Medical and Pediatric Oncology 20:130-135 (1992)

Value of Cytogenetics in the Differential Diagnosis of the Small Round Cell Tumors of Childhood Laura Sainati, MD, Mario Stella, PhD, Anna Montaldi, PhD, Sonia Bolcato, PhD, Nicola Cuercini, B S ~ , Franca Bonan, 6sC,Vito Ninfo, Luigi Zanesco, MD, and Ciuseppe Basso, MD

MD,

~~

Small round cell tumors are among the most common problems in the differential diagnosis of cancer, even when more sophisticated histological techniques are utilized (Ezinger and Weiss: In Soft Tissue Tumors. St. Louis: CV Mosby, 1988, pp 668-683). Six cases of small round cell tu-

mors are described the diagnosis of which was particularly difficult. Cytogenetic analysis provided useful information in all of them in making the definitive diagnosis. The reported cases stress the value of cytogenetic methods in approaching difficult diagnostic problems.

Key words: cancer, histology, neoplasms, cytogenetics

INTRODUCTION

Patients

The tumors commonly referred to as “small round cell tumors” comprise a heterogeneous group of neoplasms with similar histology. This often makes them difficult to classify by light microscopy [2,3] or by complementary techniques such as immunohistochemistry and electron microscopy [4]. In cancer management a precise diagnosis is especially important in order to decide the specific therapy and to formulate the prognosis. Recently, nonrandom chromosome aberrations have been reported in some of these tumors, namely in neuroblastoma [ 5 ] , Ewing sarcoma [6], Askin tumor [ 7 ] , peripheral neuroepithelioma [81, and rhabdomyosarcoma [9,101. In the last 2 years we have routinely performed cytogenetic analyses on solid tumors. The present study reports six cases of small round cell tumors in children, successfully karyotyped, in whom cytogenetic analysis played an important role in making the ultimate diagnosis.

The patients from whom tumor specimens were taken are described in Table I. The six children were referred to our hospital with a suspected solid tumor or acute leukemia. Case 1. A 15 month-old girl presented with right exophthalmos. CT scan revealed right orbital and suprarenal masses. A bone marrow aspiration showed a partial infiltration of undifferentiated blasts, sometimes arranged in small clumps. Catecholamine precursors were within the normal range. The histologic diagnosis, by biopsy of the orbital tumor was rhabdomyosarcoma. Case 2. A 20 month-old boy presented with an abdominal mass. The bone marrow was highly infiltrated by undifferentiated blasts, some arranged in clumps. The catecholamine precursors were within the normal range. Histologically, the mass was very undifferentiated; the diagnosis was “undifferentiated sarcoma.” Case 3. A 3 year-old boy presented with an endopelvic mass. Catecholamine excretion was slightly elevated.

MATERlALS A N D METHODS

During the last 2 years, in our laboratory of the Paediatric Department of Padua University, we have performed cytogenetic analyses on 24 cases of childhood solid tumors. In six of them no cytogenetic abnormality was detected and in 12 the karyotype was in accord with the diagnosis. The six others are the subject of this report. 0 1992 Wiley-Liss, Inc.

From the Dipartimento di Pediatria and Istituto di Anatornia Patologica dell’Universit8 di Padova and Servizio di Genetica Urnana dell’ospedale di San Bortolo, Vicenza, Italy. Received August 29, 1990; accepted April 3, 1991. Address reprint requests to Dr. Laura Sainati, Laboratorio di Ematooncologia, Dipartimento di Pediatria, Universiti di Padova, Via Giustiniani 3, 35128 Padova, Italy.

Cytogenetics in Diagnosis of Childhood Cancer

131

TABLE I. Clinical and Pathological Features of the Six Patients* Diagnosis Initial Final

Patient

Sex

Age

Location

Stage

1

F

15 mo

1V

RMS

NB

8 mo, dead

2

M

20mo

IV

us

NB

15 mo, dead

3

M M F M

3 yrs 22 yrs 14 yrs 14 yrs

Orbit, suprarenal gland, bone marrow Abdomen, bone marrow Pelvis Thorax Anterior mediastinurn Thorax, bonemarrow

111

NB RMS

NE ES RMS RMS

5 yr, alive 12 yr, alive 10 mo, alive I mo, dead

4 5

6

IV IV

ES

Survival

*No histologic diagnosis; survival from the diagnosis. Abbreviations: RMS = rhabdomiosarcoma; NB = neuroblastoma; US = undifferentiated sarcoma; NE = neuroepithelioma; ES = Ewing sarcoma.

Histology showed a round small cell tumor and the diagnosis was neuroblastoma. Case 4. A 24 year-old man presented with a thoracic tumor. When 12 years of age, he was treated for an Ewing sarcoma of the fibula with 12 courses of chemotherapy (Adriamycin, vincristine, and cyclophosphamide) and radiotherapy of the tumoral area (5,000 rads). Five years later he developed a soft tissue relapse in the sub-clavicle region. It was completely removed by surgery and the patient underwent a chemo- and radiotherapy program. He has been well and in complete remission since; his thoracic mass was casually discovered during a routine x-ray exam. The histology aspect of the thoracic mass was quite undifferentiated, but the ultrastructure showed the presence of muscle differentiation (myofilaments arranged in bands). The diagnosis remained unclear. Case 5. A 14 year-old girl was referred to our hospital with suspected acute leukemia, The peripheral blood count showed Hb 7 g dl, PLT 21,000 pl, WBC 15,000 p1 with blasts in the differential count. Bone marrow aspirate showed an infiltrate of large blasts (73%),with reticular chromatin and prominent nucleoli. The blasts were negative for tdt enzyme and had neither lymphoid nor myeloid antigens. They were also negative for the leucocitary antigen (CD4.5) (Coulter Clone, Hileia, Florida). Absence of this marker prompted an extension of the immunological study to the cytoskeleton proteins and the blasts reacted strongly positive to the desmin protein (Dakopatts, Denmark). Case 6. A 14 year-old boy was referred to the hospital with acute leukemia suspected. Physical examination revealed an enlarged axillary lymphnode, but no hepatosplenomegaly . His peripheral blood count was Hb 9.5 g dl, PLT 68,000 pl, WBC 3,200 pl. Bone marrow smears showed large blasts, characterized by round or oval hyperchromatic nuclei with reticular chromatin and basophilic cytoplasm. The immunologic phenotype

didn’t express any hemopoietic antigen and the blasts strongly stained for desmin. The total body CT scan revealed a small thoracic mass adjacent to the 4th-5th rib. The lymph-node biopsy gave the diagnosis of Ewing sarcoma. Cytogenetic Studies

Chromosomal analysis was performed before therapy on cells obtained in two cases from the primitive tumor (cases 3 and 4) and from an enlarged lymphonode in a third (case 6). Cells from the bone marrow aspirate of three patients (cases 1, 2, 5 ) with marrow infiltration were processed for cytogenetic studies. Tumor tissue was chopped into very small pieces with scissors and knives and treated with collagenase for 10 minutes. The cells obtained from the masses and the bone marrow cells, were seeded into 25 cm2 culture flasks in RPMI 1640 medium supplemented with 15-20% fetal calf serum, penicillin (50 IUlml), and streptomycin (SO pg/ml). Chromosome preparations were from 2 hours up to 8 days after incubation. Cell cultures were exposed to colcemid at final concentration of 0.01 pg/ml for 1 to 4 hours. They were then resuspended in 0.075 M KCl for 8 minutes and fixed in three changes of 3:l methanol and glacial acetic acid. Chromosome spreads were prepared using standard airdrying techniques. For chromosomal analysis, all were examined by GTG-banding. In some instances, QFQ-banding techniques were also employed for estimation of the breakpoints and CBG-banding was used to evaluate the centromeric heterochromatin. Phytohemagglutinin-stimulated lymphocytes from peripheral blood served as controls for all patients to establish the normal constitutive karyotype. Twenty to SO metaphases were analyzed to determine the modal chromosome number and representative karyotype of each tumor. The karyotype was determined by

132

Sainati et al.

arranging all photographed cells that were technically satisfactory, according to the International System for Human Cytogenetic Nomenclature [ 111. Electron Microscopy

Cells or tissue samples of the patients were fixed in 3% glutaraldehyde and processed for electron microscopy with the usual techniques [ 121. The ultrathin sections, stained with uranil and lead, were examined by using an Hitachi 800 electron microscope. Immunologic Markers

Immunologic markers were examined on cytospins obtained from cell suspensions by utilizing the immunoperoxidase technique as previously described [ 131. RESULTS

The complete karyotypes of the six cases are shown in Table 11. The first two cases showed several chromosomal abnormalities with a common abnormality on the short arm of chromosome 1 at band p34 (Fig. 1) and a double minute chromosomes (DMs). Initial histological investigation of these tumors had resulted in the diagnosis of rhabdomyosarcoma and undifferentiated sarcoma, respectively. Because of the discordance between the karyotypes and the histological diagnoses, the histopathology was reviewed in both and the conclusive diagnosis of neuroblastoma was made in both cases. This diagnosis was confirmed by immunohistochemistry . (Both cases were positive for neuron specific enolase and for neurofilaments .) From the tumoral cells of case 2 a cell line was established; further studies performed on the cell line confirmed the diagnosis of neuroblastoma and showed amplification of the N-myc oncogene. The two patients had poor response to chemotherapy and died of tumor progression and multi-organ dissemination 8 and 15 months, respectively, after the diagnosis.

The karyotypes of cases 3 and 4 showed an identical translocation: t( 11;22)(q24;q12) (Fig. 1). The histological diagnosis of case 3 was neuroblastoma; the electron microscopy showed the presence of neurogranules in the tumoral cells. Because of the cytogenetic results, the slides were reviewed and a conclusive diagnosis of peripheral neuroepithelioma was made. The tumor, after a partial response to chemotherapy, was radically removed and the patient is alive and well 5 years after diagnosis. The diagnosis of case 4 was very controversial. The histology showed an undifferentiated neoplasm; the electron mycroscopy showed ultrastructural signs of muscular differentiation; the clinical suspicion of Ewing sarcoma relapse was high, cytogenetic results definitely confirmed it, and the EM finding has been considered to indicate a normal muscular tissue. Both the karyotypes of cases 5 and 6 showed a translocation (2;13) (q35;q14) (Fig. 1). Electron microscopy showed in case 6 tumoral cells (lymph node) the presence of thick and thin filaments. Some filaments were arranged to form definite Z-bands. The ultrastructure of DV cells (bone marrow) showed no sign of differentiation. DISCUSSI 0N

Progress in the treatment of cancer is tightly linked to an accurate diagnosis. The recent improvements in prognosis of children’s cancer are probably due to the fact that therapy can be adapted to the specific entities, each of which is treated differently. This requires precise diagnosis, but small round cell tumors often present undifferentiated histological features [ 141, and the occurrence of discordant pathologic opinion is rather high [3]. For this reason immunohistochemistry , electron microscopy, and cell cultures have been recently suggested as necessary complementary techniques to obtain the correct diagnosis [4]. Cytogenetic analysis has only recently been applied to

TABLE 11. Cytogenetic Findings in Six Cases of Small Round Cell Tumors* Patient

Sample

No. cells analyzed

1

BM/24 hr

40

46,XX(50%)/ 49,XX,- 1,- 17,+2O,+der(l)t(I;?)(p34;?),+der(l)t(l;?)(p34:?),+iso(8q),+mar(5O%)+DMS

2 3

BMi24 hr TC/8 hr TCOhr BMi24 hr LN/48 hr

50 40 15 25 25

46,XY(10%)146,XY,-l,-12,i-der(J)t(l;?)(p34;?),+der( 12)t(12;?)(pl3;?)(90%)+DMS

4 5 6

Karotype

46,XY,t(l1;22)(q24;q12) 50,XY,+8,+8,+ 12,+15,t( 11;22)(q24;q12), 53,XX+8,+11,+13,+13,t(2;13)(q35;q14),de1(6)(q21),+3mar 47,XY,t(2;13)(q35;ql4),+mar

* BM, bone marrow; LN, lymph nodes; TC, tumor cells.

Cytogenetics in Diagnosis of Childhood Cancer I)

7

Case 1

der(l)t(l;?)

1

Case

1

[

Case3 q

4

-ti

11

t(ll.22)

11

t(11:221

22

t(11.22)

2

t(2.131

13

t(2.13)

2

112.131

13

l(2.131

Case 5

Fig. 1. Karotypes of cases 1-6.

133

134

Sainati et al.

solid tumors and is still associated with a low rate of success due to the difficulty in consistently obtaining analyzable chromosomal preparations [ 151, However, an increasing number of nonrandom karyotypic abnormalities are being reported especially in pediatric neoplasms 1171. In the last 2 years, we have routinely performed cytogenetic analyses in solid tumors, giving these studies priority over other studies (e.g., cellular cultures and molecular biology). Karyotype analyses was useful in the majority of cases in better defining the diagnosis. In the six cases reported the cytogenetic analysis was necessary to correctly classify the neoplasms. Recently, structural abnormalities of chromosome lp, with a breakpoint at or distal to lp22, have been found in 70-80% of stage I11 or IV neuroblastomas, often associated with double minute chromosomes (DMs) and or homogeneously staining regions (HSRs) [ 18,191. HSRs represent the sites of amplification of complex rearranged genomic units including a core of several copies of the N-myc oncogene [20], and DMs represent their fragmented products [ 5 ] . Therefore even if abnormalities of chromosome 1 are frequent in solid tumors [16], structural abnormalities of Ip, especially in the presence of DMs 02 HSRs, strongly support the diagnosis of neuroblastoma [5,18]. We detected 1p34 rearrangements and double minute chromosomes in two cases of advanced tumors whose initial diagnoses were rhabdomyosarcoma and undifferentiated sarcoma respectively. These cytogenetic results led to a review of the slides and a diagnosis of neuroblastoma was made in both cases. Furthermore, the bad clinical outcomes confirm the association between these abnormalities and a type of neuroblastoma with an aggressive behavior and a poor prognosis [18,19].

The reciprocal translocation ( I 1 ;22)(q24,q 12) was first described in Ewing cell lines and in Ewing tumor cells [23]. Since then, this translocation has been reported in more than 90% of successfully karyotyped Ewing sarcomas [6]. Whang-Peng et al. identified an identical translocation in peripheral neuroepithelioma and in the Askin tumor [7,8], but this has never been found in other tumors [15]. The translocation is thus considered specific for these entities for which a common neuroectodermal origin has been suggested [2 11. We detected a t( 1 I ;22)(q24p;q 12) in two patients (cases 3 and 4). Case 3 had a histological diagnosis of neuroblastoma supported by a slightly increased level of catecholamine precursors and EM results. Histologic specimens were reviewed and a diagnosis of neuroepithelioma was made. Case 4 had already been treated for a Ewing sarcoma. The diagnosis of a Ewing relapse was very doubtful because of the undifferentiated histological appearance of

the tumor associated with ultrastructural signs of muscular differentiation. The cytogenetic results strongly supported the diagnosis of a thorax relapse of the original Ewing sarcoma. Seidal et al. [9] first identified the translocation t(2; 13)(q35;q14) in a case of alveolar rhabdomyosarcoma. Since then, a few others with the same abnormality have been reported, most of them in the alveolar subtype [22,10]. We detected the t(2;13) in two patients. In case 5 we could not get histological information because of the poor clinical condition of the patient and the diagnosis was based on immunocytology alone. The positive staining for desmin strongly suggested a neoplasm of muscular origin, even if is not specific for cross-striated muscle [4]. The finding of t(2;13) in the neoplastic cells provided useful information supporting the immunological results. It also suggested the precise alveolar subtype of rhabdomyosarcoma. In fact, until now, only two cases have been reported of nonalveolar rhabdomyosarcoma with t(2;13) [ 101. However, at the International Symposium on childhood RMS (Columbus, November '89) it was proposed that every rhabdomyosarcoma in which even small and rare foci of alveolar structure are found should be classified as alveolar. It is possible that the two cases reported by Douglass could be alveolar with these new indications. The histologic diagnosis of the other case (case 6) with t(2;13) was Ewing sarcoma. This diagnosis after slide review was modified to alveolar rhabdomyosarcoma, in accord with the ultrastructural findings of filaments and bands [ 141 and with cytogenetic results. Particularly, trisomy of chromosome 8 is frequently found in malignant tumors and has been associated with disease progression [24,25]. We found an increased number of copies of chromosome 8 in two of our cases, one of which was in relapse (case 4) and the other at the time of the initial diagnosis, but with a very aggressive disease (case 5 , rhabdomyosarcoma with a leukemic presentation). In conclusion, our results confirm the previously reported association between chromosomal abnormalities and a specific tumor and the biological behavior. Furthermore, with our report we stress the role of karyotyping in solid tumor analysis. Even if obtaining a solid tumor karyotype is still a challenge due to the low rate of success [ 151, we think cytogenetic analysis should be considered as a very useful tool in solid tumor diagnostic approaches, particularly in those types that have a wide range of histological appearances or those that have an undifferentiated appearance, like the pediatric small round cell group. In fact, the frequency of disagreement in histologic diagnosis of the soft tissue tumors is still rather high: 34% in the Southeastern Cancer Study Group experience [3].

Cytogenetics in Diagnosis of Childhood Cancer

ACKNOWLEDGMENTS

We thank Dr. G.J. D’Angio for critically reviewing the manuscript and Dr. J. Biegel for her helpful suggestions. This work was partially supported by an AIRC MPI 40/60% grant. Part of this work was presented at the International Post-Graduate Course on Soft Tissue Tumors, April 20, 1990, Riva del Garda, Italy.

1 1.

12. 13.

14.

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