm scattered in a hem-
orrhagic background. These cells had fine chromatin and no nucleoli (Fig. 2A). The background showed a few necrotic fragments with occasional polymorphs. With the cytologi- cal diagnosis of WT, the kidney excised showed a large mass with compressed kidney at the periphery. It had a spindle-cell component with features consistent with cellu- lar CMN at histology.
One aspirate from a middle-aged woman with renal mass showed dyshesive, uniformly scattered small round cells measuring 8.2 µ in diameter. These cells exhibited bland chromatin and scanty cytoplasm (Fig. 2B). The background was hemorrhagic. There was no tubular/acinar differentia- tion or any fibrillary/neuropil background. A cytological diagnosis of a malignant round-cell tumor was made. The lesion was excised. It was a solid mass of 16 × 13 cm, with an attached portion of adrenal gland compressing the renal parenchyma. Multiple sections confirmed a diagnosis of ACC.
Aspirates from 10 cases diagnosed as NB yielded abun- dant cellular material (Fig. 3). The cells were arranged in fragments of small groups of small round cells with an average diameter of 7.2 p. This single cell population was noted in the prominent fibrillary background in all 10 cases. Cell clusters with central fibrillar matrix forming Homer- Wright rosettes were noted in 2 of the 10 cases. The nuclei had a classic “salt-and-pepper” chromatin pattern. No dis- tinct nucleoli were observed. Subsequently, Tru-Cut biop- sies ratified the diagnosis in all.
Aspirates in all three cases of GNB showed clusters of small round cells (neuroblasts) in fibrillary matrix forming rosettes and ganglion cells having prominent nucleolus (Fig. 4). The small cells measured 7.3 µ in contrast to ganglion cells measuring 18 p.
Discussion
SRCTs are a group of malignancies characterized by the predominance of round to oval cells at cytology and histol- ogy. The group was comprised of non-Hodgkins’ lym- phoma, WT, NB, retinoblastoma, primitive neuroectoder- mal tumour, Ewings’ sarcoma rhabdomyosarcoma, and desmoplastic SRCTs.
The role of FNAC in the diagnosis of these small round- cell lesions has gained much importance during the past decade. Various cytomorphological details have been stud- ied in detail and although they are diagnostically useful, reservations have been expressed by various investigators about the contribution of cytology alone in the diagnosis of these neoplasms.
Aktar et al.2 and Layfield3 expressed the importance of a complete history, physical examination, and radiological and laboratory evaluations in arriving at a definitive diag-
nosis of SRCT. The diagnostic accuracy can be enhanced by other tools such as immunohistochemistry, electron micros- copy, and tissue culture. However, in the absence of these techniques, the cytopathologist still will need to offer a cytodiagnosis based on morphological features combined with available clinical history and imaging findings so that appropriate treatment could be initiated.
The present study has made an attempt to evaluate the cytomorphological features with morphometric analysis correlated with relevant clinical data and imaging findings in the diagnosis of SRCTs in the region of the kidney. The small blue-cell tumors in this study were characterized predominantly by round cells with cell diameters ranging from 6 to 10 µ (mean, 8 µ) in alcohol-fixed smears (where the red blood cells measured 4 u) and had a large round hyperchromatic nucleus and indistinct cytoplasm, so that the cells appeared small and blue in routine cytological stains.
Preoperative diagnosis of WT is of diagnostic importance because of its high cure rate. The primitive small round cells with high nuclear/cytoplasmic (N:C) ratios could differen- tiate into a larger cell component when it indicates epithelial differentiation. These cells could differentiate further to form tubules when they are described as blastemal elements with tubular differentiation.5 One could even differentiate into glomeruloid bodies in extreme cases. A cell block preparation should be prepared that would be a useful adjunct to smears to identify various cell components as noted in three of our cases. The results from this study have shown predominantly a blastemal component with epithelial differentiation in all 21 cases. Further, the morphometry helps in identifying blastemal cells that measure a mean dimension of 8.2 µ and cells of the epithelial cell compo- nent are larger and measure about 9.2 p.
A predominant blastemal component may be mistaken for an intrarenal NB even when the needle is well guided into the kidney mass with a CT or ultrasound. A careful search for epithelial and stromal components favors WT whereas a fibrillary background confirms the diagnosis of intrarenal NB.6 But in rare instances, neural differentiation could be well seen in WT resulting in a diagnostic dilem- ma.7 A presence of rhabdomyoblasts with blastemal com- ponents could be seen in both WT with skeletal muscle differentiation and embryonal rhabdomyosarcoma. A bi- modal population of cells and localization of the tumor by imaging techniques to the kidney would favor the diagnosis of WT.
An FNAC may be indicated to establish a diagnosis so that appropriate therapy can be given to shrink the tumor for making it operable in those cases where the renal tumor may be inoperable as indicated by findings on imaging proce- dures or when there may be clinical contraindication for
major surgery-a situation that is more likely to arise in children in developing countries. It should be noted that according to the current NWTS guidelines for staging ,8,9 an FNAC done by posterior approach does not convert the case into stage II (J. Vijay, personal communication, 2000). If it is done by anterior approach, the case may be considered to be stage III because of the possibility of generalized peri- toneal contamination. FNAC also may be used for diagnosis of metastatic WT.
Aspirate from the case of cellular CMN mimicked WT because of the presence of a round-cell population, which was mistaken for a blastemal component. On slide review, we realized that the oval cells of mesoblastic nephroma10 had appeared “round” due to the “end on” view of the cells. A thorough search for blastemal components was, however, not present, which should have guarded us from making a wrong diagnosis of WT.
Neuroblastoma (NB) is the third most-common malig- nant neoplasm in children with the peak incidence in the first 3 yr of life. It was the second-most common neoplasm in this study with a female predominance, which also was noted earlier by Aktar et al.2 The most important feature is the filamentous background, which has been described as neuropil, cytoplasmic processes, fibrillar masses, and fibril- lary matrix.11,12 Aktar et al.2 found cytoplasmic processes in 10 of 16 cases and considered it a prerequisite for the cytological diagnosis of NB. Similarly, we found a fibrillary background in cell clusters in all of the 10 cases whereas we found cytoplasmic processes only in 4 of 6 cases. Homer- Wright rosette is another important cytological feature but its occurrence is variable. The latter was noted by Das et al.12 in 3 of 5 cases, whereas Aktar et al.2 had a relatively low frequency being observed in only 3 of 16 cases. We noted typical Homer-Wright rosettes only in 2 of 10 cases. Rosette alone can not be considered as a specific feature of NB at cytology because they may be seen in other round- cell malignant tumors such as Ewing’s sarcoma, but when seen in addition to fibrillar matrix they would definitely aid in the cytological diagnosis of NB. Some authors13 describe the term differentiated NB when these neuroblastic tumors show some degree of differentiation toward ganglion cells and that they contain <5% of mature ganglion cells. This degree of differentiation could be depicted in the form of increased size of the cells, presence of nucleoli, and increas- ing amount of acidophilic cytoplasm while others advocate the use of the term GNB even if only one ganglion cell is seen.13,14 So it remains debatable to what point the desig- nation of GNB is appropriate, especially at cytology. Al- though studies and case reports on GNB are very few,13-15 the cytomorphology is well documented. The presence of neuroblasts at different stages of maturation with mature ganglion cells is the characteristic feature. Our case of GNB
had ganglion cells with neuroblasts and Homer-Wright rosettes.
A monotonous nature of uniformly dispersed small round cells with bland chromatin in a histologically diagnosed case of ACC as in this study may simulate an SRCT. Even imaging techniques on occasions do not guide the patholo- gist as to the renal/adrenal location with confidence. One needs to know the site and size of the lesion in interpreting a small round-cell aspirate in an adult, especially when there is the slightest doubt about an adrenocortical neoplasm.
Although lymphomas are common small malignant tu- mors in the abdomen, they are relatively rare in the region of the kidney, as was noted in this study. Metastatic lesions of these SRCT in the region of the kidney are also very unusual. Intra-abdominal desmoplastic SRCT and primitive neuroectodermal tumors could be seen in the retroperito- neum but its aspirates do not help in making a definite cytological diagnosis. A final diagnosis is possible with histology alone.
In conclusion, an aspirate with malignant round cells that are larger or near double the size of red blood cells or measuring less than 10 u in diameter in alcohol-fixed smears needs to be considered as a small blue-cell tumor. Definite cytodiagnosis is possible in cases of WT, NB, and GNB. Morphometry that is well correlated with cytology, clinical history, physical findings, and radiological guidance is helpful in arriving at a cytodiagnosis of SRCT. We conclude that FNAC assisted by morphometry could be used reliably in the precise presurgical diagnostic workup of SRCTs in the region of the kidney and is mandatory for preoperative chemotherapy and in patients who are at high surgical risk.
References
1. Smith NM, Keeling JW. Paediatric solid tumour in Chapter 9. In: Antony PP, MacSween RNM, Lowe DG, editors. Recent advances in histopathology, no. 17. Edinborough: Churchill Livingstone; 1997. p 191-218.
2. Aktar M, Ali MA, Sabbah RS, Bakry M, Sackey K, Elinor J. Aspira- tion cytology of Neuroblastoma: light and electron microscopic cor- relations. Cancer 1986;57:797-803.
3. Layfield LJ, Richman A. Fine needle aspiration cytology: utilization in paediatric pathology. Dis Markes 1990;8:301-315 (as quoted by Das DK, et al. in Acta Cytol 1997;41:1035-1047).
4. Akatar M, Ali MA, Sabbah R, Bakry M, Vash JE. Fine needle aspiration biopsy diagnosis of round cell malignant tumours of child- hood. A combined light and electron microscopic approach. Cancer 1985;55:1805-1817.
5. Aktar M, Ali MA, Sackey K, Sabbah R, Burgess A. Aspiration cytology of Wilms’ tumour: correlation of cytologic and histologic features. Diagn Cytopathol 1989;5:269-274.
6. Serrano R, Rodriguez-Peralto JL, Orbe GG, et al. Intra-renal neuro- blastoma diagnosed by FNAC report of 2 cases. Diagn Cytopathol 2002;27:294-297.
7. Magee JF, Ansari S, McFadden DE, Dimmick J. Teratoid Wilms’ tumour: a report of two cases. Histopathology 1992;20:427-431.
RAVINDRA AND KINI
8. Beckwith JB. National Wilms’ Tumour Study: an update for pathol- ogists. Paediatr Develop Pathol 1998;1:79-84.
9. Sarinen UM, Wilkstrom S, Korkimies O, Sariola H. Percutaneous needle biopsy preceeding preoperative chemotherapy; the manage- ment of massive renal tumours in children. J Clin Oncol 1991;9:406- 415.
10. Kaw YT. Cytologic findings in congenital mesoblastic nephroma. A case report. Acta Cytol 1994;38:235-240.
11. Silverman JF, Dabbs DJ, Ganick DJ, Hoolbrook CT, Giesinger KR. Fine needle aspiration cytology of neuroblastoma including peripheral neuroectodermal tumour with immunohistochemical and ultrastruc- tural confirmation. Acta Cytol 1988;32:367-376.
12. Das DK, Bhambani S, Chachra KL, Murthy KS, Tripati RP. Small round cell tumours of the abdomen and thorax. Role of fine needle aspiration cytologic feature in the diagnosis and differential diagnosis. Acta Cytol 1997;41:1035-1047.
13. Otal Salaverri C, Campora RG, Vazquez AH, Puertas EL, Davidson HG. Retroperitoneal ganglioneuroblastoma. Report of a case diag- nosed by fine needle aspiration cytology and electron microscopy. Acta Cytol 1989;33:80-84.
14. Dehner L. Paediatric surgical pathology. Baltimore: Williams & Wilkins; 1987. p 869-938.
15. Kumar PV. Fine needle aspiration cytologic diagnosis of ganglioneu- roblastoma. Acta Cytol 1987;31:583-586.