Keywords
Bone marrow biopsy - Ewings/primitive neuroectodermal tumor - marrow infiltration
- neuroblastoma - nonhematopoetic small round cell tumors
Introduction
Malignant small round cell tumors is a term used for tumors composed of malignant
round cells that are slightly larger or double the size of red blood cells (RBCs)
in air-dried smears and patternless sheets of small round cells with dense cellularity
and high N:C ratio on hematoxylin and eosin (H and E). Bone marrow (BM) is commonly
infiltrated by hematopoietic tumors. Metastasis of nonhematopoietic tumor cells to
BM was first reported in 1834; however, a series of such cases was not published until
1936.[1] In 1958, McFarland and Dameshek [2] described a simplified technique for BM biopsy and demonstrated that one could discover
unsuspected malignant disease and in many cases confirm the finding obtained by aspiration.
The extension of a cancer is a major prognostic factor (Stage IV disease) which determines
the therapeutic strategy.
Small round cell tumors present more commonly during childhood and adolescent. Nonhematopoietic
small round cell tumors include: Ewing sarcoma/primitive neuroectodermal tumor, retinoblastoma,
rhabdomyosarcoma, nephroblastoma, mesenchymal chondrosarcoma, small cell osteosarcoma,
poorly differentiated chordoma, melanotic neuroectodermal tumor, desmoplastic small
round cell tumor, and germ cell tumors.[3] The recognition of metastasis in random biopsies presents challenges to pathologists
when diagnosing the primary focus and needs an extensive workup.[4]
The recent improvements in immunohistochemistry and molecular biology methods enable
to detect tumor cells in various sites such as lymph nodes, BM, and blood with a considerably
increased sensitivity as compared to conventional approaches.[5] This study was undertaken to comprehensively analyze BM metastasis of nonhematological
round cell tumors diagnosed at a single tertiary care center in South India over the
2½ years.
Materials and Methods
This retrospective study was conducted from January 2014 to June 2016. A total of
206 diagnosed cases of small round blue cell tumors (excluding lymphomas), referred
for BM examination were included in the study. Age and sex distribution, clinical
findings and investigations (including ultrasound, X-rays, computed tomography scan,
magnetic resonance imaging, and biopsy findings of primary tumor) of all patients
were noted. An ethylenediaminetetraacetic acid-blood sample was obtained for complete
blood picture and peripheral blood film examination. Peripheral blood smears were
stained by Leishmann stain. BM aspiration (BMA) was performed by 16G lumbar puncture
needle and subjected to staining by Giemsa stain. BM biopsies (BMBx) were obtained
using the conventional technique with a Jamshidi needle from the posterior superior
iliac spine under local anesthesia. The biopsies were of adequate (1.5–2 cm) length
and fixed in 10% formalin solution and decalcified using 10% formal − formic acid
for 4–6 h followed by routine processing and paraffin embedding.[5] Serial sections of 4–6 μm thickness were cut and stained by H and E and supplemented
with immune stains where ever required. Hematological parameters including hemoglobin,
total leukocyte count and platelet count, and peripheral blood film examination with
RBC morphology or presence of any atypical cells on peripheral blood were noted. A
detailed examination of BM smears was done particularly for presence of atypical cells.
BMBx sections with at least five well preserved marrow spaces were studied for cellularity,
normal hematological elements, presence of infiltration by round cells (nonhematopoietic),
histologic pattern and morphology of infiltration (island/nests, diffuse sheets),
reticulin fibrosis, necrosis, and other secondary changes. Immunohistochemistry (IHC)
panel included CD56, CD99, synaptophysin, chromogranin, S100, desmin, myogenin, and
vimentin depending on the morphology.
Results
Demographic and clinical aspects
BMA (n = 206), imprints BMA, and BMBx (n = 206) were studied for involvement by round cell tumors from January 2014 to June
2016. The cases included age range from 45 days to 25 years with a median age of 12
years. There was a male predominance with male: female 1.5:1.
Bone marrow aspirate and biopsy findings
A total number of BMA were 4137 of which 206 (5%) had an established clinical diagnosis
of round cell tumor. Among these, 33/206 (16.01%) cases were positive for BM involvement
(BMI). Erythroid hyperplasia was seen in 10/206 (4.8%), reactive marrow in 10/206
(4.8%) and marrow within normal limits in 186/206 (90.2%). Leukoerythroblastic picture
was seen in 9/206 (4.3%) cases on peripheral blood examination.
In 33 cases, BMA showed involvement by round cell tumor, in two cases, tumor was picked
up in the imprint smear and biopsy, while in two other cases, both aspirate and imprint
smears were negative but was positive on biopsy. Of the 33 positive cases in BMA,
22 cases were neuroblastoma (66.6%), 8 cases Ewing’s sarcoma (24.2%), and 3 cases
of rhabdomyosarcoma (9.1%). Distribution of the cases with BMI and IHC is shown in
[Table 1].
Table 1
Distribution of cases with bone marrow infiltration on bone marrow aspiration, bone
marrow imprint, bone marrow biopsy
|
Type of SRCT
|
Clinical Dx of SRCT (n=206)
|
Number of cases with BMA involvement (n=33/206)
|
Number of cases with BM imprint involvememt (n=35/206)
|
Number of cases with BMBx involvement (n=37/206)
|
|
BM – Bone marrow; BMBx – BM biopsy; SRCT – Small round cell tumors; BMA – BM aspiration;
PNET – Primitive neuroectodermal tumor; BMI – BM involvement
|
|
Neuroblastoma
|
84
|
22
|
22
|
24 (including 2 cases that were -ve on BMA and BMI)
|
|
Ewings sarcoma/PNET
|
80
|
8
|
9 (including 1 case that was –ve on BMA)
|
9 (including 1 case that was –ve on BMA)
|
|
Rhabdomyosarcoma
|
35
|
3
|
4 (including 1 case that was –ve on BMA)
|
4 (including 1 case that was –ve on BMA)
|
|
Medulloblastoma
|
4
|
-
|
-
|
-
|
|
Wilms tumor
|
2
|
-
|
-
|
-
|
|
Hepatoblastoma
|
1
|
-
|
-
|
-
|
Bone marrow biopsy findings
A total number of BMBx were 206 which had an established clinical diagnosis of round
cell tumor. Among these, 37/206 (17.9%) cases were positive for BMI. Stromal changes
such as fibrosis were seen in nine cases. Six out of 9 cases were positive for BMI
by round cell tumors. Necrosis was seen in two cases of Ewings sarcoma. The remaining
cases showed normal marrow.
Immunohistochemistry
IHC was done on BMBx using the panel comprising of leukocyte common antigen, CD99,
CD56, desmin, vimentin, synaptophysin, and S100 depending on the morphology. IHC was
done in cases with high suspicion on histomorphology and all the cases of BMBx (37
cases) with established diagnosis of primary round cell tumor.
Discussion
Peripheral blood
Of the 37 positive cases, 6 (16.2%) had leukoerythroblastic picture with immature
granulocytes and nucleated red cells in the peripheral smear. Similarly, Contreras
et al.[6] found leukoerythroblastic picture in 22% of their cases and Leland and MacPherson
[7] in 33% of their cases. In our study, nucleated red cell and immature granulocytes
were seen when the hemoglobin was reduced below 7.0% gm. The presence of leukoerythoblasticanemia
correlates with the degree of reactive BM fibrosis than with the extent of malignant
infiltration.[8] In our study, all the six cases showed marrow fibrosis.
Bone marrow
BM study is essential not only for hematological malignancies but also is equally
important for nonhematological small round cell tumors. Bone is a common site for
metastasis for these tumors and the presence of isolated tumor cells in the BM at
the time of diagnosis indicates an increased risk for subsequent development of distant
metastasis. In our study, we found BM infiltration by round cell tumors as 37/206
(17.9%) on BMA. Similarly, Asif et al.[3] from Pakistan found BMI in 19.5% cases (n = 82). Mishra et al.[9] found 10% (n = 50). This is in contrast with a study done by Rafiq et al.[10] at King Edward Medical University which showed infiltration in 50% cases; however,
this may not be a true representation of disease as their cases were only 14. In our
study, BM infiltration was seen maximum by neuroblastoma 24/84 (28.5%) [Figure 1], followed by [Figure 2] Ewings sarcoma 9/80 (11.2%) [Figure 3] and then RMS 3/35 (8.5%) as seen in other studies, such as Naghmietal. Similarly
in a study done by Kumar et al.,[11] BMI was highest in neuroblastoma 9/14 (64%), Ewing’s sarcoma 14/47 (29.7%), retinoblastoma
3/7 (42.8%), and in rhabdomyosarcoma 5/20 (20%). The lower number of Ewing’s sarcoma
in our studies, as compared to the other study mentioned, is probably due to the fact
that not all the cases of Ewing’s sarcoma are referred to us for BM biopsy. In a study
by Mishra et al., the most common tumor in children that metastasized to marrow was neuroblastoma
and Ewing’s sarcoma each being 40%.
Figure 1: A case of neuroblastoma with bone marrow infiltratio (a) trephine imprint
showing atypical round cells (×400) (b–d) Bone marrow biopsy displaying paratrabecular,
interstitial and diffuse pattern of infiltratio by round cells, respectively (×100)
(e) immunohistochemistry with CD56 showing cytoplasmic and membranous positivity (×100),
(f and g) the round cells showing positivity for vimentin and neuron–specifi enolase,
respectively (×100)
Figure 2: A case of rhabdomyosarcoma with bone marrow infiltratio (a) bone marrow
aspiration smears showing singly scattered large atypical round cells with scant cytoplasm
and large bizarre nuclei with dispersed chromatin (×400) (b and c) bone marrow biopsy
displaying paratrabecular and interstitial pattern of infiltratio by round cells,
respectively (×400 and × 100). (d–f) immunohistochemistry with myogenin, vimentin,
and CD56 showing positivity (×100)
Figure 3: A case of Ewings/primitive neuroectodermal tumor with bone marrow infiltratio
(a) bone marrow aspiration smears showing rosettes and singly scattered round cells
with scant cytoplasm and round nuclei with dispersed chromatin (×400) (b) bone marrow
biopsy displaying paratrabecular pattern of infiltration by round cells (×400) (c
and d) immunohistochemistry with CD99 showing membrane and cytoplasmic positivity
and negativity with CD56 (×400)
In our study, 33 cases showed involvement by round cell tumor on BMA, in 2 cases tumor
was picked up in the imprint smear and biopsy, while in 2 other cases both aspirate
and imprint smears were negative; however, biopsy was positive, with an overall positive
cases being 37 of the total 206 cases of small round cell tumors.
There have been several previous studies showing superiority of the BMBx over aspirate
smears in diagnosis of metastatic tumor [12]
[13] as deposits in the marrow are focal and may often elicit a fibrotic response, and
therefore, aspirates may be negative. Multiple sections of the biopsy enable a much
larger volume of the marrow to be examined and allow infiltration to be recognized.[14] Singh et al.[12] reported that BMBx was superior to aspiration (97% vs. 72%), Mishra et al. showed (100% vs. 91.3%) similar findings were observed in our study where biopsy
versus aspiration positivity is (100% vs. 89.1%) [Table 2]. However, a study conducted by Sharma et al.[15] showed an occasional case where metastasis was detected only in aspiration. Bearden
et al.[16] reported that BMAs and BMBx were complementary in diagnosis of various solid tumors.
Table 2
Comparison of the present study with the other studies
|
Features
|
Present study (n=206), n (%)
|
Naghmi Asif et al. (n=82), n (%)
|
Mishra et al. (n=50), n (%)
|
Rafiq et al. (n=14), n (%)
|
|
BM – Bone marrow; BMBx – BM biopsy; BMA – BM aspiration
|
|
Age range
|
1.5 months to 25 years
|
6 months to 22 years
|
1-65 years
|
1-25 years
|
|
BMA involvement by small round cell tumor
|
33/206 (16.01)
|
16/82 (19.5)
|
9/50 (18)
|
7/14 (50)
|
|
BMBx involvement by small round cell tumor
|
37/206 (18)
|
14 (17)
|
9/50 (18)
|
7/14 (50)
|
|
Neuroblastoma
|
24/84 (28.5)
|
2/5 (40)
|
2/5 (40)
|
3/3 (100)
|
|
Ewing's sarcoma
|
9/80 (11.2)
|
3/6 (50)
|
6/28 (21)
|
1/2 (50)
|
|
Rhabdomyosarcoma
|
3/35 (8)
|
3/8 (37)
|
0/9 (0)
|
2/3 (66)
|
Immunohistochemistry was used as an adjunct to the morphological findings in the marrow
in elucidating the primary site, especially in clinically unsuspected cases. Depending
on the morphology of the tumor appropriate immunohistochemistry panel was used to
narrow down the differentials.
The presence of marrow infiltration is the most adverse prognostic factor and is associated
with the disease progression and a poor clinical outcome. As these patients are at
high risk of progression, they may benefit from intensification of the therapy.[17] It is therefore stressed to routinely consider BM examination (aspiration and biopsy)
for proper staging and management of these patients. BM metastasis, however, may not
be evident only on routine microscopic examination and thus looking at different studies
[18] our recommendation is to move on to immunohistochemical staining and molecular studies
of BM samples for the detection of minimal disseminated disease at the time of diagnosis.
Conclusion
Detection of metastasis in the BM has both therapeutic and prognostic significance.
Clinical history, radiological findings, morphology, and immunohistochemistry with
a panel of antibodies are useful to arrive at a definitive diagnosis. BMAs and BMBx
are complementary in the diagnosis of small round cell tumors.
