Introduction

Abnormal immature T-cell proliferation results in T-cell acute lymphoblastic lymphoma/leukemia (T-ALL/LBL), a malignant disorder. The thymus and bone marrow are home to aberrant T-cell lymphoblasts that are growing rapidly. It is regarded as a subtype of ALL, the most prevalent form of cancer in children, however it can also strike adults, making for 15% of cases of acute leukemia. 15% of ALL cases in children and 25% of ALL cases in adults are caused by T-ALL.^[1] An approximate estimate of the yearly incidence is one per 100,000 individuals. The diagnosis depends on establishing clonality or abnormal pattern of maturity, immature nature of the disease and the T-cell lineage.^[2]

Increased N:C ratio, uneven nuclear membranes, and fine nuclear chromatin with little cytoplasm are all seen in T-ALL blasts. Nuclei can be uneven, oval, or circular in shape. Frequent mitosis is indicative of fast proliferation. Therefore, immunophenotyping is necessary to verify the T-cell lineage of blasts and differentiate them, from reactive situations such as lymphocytosis and mature T-cell/peripheral leukemia as differentiating these conditions from T-ALL solely based on morphological basis is not possible. This is where immunohistochemistry (IHC) and flow cytometry (FC) come into play.

Immaturity markers like CD34, HLA-DR and terminal deoxynucleotidyl transferase (TdT) are frequently employed in flow cytometry to distinguish between T-ALL/LBL and mature T-cell leukemia. A surface glycoprotein called CD34 is expressed on a range of progenitor and stem cells during hematopoiesis as well as endothelial cells. However, compared to B lineage blasts, T-cell expression of CD34 is more erratic.^[3], ^[4] Antigen-presenting cells are known to express the cell surface receptor HLA-DR. In immature T cells in the thymus, HLA-DR expression is normally nonexistent or low, but it can be increased following activation. TdT, an intra-nuclear DNA polymerase, is typically employed to convey the immature character of T lineage cells. It is only expressed at the most prominent stages of lymphopoiesis.^[5]

Another marker that is occasionally utilized is CD117 in the differentiation of ETP-ALL from other T-ALL and myeloid neoplasm. A surface marker protein used to detect hematopoietic stem cells (HSCs), it is also known as stem cell factor receptor (SCFR). Although T-cell precursors exhibit a low degree of expression, mature T-cells do not express it. Reports of T-ALL lacking every immaturity marker have also been made.^[1]

According to the 2017 WHO classification, early T-cell precursor acute lymphoblastic leukemia/lymphoma (ETP-ALL) is a unique and uncommon condition characterized by early T-cell markers and stem cell-like characteristics. ETP-ALL is a high-risk subtype of T-ALL that is frequently linked to a dismal prognosis and an increased chance of relapse. It includes 12–16% of pediatric cases and 17–22% of cases of T-ALL in adults. Although it exhibits myeloid or stem cell markers like CD34, CD117, CD13, and CD33, it usually displays absent or <5% CD1a and CD8 expression and CD5 expression in <75%. Rather than having the characteristics of an adult T-cell, ETP-ALL has that of an early hematopoietic stem cell.^[6], ^[7]

Compared to other subtypes, it necessitates different therapy and tactics because of its distinct immunophenotypic and genetic characteristics. Thus, the purpose of this study was to investigate the immunophenotype of T-ALL in the Indian population, with a focus on ETP-ALL, particularly in the northeastern region of the nation.

Materials and Methods

This is a retrospective study of T-ALL cases that were diagnosed in the Department of Pathology, JNIMS, Imphal, Manipur, over a three-year period (June 2020–June 2023) as a tertiary care facility. 15 T-ALL patients were assessed during this time. All T-ALL patients with >25% blasts in peripheral blood or bone marrow met the inclusion criteria, which also included meeting WHO standards for identifying T-cell lineage, such as strong cCD3 by FC/IHC and total lack of myeloperoxidase (MPO). Ambiguous,mixed T-/myeloid acute leukemia cases were excluded.

Results

Hematological profile in clinical practice 17 was the median age (range: 4–60 years). Eleven of the fifteen cases were male, and four were female, or 73.3% and 26.6% of the total. Of the four female cases, two were found to have ETP-ALL. 63% was the mean blast count.

Immunophenotypic profile

Expression of T lineage markers: 66% of cases showed expression of CD5, 100% of cases showed expression of CD7 and cCD3. The expression of distinct markers is displayed in [Table 3]. Expression of immaturity markers: CD34 was the most often expressed immaturity marker (expressed in 60% of cases), and 40% of the cases had negative results for all three immaturity markers. [Table 4] shows the expression of all immaturity markers ([Figure 2]).

Expression of aberrant markers: 6/15 cases (40%) had aberrant B cell antigen (CD10/CD79a) expression, while five out of fifteen cases (33.3%) had abnormal expression of one or more myeloid antigens (CD13/CD33/CD117/CD38). CD10 (40%) was the most often expressed aberrant B cell antigen, followed by CD79a in 1/15 (6%) cases. CD13 was the most often expressed aberrant myeloid antigen, appearing in 5/15(33.3%), followed by CD117 in 3/15(20%) and CD33 in 2/15 (13%).

Comparison of the expression of immaturity markers and aberrant antigens between the CD34 positive and negative groups: Two groups were studied ([Table 5]). We have shown that the lack of CD13 was significantly correlated with CD34 negative T-ALL (P=0.040), and that the expression of aberrant Bcell antigen (CD10, CD79a) was significantly correlated (P=0.028) with CD34 negative groups as well. [Figure 2]. A dot plot from a T-ALL case depicting the population of interest, or the red bursts. The third plot above demonstrates moderate to brilliant CD45 expression.CD19 is shown as negative in the bottom row and positivity for CD34, CD38, HLA-DR, and CD117. [Figure 3] displays negativity for CD3, CD56, CD8, and CD22 and positivity for CD7, CD5, and cCD3.

Acute lymphoblastic leukemia from early T-cell precursors (ETP-ALL): [Figure 4] 2/15 cases, or 13%, meet the WHO criteria for ETP-ALL. These individuals exhibit myeloid and hematopoietic stem cell markers, such as CD13, CD33, CD34, and CD117 HLA-DR, as depicted in the [Figure 3]. However, MPO was negative and only expressed CD7 dim CD5 (less than 75% positive cells), while other T lineage markers were negative.

Parameter	N=15	Percentage
Cellularity	Hypercellular for age	2	13.3
Normocellular for age	4	26.6
Hypocellular for age	9	60
Megakaryocyte	Adequate	4	26.6
Decrease	11	73.3
Increase	0
Erythropoiesis	Reduced	5	33.3
Adequate or increased	10	66.6
Mean Blast percentage of all nucleated cell	63%

Table 2 Bone marrow findings

S. No.	T-Lineage marker (n=15)	Expression (n,%)
1	SCD3	(2, 13%)
2	cCD3	(15,100%)
3	CD5	(10,66%)
4	CD7	(15,100%)
5	CD4	(2,13%)
6	CD8	(4,26%)
7	Double +	(1,6%)
8	Double-	(10,66%)

Table 3 Expression of various markers

S No.	Immaturity markers(n=15)	No. of cases showing expression, (%)
1	CD34	9,(60%)
2	HLADR	2,(13%)
3	CD117	3,(20%)
4	CD34/HLADR/CD117+	2,(13%)
5	CD34+/HLADR+/CD117-	0
6	CD34+/HLADR-/CD117+	1,(6%)
7	CD34-/HLADR+/CD117+	0
8	CD34+/HLADR-/CD117-	7,(46%)
9	CD34-/HLADR+/CD117-	0
10	CD34-/HLADR-/CD117-	6,(40%)
11	CD34-/HLADR-/CD117+	0

Table 4 Expression of all the immaturity markers

Parameter	CD34+ (9/15)	CD34- (6/15)	p-value
CD13			.04
Negative	4	6
positive	5	0
CD33			.48
Negative	7	6
Positive	2	0
CD117			.22
Negative	6	6
positive	3	0
CD10			.028
Negative	6	3
Positive	3	3
CD10, CD79a			.028
Negative	6	3
Positive	3	3
HLADR			.48
Negative	7	6
Positive	2	0
CD56			1.0
Negative	9	5
Positive	0	1

Table 5 Comparison between CD34 positive and negative groups

Discussion

Clinicopathological and immunophenotypic parameters were examined in 15 instances that were included in the current investigation. The greatest frequency of T-lineage markers expressed by the blasts in our investigation was 100% for cCD3 and CD7, followed by 66% for CD5 ([Table 4]). These results were consistent with those reported by Gupta et al.^[8]

Of the cases in this study, 86.6% had negative HLA-DR, 80% had CD117, and 40% had CD34. The frequency of lack of CD34 ranges from 30.7 to 100% in published literature, while the frequency of absence of HLA-DR ranges from 58.3 to 100%.^[4], ^[9], ^[10], ^[11] In our investigation, 40% of the cases tested negative for each of the three immaturity markers, namely CD34–/HLA-DR–/CD117–. We located one study that demonstrates immaturity indicators' negative effects.^[1]

The difference between peripheral T-cell leukemia and T-ALL is determined by the presence of certain markers, such as CD7, CD5, CD1a, and aberrant myeloid/B-cell antigens. We were unable to incorporate the thymic phenotype-suggesting and T-ALL-diagnostic expression of CD1a into our investigation. T-ALL is rarer to have HLA-DR positive than peripheral T-cell leukemia.^[12] A single feature cannot diagnose any particular entity hence a methodical approach is required, taking into account factors including the clinical history, the properties of the blasts in the aspirate and biopsy, and most crucially, immunophenotyping.TCR rearrangement and Vβ repertoire are the gold standard, however they are not widely available and also expensive.

The well-known characteristic of aberrancy in leukemias aids in the identification of malignant cells, which are identified by the presence of antigen from a different lineage. Another important factor in differentiating peripheral T-cell leukemias from T-ALL is the presence of B-cell or myeloid antigen.^[2] Additionally, they are helpful in prognostication and in differentiating T-ALL cells from normal hematogones while monitoring patients for MRD.^[13] Compared to Chen et al.^[14] this study reveals abnormal expression of B-cell antigen in 40% of patients. 33.3% of cases have myeloid aberrancy, which is consistent with earlier research that found it to range between 9 and 50%.^[15], ^[16] CD10 was the most often expressed B-cell antigen, followed by CD79a. In contrast, other studies found that the most often expressed aberrant B-cell antigen was CD79a.^[17] The majority of research in the literature, found that CD13 was the most often expressed aberrant myeloid antigen, appearing in 5/15(33.3%), followed by CD117 in 3/15(20%) and CD33 in 2/15 (13%) individuals.^[8], ^[9], ^[18], ^[19], ^[20], ^[21]

It is noteworthy that T-cells without CD34 were strongly linked to the absence of CD13 (P=0.040). Additionally, CD34 negative groups exhibited a significant correlation with the expression of aberrant B cell antigens, such as CD10 and CD79a (P= 0.028). The first leukemic cells of the lymohoblastic lineage often exhibit B-cell antigens, and instances where CD34 expression is lacking are likely due to phenotypically more immature blasts. This association suggests that a lot of cases have very early precursors. Additionally, the lack of CD13 was substantially correlated with CD34-negative T-ALL (P=0.040). Our results were in line with those of Gupta et al.^[1] and in opposition to those of Chen et al., who discovered a strong correlation between myeloid antigen expression in the CD34 positive group (36.58%) and the CD34-group (15.38%) (P<0.01).^[12] It is thought that the first thymic precursors are dual T and myeloid potential, and they are CD13 positive.^[21] Furthermore, the association between it and CD34 negative points to a very early precursor as the possible etiology of many leukemia cases in our study as well. Based on the six and eleven marker combination systems described in the literature,^[6] the diagnosis of ETP-ALL was made.

Conclusion

T-ALL is an uncommon and severe illness. The lack of follow-up and the small sample size were the limitations. The prognosis for T-ALL and ETP-ALL is not good, with ETP-ALL faring worse. Determining the T-cell lineage aids in prognosis. Because of its rarity and the need for a thorough diagnosis, this study is being done. Bigger follow-up studies are needed for subsequent investigations.

Source of Funding

None.

Conflict of Interest

None.