# Challenges and Clinical Relevance in Diagnosing Metastatic Cells From Non-Hematopoietic Malignancies in Bone Marrow Aspirates

Elise Kaspi, Charlotte Grosdidier, Yaël Berda-Haddad, Maud Arpin, Sylvie Cointe, Shirley Fritz, Amandine Bonifay, Marie Koubi, Carine Jiguet-Jiglaire, Patrice Roll, et al.

► To cite this version:

Elise Kaspi, Charlotte Grosdidier, Yaël Berda-Haddad, Maud Arpin, Sylvie Cointe, et al. Challenges and Clinical Relevance in Diagnosing Metastatic Cells From Non-Hematopoietic Malignancies in Bone Marrow Aspirates. Cancer Medicine, 2025, 14 (3), 10.1002/cam4.70645. hal-05024332

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RESEARCH ARTICLE

OPEN ACCESS

# Challenges and Clinical Relevance in Diagnosing Metastatic Cells From Non-Hematopoietic Malignancies in Bone Marrow Aspirates

Elise Kaspi¹ ID | Charlotte Grosdidier² | Yaël Berda-Haddad³ | Maud Arpin³ | Sylvie Cointe³ | Shirley Fritz³ |
Amandine Bonifay³ | Marie Koubi⁴ | Carine Jiguet-Jiglaire⁵ | Patrice Roll¹ | Diane Frankel¹ ID

¹Cell Biology Department, APHM, INSERM, MMG, Timone Hospital, Aix Marseille Universite, Marseille, France | ²Service of Medical Biology, APHM, North Hospital, Marseille, France | ³Medical Biological Laboratory—Hematology and Flow Cytometry Department, Timone Hospital, APHM, Marseille, France | ⁴Department of Internal Medicine, CHU Nord, Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France | ⁵APHM, CNRS, INP, Inst Neurophysiopathol, GlioME Team, Réseau PrEclinique et TRAnslationnel de Recherche en Neuro-Oncologie, CHU Timone, Service d'Anatomie Pathologique et de Neuropathologie, Aix-Marseille Universite, Marseille, France

**Correspondence:** Diane Frankel (diane.frankel@univ-amu.fr)

**Received:** 30 September 2024 | **Revised:** 21 January 2025 | **Accepted:** 23 January 2025

**Funding:** The authors received no specific funding for this work.

**Keywords:** bone marrow aspirate | bone marrow aspiration | carcinoma | Ewing sarcoma | melanoma | metastasis

## ABSTRACT

**Introduction:** The causes of cytopenias are numerous, and the bone marrow aspirate helps to identify them. In rare cases, these cytopenias are due to bone marrow metastases from solid cancers. The techniques used in hematology laboratories are limited in characterizing these cells. Interaction with the cytopathology laboratory becomes critical for characterizing tumor cells and completing a comprehensive diagnosis from the bone marrow aspirate.

**Methods:** This article describes a series of 38 bone marrow aspirates from 36 patients with bicytopenias who underwent bone marrow aspiration and for whom the hematologists sent the sample to the cytopathology laboratory to complete the diagnosis by immunocytochemistry and FISH if necessary.

**Results:** The mean age of patients is 66years, and the sex ratio is 2.8. Metastases were found in 11 cases of lung carcinoma, 4 cases of prostate carcinoma, 2 cases of breast carcinoma, 1 case of kidney carcinoma, 1 case of glioblastoma, 1 case of Ewing's sarcoma, and 1 case of melanoma. Among them, bone marrow aspiration was the only method to establish the initial diagnosis for seven patients. In six cases, immunocytochemistry confirmed the presence of carcinoma cells but could not identify their origin. In seven cases, tumor cells were insufficient to be characterized by immunocytochemistry.

**Conclusion:** Collaboration between laboratories is essential for the management of bone marrow aspirates containing non-hematopoietic metastases. Bone marrow aspiration may be sufficient to diagnose solid tumors, enabling faster initiation of treatment for patients already at an advanced stage of their disease.

## 1 | Introduction

Bone marrow aspiration is a commonly performed procedure for the diagnosis and therapeutic monitoring of hematologic disorders. This minimally invasive procedure, which

requires only local anesthesia, can be easily conducted during a routine medical consultation. Bone marrow aspirate provides both quantitative and qualitative information on hematopoietic cell populations, aiding in comprehensive clinical assessment. Standard laboratory techniques such as cytology, flow

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2025 The Author(s). *Cancer Medicine* published by John Wiley & Sons Ltd.</footnote>
cytometry, and cytogenetic analysis are essential for the characterization and follow-up of hematologic neoplasms [1]. Bone marrow aspiration is a common diagnostic tool for unexplained cytopenias, with hematologic malignancies being the primary underlying cause [2]. However, in a small subset of cases (1%–2%), bone marrow metastases may mimic a hematologic disorder and result in cytopenias. Bone marrow metastases occur in less than 10% of patients with metastatic cancers, with lung, breast, and prostate carcinoma exhibiting a higher prevalence [3]. The growth of metastatic carcinoma within the bone marrow leads to trabecular bone destruction, resulting in osteolytic lesions and subsequent cytopenias, predominantly anemia and thrombocytopenia.

Accurate identification of metastatic cells in bone marrow aspirates is critical for hematologists. While immunocytochemistry is essential for characterizing these cells, it is often not available in a hematology laboratory, as it is in a cytopathology laboratory. Therefore, effective collaboration between hematology and cytopathology laboratories is required to confirm the presence and nature of metastatic cells.

This article shows how such interdisciplinary collaboration, supported by cytological expertise, can significantly improve the detection of metastatic cells in bone marrow aspirates and improve the diagnostic management of patients at an advanced stage of their disease.

## 2 | Methods

### 2.1 | Sample Collection

This study included bone marrow aspirates received in the Hematology Laboratory of the North Hospital and La Timone Hospital (Assistance Publique des Hôpitaux de Marseille, France) from patients either with non-hematologic cells or with newly diagnosed cytopenias in a context of known solid tumors. Smears and/or bone marrow aspirates collected in EDTA tubes were received. At least three smears were stained with May-Grünwald-Giemsa (MGG). When available, additional smears and EDTA samples were sent to the Cell Biology Laboratory at La Timone Hospital (Assistance Publique des Hôpitaux de Marseille, France) for further analysis.

### 2.2 | Immunocytochemistry to Characterize Metastatic Cells

For cases where only smears were available, manual immunocytochemistry was performed until December 2023 using the SensiTEK HRP kit (ScyTek Laboratories, Logan, USA) on slides previously fixed in paraformaldehyde 4% for 10 min. Slides were incubated with peroxidase-blocking solution for 30 min. After washing, slides were incubated with SensiTEK HRP kit for 10 min. Primary antibodies (Table 1) were incubated for 30 min. The biotinylated secondary antibodies were incubated for 15 min, followed by streptavidin/HRP for 20 min and DAB Quanto chromogen (Diagomics, Blagnac, France) for 5 min.

<table><caption>**TABLE 1** | Antibodies used for immunocytochemistry to detect metastatic cells.</caption><thead><tr><th>Antibody</th><th>Clone</th><th>Manufacturer</th><th>Dilution</th><th>Type</th></tr></thead><tbody><tr><td>BerEP4</td><td>BerEP4</td><td>Dako</td><td>1/100</td><td>A</td></tr><tr><td>TTF1</td><td>8G7G3/1</td><td>Dako</td><td>1/100</td><td>A/M</td></tr><tr><td>P40</td><td>Polyclonal</td><td>Biocare Medical</td><td>1/100</td><td>M</td></tr><tr><td>P40</td><td>SP225</td><td>Roche</td><td>None</td><td>A</td></tr><tr><td>PAX8</td><td>MRQ-50</td><td>Roche</td><td>None</td><td>A/M</td></tr><tr><td>GATA-3</td><td>L50-823</td><td>Roche</td><td>None</td><td>A/M</td></tr><tr><td>Pancytokeratin</td><td>Polyclonal</td><td>Ventana</td><td>None</td><td>M</td></tr><tr><td>AE1/AE3</td><td>Polyclonal</td><td>Roche</td><td>None</td><td>A</td></tr><tr><td>Epithelial Membrane Antigen</td><td>MOC31</td><td>Dako</td><td>1/50</td><td>M</td></tr><tr><td>CD45</td><td>2b11+PD7</td><td>Diagomics</td><td>1/500</td><td>A/M</td></tr><tr><td>PSA</td><td>35H9</td><td>Leica</td><td>1/100</td><td>A/M</td></tr><tr><td>Chromogranin</td><td>DAK-A3</td><td>Dako</td><td>1/200</td><td>A/M</td></tr><tr><td>Synaptophysin</td><td>DAK-SYNAP</td><td>Dako</td><td>1/100</td><td>A/M</td></tr><tr><td>CD56</td><td>MRQ-42</td><td>Roche</td><td>None</td><td>A/M</td></tr><tr><td>CK7</td><td>OV.TL12/30</td><td>Dako</td><td>1/100</td><td>A/M</td></tr><tr><td>SOX10</td><td>EP268</td><td>MMF</td><td>1/100</td><td>A/M</td></tr><tr><td>NKX3.1</td><td>Polyclonal</td><td>Diagomics</td><td>1/100</td><td>A/M</td></tr><tr><td>NKX2.2</td><td>EP336</td><td>Diagomics</td><td>None</td><td>A</td></tr></tbody></table>

Abbreviations: A/M, Manual or automated technique; A, Automated technique on Benchmark Ultra; M, Manual technique using the SensiTEK HRP kit.

Nuclei were counterstained with Mayer's hemalum solution. Slides were mounted with Aquatex.

When bone marrow aspirate in EDTA tubes was available, smears or cytospins were performed after red cell lysis. In all cases, TOMO slides (Matsunami Glass, Bellingham, USA) were used to prevent the detachment of cells. Immunocytochemistry was performed on BenchMark ULTRA using the ultraview kit (Ventana, Roche, USA) from December 2023. The list of antibodies and the conditions used are provided in Table 1.

### 2.3 | Flow Cytometry Analysis

Lymphocytes from bone marrow were analyzed by flow cytometry using the Navios 3-laser instrument (Beckman Coulter, Miami, FL, USA). Fluorescent antibody reagents were procured from Beckman Coulter Immunotech (Marseille, France): CD45-FITC (B3821F4A), CD3-PC5 (UCHT1), CD4-PE (SFCI12T4D11), CD8-ECD (SFCI21Thy2D3), HLA-DR-PB (IMMU357), CD19-ECD (J3.119), CD16-PE (3G8), CD56-PE (N901-NKH1), and CD5-PC7 (BL1a). Briefly, 100 µL of bone marrow was incubated with combinations of optimally titrated monoclonal antibodies for 15 min at room temperature in the dark. Finally, the samples
<figure><img src="image_2.png" /><figcaption>**FIGURE 1** | Carcinomas diagnosed from bone marrow aspirate with MGG staining and immunocytochemistry: Small cell lung carcinoma (SCLC) with May-Grünwald-Giemsa (MGG) staining on (A) smears and (B) cytospin slide obtained from EDTA sample. Immunocytochemistry confirming SCLC with the positivity of (C) TTF1, (D) NCAM/CD56, (E) synaptophysin. (F) Lung adenocarcinoma (MGG staining). Prostate carcinoma with MGG staining (G) confirmed by immunocytochemistry using (H) NKX3.1 and (I) EpCAM. Breast carcinoma with MGG staining (J and K) confirmed by immunocytochemistry using (L) EpCAM and (M) GATA-3. Renal carcinoma with MGG staining (N) and confirmed with (O) PAX8 and EpCAM (not shown).</figcaption></figure>

were treated with a lysis buffer ([NH₄Cl] = 8.3 g/L), then washed with 2 mL PBS-BSA1%-azide, centrifuged at 300g for 5 min, and resuspended in 500 µL BPS/BSA buffer. Data acquisition was conducted using Navios software, and the data were analyzed with Kaluza C software.

## 2.4 | Fluorescence In Situ Hybridization

Smears were first washed with 2XSSC at 37°C for 2 min and then treated with pepsin-HCl (0.001 g/L-0.01 N) at 37°C for 13 min. After washing in PBS, post-fixation was performed in formaldehyde-MgCl2 (1% -0.05 M) for 5 min. Dehydration was carried out in baths containing increasing percentages of ethanol (70%, 85%, and 100%), and slides were air-dried for 30 min. Smears were hybridized with the Vysis LSI EWSR1 Break Apart FISH Probe Kit (Abbott, IL, USA) for 17 h on Thermobrite (Leica, Nanterre, France). Post-hybridization washes were performed in 0.4 SSC at 72°C for 2 min and 2 SSC-0.01% Tween 20 at room temperature. Smears were air-dried for 30 min before counterstaining with DAPI (Leica, Nanterre, France). Nuclei were

observed with an Axio Imager Z2 microscope (Zeiss, France) and analyzed with Metafer software (Metasysems, Cormeilles-en-Parisis, France).

## 2.5 | Consent

The project was reviewed and approved by the local ethics committee of treated with a lysisor their diagnosis and treatment. In accordance with the French data protection authority (Commission Nationale de l'Informatique et des Libertés), general information on research activities is provided in clinical units to departments and to the persons concerned, who may exercise their right to object, which is verified prior to data collection.

## 3 | Results

The Cell Biology Laboratory analyzed 38 bone marrow aspirates from 36 patients to characterize metastatic cells in a known or unknown neoplastic context. The mean age of the patients was 66 years (range 21-90 years) and the sex ratio was 2.8.
<figure><img src="image_3.png" /><figcaption>**FIGURE 2** | Melanoma diagnosed with MGG and immunocytochemistry. (A) MGG staining showing large sheets of tumor cells. (B) Immunocytochemistry showing nuclear positivity with SOX10.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**FIGURE 3** | Ewing sarcoma. (A–C) Clusters or isolated small cells with a high nucleo-cytoplasmic ratio (MGG staining). (D–G) Flow cytometry graph showing negative cells for CD45, CD19, and CD3 markers and positive cells for CD56. Immunocytochemistry showing positivity for (H) CD99 and (I) NKX2.2. (J) FISH with an EWSR1 break-apart probe showing a separation of the red and green probes and confirming EWSR1 fusion.</figcaption></figure>
Metastatic lung carcinoma was diagnosed in 11 cases (29%), in-
cluding small cell lung cancer in 10 cases (Figure 1A–E) and
lung adenocarcinoma in one case (Figure 1F). Prostate carci-
noma was diagnosed in four cases (10.5%) (Figure 1G–I), breast
carcinoma in two cases (5.2%) (Figure 1J–M), renal carcinoma
(Figure 1N,O), melanoma (Figure 2), glioblastoma, and Ewing
sarcoma (Figure 3) in one case each (2.6%). The last two cases
were confirmed by bone marrow biopsy (Table 2). In 7 of these 21
cases, bone marrow aspirate was the only method or the fastest
to establish the initial diagnosis. For example, in a 90-year-old
patient, a prostate biopsy was not feasible due to high perfor-
mance status. However, a bone marrow aspirate revealed meta-
static prostate carcinoma, and genetic testing for BRCA1/2 was
performed on the collected EDTA sample. Treatment was initi-
ated based on the results of the bone marrow aspiration. A bone
marrow aspirate from a 21-year-old patient showed small and
round cells, with a high nucleo-cytoplasmic ratio, often in clus-
ters (Figure 3A–C). Flow cytometry analysis showed negativity
for CD45, CD19, and CD3 markers but positivity for the CD56
marker (Figure 3D–G). Immunocytochemistry was positive for
CD99 and NKX2.2 (Figure 3H,I). FISH analysis on the aspirate
using an EWSR1 break-apart probe confirmed the presence of
an EWSR1 fusion, a hallmark of Ewing sarcoma (Figure 3J).
Bone marrow aspiration results were obtained 1 week earlier
than those from a bone marrow biopsy, allowing for earlier ini-
tiation of treatment.

In six other bone marrow aspirates from five patients, immuno-
cytochemistry revealed metastatic carcinoma cells positive for
cytokeratins and BerEP4/EpCAM. Four of these patients had
previously been diagnosed with a carcinoma of known origin (2
breast, 1 prostate, and 1 renal). However, immunocytochemis-
try failed to identify these possible origins based on the known
primitive tumor (negative for GATA-3, NKX3.1, or PAX8 respec-
tively) or to identify other origins. In the remaining case, the
bone marrow aspirate was the initial evidence of carcinoma, and
a subsequent chest tomography scan revealed a mass that had
developed at the expense of the gastric wall. Unfortunately, the
patient died 11 days after the bone marrow aspiration without
biopsy confirmation.

In seven cases, cells suspicious of non-hematopoietic neoplasia
were observed on the smears. However, immunocytochemical
analyses were inconclusive due to the insufficient number of
these cells. Among them, a patient with melanoma undergoing
immunotherapy presented with bicytopenia. The challenge was
to differentiate between a bicytopenia due to metastasis and a
side effect of the immunotherapy. Two consecutive bone mar-
row aspirations were performed, and metastatic cells were de-
tected in limited numbers only in the first aspirate. However,
the subsequent bone marrow biopsy performed several weeks
later revealed metastatic prostate cancer in this patient.

In four cases with a history of solid cancers (lung adenocarci-
noma, small bowel neuroendocrine tumor, renal cell carcinoma)
and bicytopenias, the bone marrow aspirate was sent by the he-
matologists to the cell biology laboratory to rule out metastatic
disease, which was done.

Fourteen patients died shortly after the bone marrow aspiration
(median 20 days, range 2 days to 6 months). The earliest death

occurred in a 34-year-old patient who had been diagnosed with
melanoma 1 month earlier (Figure 2). Among the 11 patients
with lung carcinoma, 4 died within 15 days of the bone marrow
aspiration, 1 died within a month, 1 died within 6 months, 3 pa-
tients were lost to follow-up, and 2 were alive 1 year and 4 years
after the bone marrow aspiration.

4 | Discussion

The diagnosis of non-hematopoietic metastatic cells in a bone
marrow sample requires the identification of cells that do not
belong to the normal hematologic lineage. This relies on the
expertise of hematologists. Flow cytometry can confirm their
non-hematopoietic origin by identifying cells lacking CD45 ex-
pression. The cytologic characteristics of non-hematopoietic met-
astatic cells vary depending on the type of tumor. Carcinomas
often occur in clusters, making them easier to detect. Small cell
lung cancer, neuroblastoma, retinoblastoma, and Ewing sar-
coma present as small, round cells with high nucleo-cytoplasmic
ratios, often in clusters. Recently, artificial intelligence tools are
being developed to assist pathologists and hematologists in di-
agnosing metastatic tumor cells in bone marrow aspirates [4].

In the context of non-hematopoietic metastases, immunocyto-
chemistry is essential for the characterization of cancer cells,
especially those of unknown primary origin. Optimization of
both the material and the technique used is essential for accu-
rate diagnosis [5]. Our laboratory has improved immunocyto-
chemistry by moving to an automated method for both smears
and cytospins from EDTA tubes. Positive-charged slides are crit-
ical to prevent cell detachment during automated procedures.
This technical issue has led to several failures in cell charac-
terization. Cytoblocks can also be prepared from EDTA tubes
if available for immunohistochemical characterization. In our
experience, bone marrow aspirates in the EDTA tubes are ideal
because they allow for the preparation of smears, cytospins, and
cytoblocks, and thus can be adapted to various laboratory tech-
niques and allow for genetic analysis. Receiving smears directly
from clinical services limits the number of antibodies that can
be tested because of the limited number of smears available.
The main challenge in characterizing metastatic cancer cells
is the insufficiency of neoplastic cells in the samples. For some
patients, cells were visible on only a few smears and often ab-
sent from smears used for immunochemistry, making charac-
terization difficult. While bone marrow biopsy is the preferred
method for characterizing non-hematopoietic cells, it requires
general anesthesia, which is not always feasible in patients with
severe thrombocytopenia, and has a longer turnaround time
due to decalcification. Bone marrow aspirates provide faster re-
sults, help start chemotherapy on time, and complement biopsies
when they are inconclusive. However, bone marrow biopsy still
remains more effective than bone marrow aspirate in detecting
metastasis [6, 7].

Our study mainly included patients with metastatic small cell
lung carcinoma, whereas other studies reported a predominance
of gastric tumors [4, 8]. These differences may be attributed to
the recruitment practices of each hospital. The Nord Hospital
in Marseille has a very active Oncopneumology Department,
which could explain the overrepresentation of small cell
<table><caption>TABLE 2 | Results of the 38 bone marrow aspirations from 36 patients.</caption><thead><tr><th>Patient (number of bone marrow aspirates)</th><th>Age (years old)</th><th>Sex</th><th>Diagnosis on bone marrow aspiration</th><th>Diagnosis on biopsy (type and date)</th><th>Death (time elapsed after the bone marrow aspiration)</th></tr></thead><tbody><tr><td>1</td><td>69</td><td>M</td><td>Lung adenocarcinoma</td><td>Lung adenocarcinoma (liver biopsy, 4 months before)</td><td>Yes (11 days)</td></tr><tr><td>2</td><td>58</td><td>M</td><td>SCLC</td><td>SCLC (left inferior lung biopsy, same date)</td><td>No</td></tr><tr><td>3</td><td>78</td><td>M</td><td>SCLC</td><td>None</td><td>Yes (9 days)</td></tr><tr><td>4</td><td>73</td><td>M</td><td>SCLC</td><td>SCLC (upper right lung biopsy, same date)</td><td>Not known</td></tr><tr><td>5</td><td>65</td><td>M</td><td>SCLC</td><td>None</td><td>Yes (13 days)</td></tr><tr><td>6</td><td>62</td><td>M</td><td>SCLC</td><td>SCLC (left mediastinal-hilar biopsy, 4 days before)</td><td>Not known</td></tr><tr><td>7</td><td>75</td><td>M</td><td>SCLC</td><td>SCLC (lymph node 7 biopsy, same date)</td><td>Yes (25 days)</td></tr><tr><td>8</td><td>52</td><td>F</td><td>SCLC</td><td>None (CT scan reveals a lung mass with metastases)</td><td>Not known</td></tr><tr><td>9</td><td>63</td><td>M</td><td>SCLC</td><td>SCLC (left carina biopsy, same date)</td><td>Yes (14 days)</td></tr><tr><td>10</td><td>63</td><td>M</td><td>SCLC</td><td>None</td><td>Yes (6 months)</td></tr><tr><td>11</td><td>54</td><td>M</td><td>SCLC</td><td>None (SCLC confirmed on right clavicle biopsy, 2 days later)</td><td>No</td></tr><tr><td>12</td><td>73</td><td>M</td><td>Prostatic carcinoma</td><td>Prostatic carcinoma (prostate, 2 years before)</td><td>No</td></tr><tr><td>13</td><td>76</td><td>M</td><td>Prostatic carcinoma</td><td>Prostatic carcinoma (prostate, 15 years before with a relapse 5 years before)</td><td>No</td></tr><tr><td>14</td><td>87</td><td>M</td><td>Prostatic carcinoma</td><td>Prostatic carcinoma (prostate, 7 years before)</td><td>Not known</td></tr><tr><td>15</td><td>90</td><td>M</td><td>Prostatic carcinoma</td><td>None, BRCA testing performed on the bone marrow aspirate</td><td>Yes (18 days)</td></tr><tr><td>16</td><td>86</td><td>F</td><td>Breast carcinoma</td><td>Breast carcinoma (right breast biopsy, 1 month before)</td><td>No</td></tr><tr><td>17</td><td>63</td><td>F</td><td>Breast carcinoma</td><td>Breast carcinoma (breast biopsy, 3 years before)</td><td>Not known</td></tr><tr><td>18</td><td>21</td><td>M</td><td>Ewing sarcoma</td><td>None (bone marrow biopsy confirmed the diagnosis 1 week after)</td><td>Not known</td></tr><tr><td>19</td><td>76</td><td>F</td><td>Glioblastoma</td><td>Glioblastoma (left temporal biopsy, 6 months before)</td><td>No</td></tr><tr><td>20</td><td>34</td><td>M</td><td>Melanoma</td><td>Melanoma (left inguinal lymph node, 1 month before)</td><td>(Yes 2 days)</td></tr><tr><td>21</td><td>58</td><td>M</td><td>Renal cell carcinoma</td><td>Renal cells carcinoma (left iliac mass, 7 years before; kidney, 11 years before)</td><td>Not known</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 2 | (Continued)</caption><thead><tr><th>Patient (number of bone marrow aspirates)</th><th>Age (years old)</th><th>Sex</th><th>Diagnosis on bone marrow aspiration</th><th>Diagnosis on biopsy (type and date)</th><th>Death (time elapsed after the bone marrow aspiration)</th></tr></thead><tbody><tr><td>22</td><td>70</td><td>F</td><td>Carcinoma</td><td>Breast carcinoma (mastectomy, 13 years before with a relapse 3 years before)</td><td></td></tr><tr><td>23</td><td>89</td><td>M</td><td>Carcinoma</td><td>Renal cells carcinoma (date of diagnosis not known)</td><td>Not known</td></tr><tr><td>24</td><td>51</td><td>M</td><td>Carcinoma</td><td>None (CT scan reveals a mass developed at the expense of the gastric wall)</td><td>Yes (11 days)</td></tr><tr><td>25 (2)</td><td>67</td><td>F</td><td>Carcinoma</td><td>Breast carcinoma (bone marrow biopsy, same date)</td><td>No</td></tr><tr><td>26</td><td>77</td><td>M</td><td>Carcinoma</td><td>None (prostate biopsy confirmed prostatic carcinoma 1 month later)</td><td>Yes (4 months)</td></tr><tr><td>27</td><td>68</td><td>F</td><td>None (too few metastatic cells)</td><td>Pancreatic carcinoma (gastric biopsy, 1 month before)</td><td>Yes (1 month)</td></tr><tr><td>28 (2)</td><td>66</td><td>M</td><td>None (too few metastatic cells)</td><td>History of melanoma. Prostatic carcinoma diagnosed on bone marrow biopsy 3 months later</td><td>No</td></tr><tr><td>29</td><td>69</td><td>M</td><td>None (too few metastatic cells)</td><td>None (prostatic carcinoma confirmed on prostate biopsy 3 weeks later)</td><td>Not known</td></tr><tr><td>30</td><td>56</td><td>M</td><td>None (too few metastatic cells)</td><td>History of cutaneous T-cell lymphoma</td><td>Not known</td></tr><tr><td>31</td><td>82</td><td>M</td><td>None (too few metastatic cells)</td><td>None (renal cells carcinoma confirmed on right iliac wing 2 months later)</td><td>Yes (2 months)</td></tr><tr><td>32</td><td>39</td><td>M</td><td>None (too few metastatic cells)</td><td>None (medulloblastoma confirmed on left femur biopsy 1 month later)</td><td>Not known</td></tr><tr><td>33</td><td>74</td><td>M</td><td>Absence of metastatic cells</td><td>Lung adenocarcinoma (lung biopsy, 2 years before)</td><td>Yes (22 days)</td></tr><tr><td>34</td><td>54</td><td>M</td><td>Absence of metastatic cells</td><td>Squamous lung cancer (lung biopsy, 4 years before)</td><td>Not known</td></tr><tr><td>35</td><td>88</td><td>F</td><td>Absence of metastatic cells</td><td>Renal cell carcinoma (lung biopsy, 3 months before)</td><td>Yes (4 months)</td></tr><tr><td>36</td><td>58</td><td>F</td><td>Absence of metastatic cells</td><td>Lung adenocarcinoma (brain biopsy, 3 years before)</td><td>Not known</td></tr></tbody></table>

Abbreviation: SCLC, small cell lung cancer.

neuroendocrine carcinoma cases in our study compared to other centers.

In our series, we diagnosed a case of Ewing sarcoma confirmed by EWSR1 fusion by FISH. Flow cytometry confirmed the non-hematopoietic origin of the metastatic cells by negative expression of CD45, CD19, and CD3, despite CD56 positivity. In a study of 46 Ewing sarcomas, CD56 positivity was found in 60% and was

significantly associated with progression-free survival [9]. The patients underwent a bone marrow biopsy, confirming these findings. Ewing sarcoma is the second most common bone tumor in children and adolescents. Bone marrow is a common site of metastasis (5%-17%) after bone (30%-40%) and lung (50%) [10]. The National Ewing Sarcoma Tumor Board recommended in 2023 that if imaging suggests metastatic involvement of the bone marrow at diagnosis, bilateral bone marrow biopsies and bone marrow
aspiration should be performed to confirm the involvement and to establish a baseline for evaluating therapeutic response [11].

Patients with bone marrow metastases have a poor prognosis due to rapid disease progression and limited response to treatment [12, 13]. In our series, 39% of patients died within days or weeks after puncture. Kucukzeybek et al. [8] reported differences in survival depending on the origin of the carcinoma between breast and gastric carcinomas. In our series of lung carcinoma, the very short mean overall survival of 14 days after puncture is probably linked to the aggressiveness of small cell lung cancer. The poor survival of patients in our cohort is also due to the selection of patients requiring a bone marrow aspiration. Indeed, bone marrow aspiration is performed in patients known to have solid tumors, either when there is an indication for a change in therapeutic line or in cases of suspected drug toxicity induced by chemotherapy or immunotherapy. These patients are typically at a more advanced stage of their disease. There are also patients for whom bone marrow metastases are discovered incidentally because the cancer was previously unknown. These patients are therefore diagnosed at an already advanced stage of their disease, and the bone marrow biopsy becomes advantageous for diagnosis. It can also be used for molecular biology analyses, which are often necessary before the initiation of chemotherapy.

## 5 | Conclusion

Collaboration between hematologic and non-hematologic cytologists is critical to the management of metastatic bone marrow aspirates. In our experience, the techniques used by the cell biology laboratory allow diagnosis without the need for additional samples or biopsies. For these patients, who have a short average survival, obtaining reliable and timely results is essential for initiating or adjusting treatment.

### Author Contributions

**Elise Kaspi:** conceptualization, writing – review and editing, methodology, formal analysis. **Charlotte Grosdidier:** formal analysis, writing – review and editing, resources. **Yaël Berda-Haddad:** formal analysis, writing – review and editing, resources. **Maud Arpin:** formal analysis, writing – review and editing, resources. **Sylvie Cointe:** resources, formal analysis, writing – review and editing. **Shirley Fritz:** resources, formal analysis, writing – review and editing. **Amandine Bonifay:** resources, formal analysis, writing – review and editing. **Marie Koubi:** resources, formal analysis, writing – review and editing. **Carine Jiguet-Jiglaire:** resources, formal analysis, writing – review and editing. **Patrice Roll:** resources, formal analysis, writing – review and editing. **Diane Frankel:** conceptualization, investigation, writing – original draft, methodology, validation, formal analysis, resources, supervision.

### Acknowledgements

We thank Nathalie Boitano, Corinne Derrien, Johan Rinaldi, and Nadine Maggio for their technical assistance.

### Ethics Statement

The project has been reviewed and approved by the local ethics committee of Assistance Publique-Hôpitaux de Marseille (approval

number n°WQ4C73). The data for this study were obtained from patients treated at Assistance Publique-Hôpitaux de Marseille for their diagnosis and treatment. In accordance with the French data protection authority (Commission Nationale de l'Informatique et des Libertés), general information on research activities is provided in clinical units to departments to the persons concerned, who may exercise their right to object, which is verified prior to data collection. This procedure exempts the written informed consent for all patients as granted by the local ethics committee of Assistance Publique-Hôpitaux de Marseille.

### Conflicts of Interest

The authors declare no conflicts of interest.

### Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

### References

1. R. S. Riley, D. Williams, M. Ross, et al., "Bone Marrow Aspirate and Biopsy: A Pathologist's Perspective. II. Interpretation of the Bone Marrow Aspirate and Biopsy," *Journal of Clinical Laboratory Analysis* 23 (2009): 259-307, https://doi.org/10.1002/jcla.20305.

2. E. P. Weinzierl and D. A. Arber, "Bone Marrow Evaluation in New-Onset Pancytopenia," *Human Pathology* 44 (2013): 1154-1164, https://doi.org/10.1016/j.humpath.2012.10.006.

3. S. Gajendra and R. Sharma, "Cytomorphological Evaluation of Non-Haematopoietic Malignancies Metastasizing to the Bone Marrow," *American Journal of Blood Research* 13 (2023): 1-11.

4. P. Chen, R. Chen Xu, N. Chen, et al., "Detection of Metastatic Tumor Cells in the Bone Marrow Aspirate Smears by Artificial Intelligence (AI)-Based Morphogo System," *Frontiers in Oncology* 11 (2021): 742395, https://doi.org/10.3389/fonc.2021.742395.

5. N. Pavlidis and G. Pentheroudakis, "Cancer of Unknown Primary Site," *Lancet* 379 (2012): 1428-1435, https://doi.org/10.1016/S0140-6736(11)61178-1.

6. L. Xiao, S. Luxi, T. Ying, L. Yizhi, W. Lingyun, and P. Quan, "Diagnosis of Unknown Nonhematological Tumors by Bone Marrow Biopsy: A Retrospective Analysis of 10,112 Samples," *Journal of Cancer Research and Clinical Oncology* 135 (2009): 687-693, https://doi.org/10.1007/s00432-008-0503-2.

7. G. Singh, J. R. Krause, and V. Breitfeld, "Bone Marrow Examination: For Metastatic Tumor: Aspirate and Biopsy," *Cancer* 40 (1977): 2317-2321, https://doi.org/10.1002/1097-0142(197711)40:5<2317::aid-cncr2820400546>3.0.co;2-z.

8. B. B. Kucukzeybek, A. O. Calli, Y. Kucukzeybek, et al., "The Prognostic Significance of Bone Marrow Metastases: Evaluation of 58 Cases," *Indian Journal of Pathology & Microbiology* 57 (2014): 396-399, https://doi.org/10.4103/0377-4929.138728.

9. S. Ash, D. Luria, I. J. Cohen, et al., "Excellent Prognosis in a Subset of Patients With Ewing Sarcoma Identified at Diagnosis by CD56 Using Flow Cytometry," *Clinical Cancer Research* 17 (2011): 2900-2907, https://doi.org/10.1158/1078-0432.CCR-10-3069.

10. N. Gaspar, D. S. Hawkins, U. Dirksen, et al., "Ewing Sarcoma: Current Management and Future Approaches Through Collaboration," *Journal of Clinical Oncology* 33 (2015): 3036-3046, https://doi.org/10.1200/JCO.2014.59.5256.

11. A. Gupta, R. F. Riedel, C. Shah, et al., "Consensus Recommendations in the Management of Ewing Sarcoma From the National Ewing Sarcoma Tumor Board," *Cancer* 129 (2023): 3363-3371, https://doi.org/10.1002/cncr.34942.
12. M. Kobayashi, T. Okabayashi, T. Sano, and K. Araki, "Metastatic Bone Cancer as a Recurrence of Early Gastric Cancer—Characteristics and Possible Mechanisms," *World Journal of Gastroenterology* 11 (2005): 5587-5591, https://doi.org/10.3748/wjg.v11.i36.5587.

13. G. Wiedswang, E. Borgen, R. Kåresen, et al., "Detection of Isolated Tumor Cells in Bone Marrow Is an Independent Prognostic Factor in Breast Cancer," *Journal of Clinical Oncology* 21 (2003): 3469-3478, https://doi.org/10.1200/JCO.2003.02.009.