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Keywords:

  • TIM3;
  • acute myeloid leukemia;
  • flow cytometry;
  • myeloblasts

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Background:

T-cell immunoglobulin mucin-3 (TIM3) has recently been described as an acute myeloid leukemia (AML) stem cell antigen expressed on leukemic myeloblasts, but not on normal hematopoietic stem cells. TIM3 is also expressed by monocytes, natural killer cells, and several T cell subsets; however, normal myeloblasts have not been well-characterized or compared to AML. A specific flow cytometric marker capable of separating leukemic myeloblasts from non-neoplastic myeloblasts would be diagnostically useful, especially in the post-chemotherapy setting.

Methods:

TIM3 myeloblast expression was assessed in 69 bone marrow and/or peripheral blood specimens, including 27 AML and 42 non-neoplastic cases (20 with a recent history of chemotherapy). TIM3 median fluorescence intensity (MFI) was evaluated within myeloblast, monocyte, T cell, and natural killer cell populations.

Results:

The median percentage of myeloblasts positive for TIM3 was lower in non-neoplastic specimens without a history of recent chemotherapy (50.3%) as compared to AML (71.4%), but not significantly different as compared to non-leukemic myeloblasts in the post-chemotherapy setting (72.4%). Mean myeloblast TIM3 MFI was higher in AML myeloblasts and non-leukemic myeloblasts in the post-chemotherapy setting as compared to non-neoplastic myeloblasts in cases lacking a history of chemotherapy. Mean monocyte, natural killer cell, and T-cell TIM3 MFI remained relatively constant in varied clinical settings.

Conclusions:

We confirm that leukemic myeloblasts overexpress TIM3 as compared to non-neoplastic controls; however, high levels of expression may also be seen among non-leukemic myeloblasts in the post-chemotherapy setting. This overlap limits the diagnostic utility of TIM3 as a specific marker of neoplasia. © 2013 International Clinical Cytometry Society


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

T-cell immunoglobulin mucin-3 (TIM3) has recently been described as a unique acute myeloid leukemia (AML) stem cell antigen that is not present on normal hematopoietic stem cells (1). TIM3 has been shown to be expressed in most leukemic stem cells (LSC) as well as CD34 positive leukemic blasts in the majority of AML subtypes (2). Although the specific mechanism underlying upregulation is unclear, targeted therapy with an anti-TIM3 monoclonal antibody has shown efficacy in reducing leukemic burden in a mouse model (2).

The role of TIM3 myeloblast expression in AML is also unclear. TIM3 is also known be expressed in natural killer (NK) cells, monocytes, and a subset of T cells (2). The TIM3/Galectin-9 (Gal-9) pathway has previously been characterized as a negative regulator of T cell-mediated immune responses (3–5), as binding of TIM3 to its Gal-9 ligand causes Th1 cell death (6). Gal-9 treatment has been shown to reduce the number of cytotoxic T cells and ameliorate progression of graft versus host disease in murine transplantation models (7). In addition to this inhibitory role, TIM3 has also been shown to have an activating function in other circumstances. TIM3 expression on monocytes/macrophages increases the number and activation of macrophages, and also enhances secretion of the TNF-alpha cytokine, indicating a possible role of TIM3 in promoting inflammation (8, 9). In AML, a prior functional study focused on natural killer cells (NK), showing enhanced interferon-gamma production by TIM-3 overexpressing NK cells against galectin-9 (Gal-9) positive AML primary tumors (10). Although this study has important therapeutic implications for AML therapy, it did not address the function of TIM3 expression on leukemic myeloblasts.

The initial studies that have reported TIM3 expression in AML have focused on evaluating TIM3 expression in LSC, which requires specialized flow cytometric sorting techniques and would not be broadly applicable to routine clinical assessment. Given that this antigen is also expressed on the non-LSC “bulk” AML cell compartment, we hypothesized that detection of TIM3 overexpression could aid in the identification of neoplastic myeloblasts. This distinction would be especially diagnostically useful during the post-chemotherapy time interval, in which the differential diagnosis is often between regenerating versus residual leukemic myeloblasts. However, TIM3 myeloblast expression in non-neoplastic myeloblasts, especially in the post-chemotherapy setting, has not been well characterized. The aim of this study was to evaluate the diagnostic utility of flow cytometric TIM3 expression in separating leukemic from non-leukemic myeloblasts in a variety of clinical settings.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Specimens Analyzed for TIM3 Expression by Flow Cytometry

The study was performed in accordance with the University of Pittsburgh Institutional Review Board protocols. Sixty-five bone marrow and four peripheral blood specimens were evaluated between January 2012 and July 2012. Cases were selected based on clinical use of the acute leukemia/myelodysplastic syndrome (MDS) immunophenotyping panel, and/or clinical indication of the specimen as a staging marrow for lymphoma. The diagnostic material (Wright-Giemsa-stained aspirate smears, H&E stained bone marrow biopsy section, flow cytometric histograms, additional immunohistochemical studies as available) was re-reviewed by one of the authors (C.G.R) to confirm the diagnosis and disease category.

The study cohort consisted of 27 AML, 22 non-neoplastic cases without a history of recent chemotherapy, and 20 negative post-chemotherapy cases. The AML included 15 with no history of recent chemotherapy, 9 with a history of chemotherapy within 3 months, and 3 with a recent history of azacitidine therapy. 13 AML cases showed a normal karyotype; 13 showed an abnormal karyotype including 3 complex karyotype (≥3 abnormalities), 2 inversion (16)/CBFB-MYH11, 1 t(15;17)/PML-RARA, 1 inversion(3)/RPN1-EVI1. One AML case did not have cytogenetic information available aside from a negative PML/RARA fluorescence in-situ hybridization study. All four of the peripheral blood specimens were within the AML category, and the concurrent bone marrow evaluations accompanying three out of four cases were also re-reviewed for diagnosis confirmation; these bone marrow specimens did not undergo TIM3 flow cytometric testing. The 22 non-neoplastic controls did not have any history of prior AML and consisted of 7 negative staging marrows for lymphoma, and 15 marrows with a clinical history of cytopenias, but no overt morphologic dysplasia and normal karyotype by classical cytogenetic analysis. The 20 negative post-chemotherapy controls ranged from day 14 to 4 months (median: 29 days) post-chemotherapy for a previously diagnosed AML, with less than 5% morphologic blasts and normal cytogenetics in those with previously abnormal cytogenetic results.

Multiparameter Flow Cytometry

Greater than 90% viability as determined by Trypan blue exclusion was required for study inclusion. Excess cells from clinical samples were adjusted to approximately 500,000 cells/100 μl, suspended in 2% fetal calf serum and incubated with the eight-color antibody cocktail in the dark for 15 min at 4° F. The following fluorochrome-labeled monoclonal antibody cocktail was used: CD14− FITC (clone M φP9), TIM-3-PE (clone 344823), CD117 PerCP-Cy5-5 (clone 104D2), CD13/33-PE-Cy7 (L138/P67.6), CD34-APC (clone 8G12), CD3-APC-H7 (clone SK7), CD56-V450 (B159), and CD45-V500 (clone HI30). All antibodies were obtained from BD Biosciences (San Jose, CA) with the exception of TIM-3-PE, which was obtained from R&D Systems (Minneapolis, MN). After incubation, red blood cells were lysed with 3 ml FACS Lyse (BD Biosciences, San Jose, CA) for 8 min. The sample was then centrifuged, washed with 3 ml phosphate buffered saline (PBS) with 0.1% sodium azide and centrifuged again. Remaining cells were fixed with 100 μl of 2% formaldehyde.

A maximum of 30,000 events were acquired using a BD FACSCanto II instrument, and analyzed using FACSDiva software (BD Biosciences, San Jose, CA). Beads were used for instrument standardization. Nearly all of the samples were acquired on the flow cytometer within 48 h, with the exception of one specimen. This case was tested after 57 h and showed no significant immunophenotypic differences in CD14, CD117, CD13/33, CD34, CD3, CD56, or CD45 expression among the myeloblast, monocytic, T cell, or NK cell populations as compared to the flow cytometric clinical evaluation.

Gating Strategy

Initially, linear FSC area versus FSC height plots was used to exclude potential doublets. Nonspecific staining was identified by visual inspection of the histograms and excluded. SSC-A plots were used to identify pertinent cell populations: CD34+blasts with low side scatter; CD117+ immature precursors with low side scatter; CD14+ monocytes with intermediate side scatter; and CD3+ T-cells with low side scatter. Natural killer cells were identified by additional gating to identify a CD45bright+/CD3−/CD56+ population.

TIM-3 positivity was evaluated specifically within the CD34+/CD13/33+ myeloblast population, or within the CD117+/CD13/33+ myeloblast population if CD34 was negative on the leukemic myeloblast population, and was quantified and recorded as a percentage of myeloblasts. The threshold for TIM-3 positivity was determined by evaluation of fluorescence minus-one (FMO) controls of 12 sequential specimens of varied diagnoses. The threshold for TIM-3 positivity on myeloblasts was set at a value two standard deviations above the mean value of nonspecific reactivity. There were greater than 100 TIM3 positive, CD13/33 positive events in most cases, except for 2 AML cases and 9 negative post AML chemotherapy cases in which the number of events ranged from 22 to 82. TIM3 median fluorescence intensity (MFI) was also measured among CD34+ or CD117+, CD13/33 positive myeloblasts, CD14+ monocytes, CD3+ T cells, and CD56+/CD3− natural killer cells, which were identified as previously described.

Statistical Analysis

TIM3 MFI expression levels in different cell types and percentages of TIM3 positive myeloblasts were compared using the Mann–Whitney U-test with the aid of GraphPad Prism version 5 (San Jose, CA). A P-value of <0.05 was considered statistically significant. Receiver operator curve (ROC) analysis was also performed with GraphPad Prism software.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Nonleukemic Myeloblasts Show Lower Levels of TIM3 Expression than AML Myeloblasts, but not in the Post-chemotherapy Setting

The median percentage of myeloblasts positive for TIM3, calculated as a percentage of CD34+ or CD117+, CD13/33+ myeloblasts, was 71.4% in AML. This percentage was significantly higher than the 50.3% TIM3 positive myeloblasts in non-neoplastic specimens (P = 0.0036) (Table 1 and Fig. 1). Of the four AML cases with very high proportions of TIM3 positive myeloblasts (greater than 90%), two were associated with the recurrent genetic abnormality inversion (16)/CBFB-MYH11. However, the 71.4% median leukemic myeloblast TIM3 positivity in AML was not significantly different than the 72.4% median TIM3 positive non-leukemic myeloblasts in negative post-chemotherapy specimens (P = 0.79), even with excluding cases with less than 100 events from both categories (P = 0.89).

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Figure 1. Percentage of TIM3 positive myeloblasts in AML, non- neoplastic, and negative post-chemotherapy specimens

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Table 1. Percentage of TIM3 Positive Myeloblasts and Median Fluorescence Intensity in Acute Myeloid Leukemia, Non-neoplastic, and Negative Post-chemotherapy Specimens
 Median % myeloblastsMedian % myeloblasts TIM3+ (range)Mean myeloblast TIM3 MFI (range)
  1. MFI, median fluorescence intensity; AML, acute myeloid leukemia.

AML13.5%71.4% (20.0–94.3)816 (189–2705)
Non-neoplastic0.8%50.3% (26.1–78.1)455 (213–826)
Negative post-chemotherapy0.4%72.4% (47.2–88.3)1024 (258–3598)

Given that some of the AML cases had a recent history of induction or consolidation chemotherapy, further analysis was performed on this subset. The nine AML cases with a history of recent chemotherapy showed 78.4% median percentage of TIM3 positive myeloblasts, which did not differ significantly from the 85.9% median percentage of TIM3 positive myeloblasts in the 15 AML cases without a history of recent chemotherapy (P = 0.81). Neither AML with a history of chemotherapy nor without a history of chemotherapy showed a significant difference in median TIM3 myeloblast percentage as compared to negative post-chemotherapy specimens (P > 0.38 for both). Both AML with and without a history of chemotherapy retained a significantly increased level of TIM3 positivity as compared to myeloblasts in non-neoplastic specimens (P = 0.018 and P = 0.0094, respectively).

Although both leukemic and non-leukemic myeloblasts expressed TIM3, 22% (6/27) AML cases showed bright intensity staining of TIM3 (>1,000 absolute fluorescence units) as compared to none of the non-neoplastic cases. The six AML cases with bright intensity staining of TIM3 included three with a recent history of chemotherapy. Interestingly, 30% (6/20) negative post-chemotherapy specimens also showed very strong TIM3 myeloblast expression. In fact, mean myeloblast TIM3 MFI was highest in negative post-chemotherapy specimens (mean: 1,024), followed by AML (mean: 816) and non-neoplastic specimens (mean: 455) (Fig. 2, 3). Leukemic myeloblasts in AML showed brighter intensity expression as compared to non-neoplastic myeloblasts without a history of chemotherapy (P = 0.01), even when excluding cases with less than 100 events analyzed (P = 0.0238). However, no significant differences in intensity were seen in comparison of AML to non-leukemic myeloblasts in the post-chemotherapy setting (P = 0.43). These results remained the same even with exclusion of cases with less than 100 events from both categories (P = 0.71). Non-leukemic myeloblasts in the post-chemotherapy setting showed higher intensity expression as compared to non-leukemic myeloblasts without a history of prior chemotherapy (P = 0.0004), even when excluding cases with less than 100 events (P = 0.0071).

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Figure 2. Representative examples of TIM3 expression in myeloblasts (black) as compared to T cells (blue), NK cells (red), and monocytes (green) in AML (left panel), non-neoplastic (middle panel), and negative post-chemotherapy (right panel) specimens. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 3. Median myeloblast TIM3 fluorescence in AML, non-neoplastic, and negative post-chemotherapy specimens.

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Diagnostic Utility of TIM3 Expression in Leukemic Myeloblasts

To evaluate TIM3 as a potential marker for leukemic myeloblasts, ROC analysis was undertaken. In evaluating AML versus non-neoplastic samples, the area under the curve was 0.7449 with a TIM3 percentage cut off of >66% discriminating between leukemic and non-leukemic myeloblasts (sensitivity: 59%, specificity: 95%). However, when the negative post-chemotherapy specimens were added to the negative control group, a value of >84.6% was required to distinguish neoplastic from non-neoplastic myeloblasts, the area under the curve decreased to 0.6398, accompanied by a markedly reduced specificity (37%) with only minimal improvement of the specificity (97.6%).

Monocytes, Natural Killer Cells, and a Subset of T Cells Show Consistent Levels of TIM3 Expression in a Variety of Conditions

The level of TIM 3 expression on non-myeloblast hematolymphoid cells was also characterized. In non-neoplastic controls, TIM3 is expressed with the strongest intensity on monocytes (mean MFI = 1950) and natural killer cells (mean MFI = 879) and the lowest intensity on T cells (mean MFI = 105). The TIM3 MFI within a specific non-myeloblast population did not vary by the clinical setting. Similar mean MFI values within monocytes, natural killer cells, and T cell populations were found in AML as compared to non-neoplastic specimens (P = 0.36, 0.23, and 0.32, respectively) as well as negative post-chemotherapy cases (P = 0.28, 0.35, and 0.45, respectively). Representative examples of myeloblast, monocytic, T-cell, and NK-cell TIM3 expression in AML, non-neoplastic marrows, and negative post-chemotherapy follow-up specimens are shown in Figure 2. By visual inspection of the histograms, monocytic cells generally showed the strongest expression, followed by natural killer cells, and a subset of the myeloblasts. T cells were generally negative, but in some cases, a subset of T cells showed TIM3 expression. Overall, TIM3 expression within non-myeloblast hematolymphoid populations appeared relatively stable across a spectrum of AML and non-neoplastic specimens.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Current AML research efforts include identification of novel markers useful in the diagnosis and follow-up of this clinically heterogenous disease. For example, the leukemic stem cell (LSC)-associated marker human myeloid inhibitory C-type lectin-like receptor (hMICL) has recently been shown to be a robust diagnostic marker for AML with a very homogenous immunoprofile in both diagnostic and relapse samples (11). TIM3 has recently been described as a novel LSC marker that is highly expressed in AML, potentially enabling separation of non-neoplastic hematopoietic stem cells from LSC (1). These investigators also demonstrated that the non-LSC “bulk” AML compartment overexpressed TIM3, with TIM3 expression levels comparable to TIM3 positive LSC. However, TIM3 expression among non-leukemic myeloblasts has not been well characterized, and it is unknown whether or not myeloblast TIM3 expression is a reliable, specific, or sensitive marker to distinguish between leukemic and non-leukemic myeloblasts in varied clinical settings.

In our study comparing 27 AML with 42 non-neoplastic cases, we confirm prior observations that TIM3 is overexpressed on leukemic myeloblasts in AML. However, we also noted increased proportions and intensity of TIM3 positive myeloblasts in the post-chemotherapy setting, which limits the diagnostic utility. One limitation of our study is the relatively small number of patients, and lack of long-term clinical follow-up. For patients with AML in long-term complete remission, it is unknown if and when non-neoplastic myeloblast TIM3 expression normalizes, which would be an interesting question to address in a future investigation. For neoplastic myeloblasts, the reason for the increased TIM3 expression in AML is not clear. It has been proposed that genetic mutations that influence myeloid differentiation may upregulate transcription of TIM3 or cause maturation arrest. Interestingly, TIM3 expression in AML has been described to be significantly higher in AML associated with core binding factor translocations or mutations in CEBPA (1, 2). Our study included only two inversion (16)/CBFB-MYH11 cases with core binding factor translocations but both showed very high proportions of TIM3 positive myeloblasts (>90%), in line with the prior literature. Interestingly, TIM3 has been reported to be absent in acute promyelocytic leukemia (APML) (2), but our one case showed 86.7% TIM3 positive myeloblasts, which was within the range seen in non-APML AML. Further study is warranted in order to explore this interesting observation.

It is intriguing to consider the possibility that upregulated TIM3 expression may help create a microenvironment favorable for proliferation of the leukemic clone. Several lines of evidence hint at this possibility. First, transcription of the Chemokine (C-C motif) Ligand 1(CCL1) gene has been shown to be increased in a subset of human T cells abundantly expressing TIM3 as compared to cells with low TIM3 expression levels (12). Blockade of CCL1 inhibits T cell-mediated suppressive function (13), and therefore, increased CCL1 could enhance T-cell suppressive function and result in a reduced anti-tumor response. It may be of interest to evaluate the levels of CCL1 transcripts in TIM3 overexpressing myeloblasts in future studies. Second, upregulated TIM3 expression has been noted in human mast cells and melanoma tumor cells in response to transforming growth factor (TGF)-beta, and it has been suggested to contribute to the lower adhering capacity of tumor cells as well as an autocrine-mediated local immunosuppression (14). Third, recent work has shown that ectopic expression of TIM3 in mouse and human T cells can upregulate TCR signaling and T cell cytokine production (15). This includes the Akt/S6 signaling pathway, which has also been found to be upregulated in AML samples in a subset of relapsing patients (16). Recent studies of anti-TIM3 blockade in combination with anti-PD1 and/or anti-CTLA-4 monoclonal antibodies against a variety of established murine tumors have shown promising disruption of the pathophysiologic inhibition of T cell activity (17). Further development of this work will be important to assess the clinical benefit of blocking TIM3 and other human proteins important in the immune response in AML and other diseases.

In summary, in characterizing TIM3 expression within AML and non-leukemic myeloblasts, we found higher proportions and intensity of TIM3 expression in leukemic myeloblasts as compared to non-neoplastic myeloblasts without a history of recent chemotherapy. However, TIM3 myeloblast expression is also upregulated after chemotherapy, and this overlap in expression limits its utility in the post chemotherapy setting in distinguishing between leukemic and regenerating, non-neoplastic myeloblasts. Although flow cytometric expression of TIM3 might be a useful adjunct in establishing a de novo diagnosis of AML, myeloblast TIM3 expression cannot be used in isolation to definitively establish the presence of residual leukemia in routine clinical practice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

We acknowledge the excellent administrative assistance provided by Janice Kizior. We have no relevant conflicts of interest to declare.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
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