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CASE HISTORY

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. LITERATURE CITED
  6. Supporting Information

A 16-year-old high school sophomore presented to his primary care physician (PCP) with a 5-day history of fever and flu-like symptoms. Despite a course of antibiotics, his symptoms did not improve, and he returned to his PCP where a strep test was performed and was positive, but a low white blood cell count (WBC) was noted. He was sent to the emergency room (ER), where his labs showed the following: WBC: 2.02 × 103/μl, absolute neutrophil count (ANC) of 750, hemoglobin and hematocrit of 10.8 gm/dl and 29%, respectively, mean corpuscular volume (MCV) of 100.6 femtoliters (FL), and platelets of 193,000/μl. He was evaluated by Hematology/Oncology, and the peripheral smear was reviewed with no abnormal cells seen, and the patient was sent home. A few days later, he represented to his PCP with continued symptoms, and was again transferred to the ER. His ANC had dropped to 500 (other hematological parameters approximately the same), and the patient was admitted for further work up and possible IV antibiotics. Review of a second peripheral smear was also read as negative by Hematology/Oncology. Flow cytometric studies performed on a bone marrow obtained on day 8 following induction therapy had similar findings and is presented below.

FLOW CYTOMETRIC STUDIES

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. LITERATURE CITED
  6. Supporting Information

Flow cytometry performed on peripheral blood showed a 2% abnormal population of unclear lineage. Positive markers included CD2, CD7, partial CD13, partial CD22 (surface only), partial CD34, partial CD117, HLA-DR, and CD45 (dim). Of note, neither myeloperoxidase nor surface or intracytoplasmic expression of CD3 was detected. Flow cytometry on the bone marrow aspirate was diagnostic, but was performed at an outside facility and is not available for review. Flow cytometric studies performed on a bone marrow obtained on day 8 following induction therapy that had similar findings.

Flow cytometry was performed using the following antibody/fluorochrome combinations listed in order FITC/PE/PerCP-Cy5.5/APC: isotype, CD14/CD13/CD45/CD34, HLA-Dr/CD22/CD45/CD33, sCD3/CD4/CD45/CD8, CD2/CD7/CD45/CD5, CD10/CD19/CD45/CD34, CD10/CD20/CD45/CD19, CD36/CD117/CD45/CD34, CD15/CD11b/CD45/CD34, CD16/CD56/CD45/sCD3, CD61/CD64/CD45/CD33, kappa/lambda/CD45/CD19, Intracytoplasmic TdT/CD22/CD45/CD3, Intracytoplasmic MPO/Lacto/CD45/CD34, CTL/CTL/CD45/CTL (CD36, CD117, CD16, CD64, Beckman Coulter, Brea, CA; Kappa and Lambda, Dako, Carpinteria, CA; remaining antibodies Becton Dickinson, San Jose, CA; CD15 BD Pharmingen). Acquisition was performed using the Canto II BD flow cytometer. Given that the patient was a child, where the incidence of B lymphoblastic leukemia is relatively high, gating on mononuclear cells was performed using forward scatter (FSC) versus side scatter (SSC), as opposed to CD45 versus SSC, to avoid exclusion of CD45 negative cells and a low threshold for FSC was set to identify cells smaller than normal lymphocytes. List mode files for the following tubes are available for download from the website: tube B4: sCD3/CD4/CD45/CD8; tube B5: CD2/CD7/CD45/CD5; tube B8: CD36/CD117/CD45/CD34; tube IC: Intracytoplasmic TdT/CD22/CD45/CD3.

DISCUSSION

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. LITERATURE CITED
  6. Supporting Information

The bone marrow shows a blast population comprising ∼33% of the total events analyzed which express CD2, CD3 (cytoplasmic only), CD7, CD13, partial CD15, partial CD22, CD34, partial CD36, partial CD117, HLA-DR, TDT, and CD45 (dim) (Fig. 1; see additional analysis as Supporting Information, web only). Of note, expression of CD4, CD5, and CD8 are not detected. In addition, by immunohistochemistry, CD1a was negative on the marrow core biopsy. These findings indicate involvement by T-lineage lymphoblastic leukemia, with a distinct phenotype.

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Figure 1. Flow cytometric findings from bone marrow obtained on day 8 following induction therapy. Blasts are highlighted in red in all images. Lymphocytes are highlighted in purple for the top two images only. The lower six plots display “mononuclear cells” only gated from the plot of FSC versus SSC. The lower right image displays cytoplasmic staining for CD3; there was lack of surface staining for CD3 (not shown).

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Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, and T-cell ALL accounts for 10–15% of pediatric ALL cases. While once a nearly uniformly fatal disease, now with modern chemotherapy regimens, close to an astonishing 80% of pediatric patients can be cured. Unlike B lymphoblastic leukemia (B-ALL), where cytogenetic analysis is a cornerstone in prognosis and treatment, it does not provide consistent insight into the biology of T-ALL. It is, therefore, difficult to predict which cases of T-ALL will respond favorably with modern chemotherapy regimens and which will have poor prognosis.

Recently, St. Jude Children's Research Hospital in collaboration with Associazione Italiana Ematologia Oncologia Pediatrica described a distinct subgroup of T-ALL with particularly poor prognosis (1). By gene expression profiling, the leukemic cells closely resembled early T-cell precursors (ETPs), which are a subgroup of thymocytes which have very recently immigrated from the bone marrow. These cells still have the potential to differentiate along T and myeloid lineages, which suggest they are closely related to multipotent hematopoietic stem cells. It is thought that this distinct group of T-ALL arises from a transformed ETP, and comprises ∼12% of all childhood T-ALL.

Gene expression profiling of 55 T-ALLs from St. Jude collected over a 5-year time frame showed a cluster of 13 cases with a unique genetic signature, showing similarity to early T-cell precursors. Overexpressed genes included CD44, CD34, KIT, GATA2, CEPBA, SPI1, ID2, and MYB. Underexpressed genes included CD1, CD3, CD4, CD8, RAG1, NOTCH3, and ZAP70. As there are no reliable cytogenetic characteristics unique to this entity, and gene expression profiling is not feasible in the clinical laboratory, a distinct immunophenotype served as a surrogate marker in 9 of these 13 cases and is critical to the recognition of this entity.

The immunophenotype that characterizes early ETP-ALL is as follows:

  • 1
    Absence (<5% positive lymphoblasts) of CD1a and CD8 expression.
  • 2
    Weak CD5 expression (in less than 75% of lymphoblasts).
  • 3
    Gain of one or more of the myeloid/stem cell antigens on at least 25% of the lymphoblasts: CD117, CD34, HLA-DR, CD13, CD33, CD11b, or CD65.

It is of interest to note that these cases of ETP-ALL would not fulfill the WHO criteria of mixed phenotype acute leukemia, T/myeloid, as there was no co-expression of cytoplasmic CD3 and myeloperoxidase. The blasts also do not show monoblastic differentiation as outlined by WHO criteria (2). Also, simply the presence of myeloid markers does not confer a poor prognosis; it is the entire constellation of immunophenotypic findings, including loss of CD1a, CD8, and weak/absent CD5 expression in combination with myeloid/stem cell antigens that confers the ETP immunophenotype.

So how did the patients identified at St. Jude with ETP-ALL do? The rate of clearance of leukemic cells after the first phase of induction chemotherapy was much worse in patients with ETP-ALL. As evidence to this, marrow evaluation at the end of induction (day 43) showed 71% (10/14) minimal residual disease (MRD) positivity among the ETP group versus 24% (28/116) of those with typical T-ALL. Forty-three percent (6/14) of those with ETP-ALL had MRD of at least 1%, indicating an extremely poor prognosis, as compared with only 5% (6/116) of the typical T-ALL group. In line with positive MRD, patients with ETP-ALL had a significantly worse outcome, with a 10-year overall survival of 19% versus 84% for typical T-ALL, and a 10-year event free survival of 22% versus 69%. In fact, by univariate and multivariate analysis, the diagnosis of ETP-ALL had the strongest negative effect on event free survival, even more than MRD status! The long-term response to therapy is equal to or worse than Ph+ B-LL or infant ALL with MLL gene rearrangement. They proposed that as these cells are very early in development and still have multilineage differentiation potential, therapy targeted at lymphoid cells is not as effective. As such, St. Jude has modified their approach to these patients to include bone marrow transplantation in first remission, after consolidation and reintensification therapy.

In a separate study, a group headed by Dr. Brent Wood examined the immunophenotype on 416 patients with T-ALL enrolled on the Children's Oncology Group study AALL03B1 (3). Twenty-five of 416 patients (6%) had the ETP-phenotype, and these patients tended to be older and have a lower white blood cell count at presentation (<50,000/μl) than typical T-ALL. Their findings confirmed the St. Jude study that patients with the ETP-ALL phenotype had a dramatically higher levels of residual disease at end of induction, with 100% having ≥0.01% disease at day 29 (compared with 46% of typical T-ALL) and 74% having ≥1% day 29 MRD (vs. 21% of typical T-ALL). Literature on the adult experience with ETP-ALL also confirms the pediatric findings. A German group headed by Dr. Martin Neumann studied 86 adult patients with T-ALL by gene expression profiling, of which 17 (20%) had and ETP gene expression profile, which showed differential expression of 2065 probe sets (4). In addition to gene profiling, these authors also examined the immunophenotype of 297 T-ALLs as well as using PCR to test expression levels of BAALC, IGFBP7, MN1, and WT1. Of these, 19 (6.4%) showed the ETP-phenotype. Key genes included upregulation of BAALC, IGFBP7, MN1, and WT1, and downregulation of HOX11, a marker of thymic T-ALL. The authors noted that MN1 and WT1 are also upregulated in cytogenetically normal acute myeloid leukemia (AML), which may indicate a closer relationship of ETP-ALL to AML versus typical T-ALLs. These authors supported classifying ETP-ALL in adults as high risk, and recommended assignment to bone marrow transplantation.

At Children's Healthcare of Atlanta (CHOA), we have seen 2 cases of ETP-ALL since January 2009, when the paper by St. Jude was published: the current case and an 11-year-old girl, both of which received bone marrow transplantation in first remission, and remain disease free at 18 and 10 months post diagnosis. We have also had a 22-year-old young man transfer to our institution in relapsed ETP-ALL, who died within 2 months of arrival to CHOA.

In conclusion, ETP-ALL is a subset of T-lymphoblastic leukemia with higher levels of minimal residual disease and a particularly poor outcome. While first recognized in the pediatric population, this entity is also applicable in adults and shows similar prognosis. The main way of detecting this subpopulation currently is by immunophenotype (CD1a, CD8, CD5weak, with expression of myeloid/stem cell markers). Recognition of this entity is critical, as bone marrow transplantation in first remission is recommended, as opposed to conventional chemotherapy.

CASE 3 DIAGNOSIS: Early T-cell Precursor-Acute Lymphoblastic Leukemia

LITERATURE CITED

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. LITERATURE CITED
  6. Supporting Information
  • 1
    Coustan-Smith E, Mulligan CG, Onclu M, Behm FG, Raimondi SC, Pei D, Cheng C, Su X, Rubnitz JE, Basso G, Biondi A, Pui CH, Downing JR, Campana D. Early T-cell precursor leukemia: A subtype of very high-risk acute lymphoblastic leukemia. Lancet Oncol 2009; 10: 147156.
  • 2
    IARC Publication. WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Publication; 2008. pp 150154.
  • 3
    Wood B, Winter S, Dunsmore K, Raetz E, Borowitz MJ, Davidas M, Winick NJ, Carroll WL, Hunger SP, Loh ML. Patients with early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) have high levels of minimal residual disease (MRD) at the end of induction—a children's oncology group (COG) study. In: Oral and Poster Abstracts #9, American Society for Hematology (ASH) Meeting, New Orleans, LA, December, 2009.
  • 4
    Neumann M, Heesch S, Schwartz S, Gokbuget N, Hoelzer D, Haferlach T, Hofmann W-K, Thiel E, Baldus CD. Early T-cell precursor-ALL in adult T-ALL. In: Oral and Poster Abstracts #911, American Society for Hematology (ASH) Meeting, New Orleans, LA, December, 2009.

Supporting Information

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. LITERATURE CITED
  6. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_20599_sm_suppinfo1.fcs185KSupporting Information 1
CYTO_20599_sm_suppinfo2.fcs194KSupporting Information 2
CYTO_20599_sm_suppinfo3.fcs170KSupporting Information 3
CYTO_20599_sm_suppinfo4.fcs119KSupporting Information 4
CYTO_20599_sm_suppinfoCase3.pdf66KFigure 1: Blasts (red) and normal lymphocytes (purple) displayed in FSC vs. SSC and CD45 vs SSC. Blasts are CD45 dim. Figure 2: Blasts fail to express surface CD3, CD4, or CD8 Figure 3: Blasts show expression of CD2 and CD7, but fail to express CD5 Figure 4: Blasts show co-expression of CD34 and dim CD13 Figure 5: Blasts are HLA-DR positive, whereas CD33 expression is largely negative Figure 6: A minor subset of blasts show CD36 expression. CD117 expression is dim. Figure 7: A minor subset of blasts show CD15 expression. Figure 8: Intracytoplasmic/intranuclear staining is positive for CD3 and TDT

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