CD44 expression in bone marrow interstitial fluid of hematological malignancies and marrow blasts

Technical Summary: CD44 and TIM-3 Expression in Bone Marrow Interstitial Fluid of Hematological Malignancies

Problem Statement
Leukemic stem cells (LSCs) are recognized as a primary driver of therapeutic resistance and relapse in hematological malignancies, particularly Acute Myeloid Leukemia (AML). While surface markers such as CD34, CD38, CD44, and T-cell immunoglobulin and mucin domain 3 (TIM-3) have been identified to define LSC phenotypes, previous prognostic significance for CD44 and TIM-3 has been established primarily through histopathological tissue analysis or serum measurements. There is a gap in understanding the concentration and clinical relevance of these markers specifically within the bone marrow interstitial fluid (BMIF), the microenvironment where leukemic cells interact with the host. This study aimed to define the expression levels of CD44 and TIM-3 in BMIF at the time of diagnosis and correlate these findings with clinical parameters in patients with hematological malignancies.

Methodology
The study was a cross-sectional analysis conducted at Chi-Mei Medical Center involving 88 patients divided into three groups:

• Group 1 (Myeloid Neoplasm): 28 patients (16 newly diagnosed AML, 12 Myelodysplastic Syndrome [MDS]).
• Group 2 (Lymphoid Neoplasm): 32 patients with various lymphomas (including Diffuse Large B-Cell Lymphoma, Myeloma, Follicular Lymphoma, etc.) undergoing bone marrow staging.
• Group 3 (Control): 28 patients without hematological malignancies (e.g., aplastic anemia, viral infections) who underwent diagnostic bone marrow aspiration.

Sample Collection and Analysis:

• Bone marrow samples were collected during the first diagnostic aspiration.
• Bone marrow interstitial fluid (BMIF) was isolated from the aspirate using centrifugation.
• Quantification: CD44 and TIM-3 concentrations were measured using Enzyme-Linked Immunosorbent Assay (ELISA) with a DuoSet development system. Monoclonal capture antibodies (anti-human CD44 and TIM-3) were used, followed by biotinylated detection antibodies.
• Statistical Analysis: Quantitative data were expressed as mean ± standard error of the mean (SEM). Comparisons between groups utilized the two-sample t-test. To mitigate the effect of extreme values, Winsorized means were applied for concentration analysis. Statistical significance was set at $P \leq 0.05$.

Key Results

1. Elevated Marker Levels in Malignancy: Patients with hematological malignancies exhibited significantly higher levels of CD44 in BMIF compared to the non-malignant control group ($40.24 \pm 5.45$ ng/ml vs. $16.36 \pm 2.44$ ng/ml).

   • CD44 levels were elevated in both myeloid ($39.37 \pm 5.02$ ng/ml) and lymphoid ($42.64 \pm 9.97$ ng/ml) malignancy groups compared to controls.
   • TIM-3 levels were also higher in the malignancy group overall ($7.00 \pm 1.32$ ng/ml) compared to controls ($3.13 \pm 0.43$ ng/ml). However, statistically significant elevation over controls was observed specifically in the lymphoid malignancy group ($8.46 \pm 2.24$ ng/ml, $P=0.04$).

2. Impact of Bone Marrow Involvement:

   • CD44: Expression was significantly higher in patients with hematological malignancies that had documented bone marrow involvement ($51.25 \pm 6.86$ ng/ml) compared to those without involvement ($14.57 \pm 1.61$ ng/ml, $P < 0.01$).
   • TIM-3: Similarly, TIM-3 expression was significantly higher in malignancies with bone marrow involvement ($8.59 \pm 1.75$ ng/ml) versus those without ($2.97 \pm 0.32$ ng/ml, $P < 0.01$).

3. Correlation with Blast Counts in AML/MDS:

   • In the AML and MDS subgroups, CD44 expression in BMIF showed a positive correlation with bone marrow blast percentages.
   • In contrast, TIM-3 expression did not demonstrate a correlation with blast percentages in AML/MDS patients.

Significance and Claims
The paper concludes that both CD44 and TIM-3 are elevated in the bone marrow interstitial fluid of patients with hematological malignancies, distinguishing them from non-neoplastic controls. The study highlights a specific clinical utility for CD44 in the context of myeloid neoplasms (AML and MDS), where its expression level in the marrow fluid correlates positively with the burden of blast cells.

The authors note that while TIM-3 is elevated in malignancies with marrow involvement, it lacks the specific correlation with blast counts in AML/MDS that CD44 demonstrates. The study positions CD44 as a potential marker reflecting the tumor burden in the marrow microenvironment for myeloid diseases. The authors maintain a modest scope, stating that while these findings suggest a role for CD44 and TIM-3 as cancer stem cell markers, further studies are required to fully define their therapeutic implications and clinical impact. The work builds upon prior research indicating CD44's role in leukemic cell adhesion and resistance, and TIM-3's association with poor survival, by providing data specifically from the interstitial fluid compartment.