Clinical features and prognosis of chronic natural killer cell lymphoproliferative disorders

ABSTRACT

Objective

To analyze the current treatment status and prognostic regression of the chronic NK cell lymphoproliferative disorder (CLPD-NK).

Methods

We retrospectively analyzed the clinical features, treatment and prognosis of 18 patients with CLPD-NK who were treated at our Hospital between September 2016 and September 2022.

Results

Eighteen patients were included: three patients were treated with chemotherapy, five patients underwent immune-related therapy, one patient was treated with glucocorticoids alone, five patients were administered granulocyte colony-stimulating factor, blood transfusion therapy, or anti-infection therapy, followed by observation and follow-up, and four patients were observed without treatment. Fifteen patients survived, including two patients who achieved complete remission (CR) and seven patients who achieved partial remission (PR), of whom one patient progressed to Aggressive NK-cell leukemia (ANKL) and sustained remission after multiple lines of treatment; three patients were not reviewed, of which one patient was still in active disease, three patients developed hemophagocytic syndrome during treatment and eventually died, one of them had positive Epstein–Barr virus (EBV) expression. The 5-years overall survival rate was 83%.

Conclusion

Most patients with CLPD-NK have inert progression and a good prognosis, whereas some patients have a poor prognosis after progressing to ANKL and combined with hemophagocytic syndrome. Abnormal NK cells invading the center suggest a high possibility of ANKL development, and immunosuppressants and hormones are effective treatments for this disease.

KEYWORDS:

• Chronic NK-cell lymphoproliferative disorders
• clinical features
• prognosis

Introduction

Chronic NK-cell lymphoproliferative disorder (CLPD-NK) is a rare lymphocytic proliferative disorder [1]. In the 2016 World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues, CLPD-NK was distinguished from aggressive NK-cell leukemia (ANKL) and was listed with T-cell large granular lymphocytes (T-LGL) and ANKL as one of the three major entities of large granulocytic leukemia (Table 1) [2]. In North America and Europe, large granulocytic leukemia accounts for 2–5% of chronic lymphatic proliferative disorders, and account for 5–6% in Asia, with CLPD-NK accounting for only 10%. CLPD-NK often occurs in middle-aged and elderly patients over 60 years of age [3], and is mainly characterized by the presence of several abnormal NK cells in the peripheral blood or bone marrow for unknown reasons, with an inert disease course and negative Epstein–Barr virus (EBV) expression. Patients may have no clinical symptoms; however, most eventually develop anemia or neutropenia, and some patients have a combination of autoimmune disease and malignancy [4].

Table 1. Clinical differential diagnosis of CPLD-NK, T-LGL, ANKL and NK/T lymphoma.

	CPLD-NK	T-LGL	ANKL	NK/T lymphoma
clinical manifestation	Insidious onset, mild symptoms (characterized by abnormal blood cells)	Insidious onset, mild symptoms (characterized by abnormal blood cells)	Acute presentation, severe systemic symptoms, combined with hemophagocytic syndrome	Acute manifestations, severe systemic symptoms, accompanied by solid lesions, combined with hemophagocytic syndrome
Peripheral blood NK cells and T cells were detected by flow cytometry	The number of abnormal NK cells was > 2× 10^9 /L and lasted for more than 6 months	The number of abnormal T cells was > 2× 10^9 /L and lasted for more than 6 months	Abnormal T lymphocyte ratio increased	Abnormal NK/T lymphocyte ratio increased
Identification of NK and T cell immune phenotypes	CD3-CD16+CD56+ (NK cell immunophenotype)	CD3+CD8+CD16-/+CD56+CD57+ (T cell immunophenotype)	CD3-CD16+CD56+ (NK cell immunophenotype)	CD3+CD16+CD56+ perforin (NK cell and T cell immunophenotypes)
Pathological smear examination	Monoclonal proliferation of large granular cells	Monoclonal proliferation of large granular cells	Monoclonal proliferation of large granular cells	Monoclonal proliferation of NK cell and T cell
Gene mutation and histological identification	KIR restricted expression, STAT3 and TET2 mutations	STAT3, TET2, STAT5 mutations, TCR gene rearrangement	EBER+, EB virus positive, KIR restricted expression	EBER+, EB virus positive
prognosis	good	good	poor	poor

Notes: Chronic NK-cell lymphoproliferative disorder (CLPD-NK), aggressive NK-cell leukemia (ANKL), T-cell large granular lymphocytes (T-LGL), Epstein-Barr virus (EBV), Epstein-Barr virus-encoded RNA (EBER)

Among the diagnostic criteria recognized by most treatment centers, the detection of monoclonal proliferating NK cells is required, the molecular phenotype of NK cells should be consistent with CD3^-/CD16⁺/CD56⁺, the number should be greater than 2 × 10⁹/L (normal is < 0.3 × 10⁹/L), and the disease duration should be > 6 months. However, patients with long-term hematological manifestations and abnormally proliferating NK cells that are monoclonal may be considered for the diagnosis of CLPD-NK, even if the number does not meet the criteria. In addition, most patients exhibit a large number of abnormal NK cells in the bone marrow. When the number of abnormal NK cells in the peripheral blood flow is not reached, bone marrow flow can aid in the diagnosis [1, 3, 5].

The pathogenesis of CLPD-NK is unknown, and most current studies believe that the combination of exogenous antigenic stimulation (e.g. various viruses) and autoimmune dysfunction ultimately leads to the abnormal proliferation of NK cells [6]. Owing to their similar pathogenesis, CLPD-NK is similar to T-LGL, another major granulocytic leukemia entity, in terms of treatment. Asymptomatic patients are followed up and observed. Long-term anemia, neutropenia, and combined autoimmune disease are indications for treatment. Most treatment centers use low-dose immunosuppressive drugs (methotrexate, cyclosporine, cyclophosphamide), and treatment lasts for 4 months after efficacy evaluation is performed [5].

CLPD-NK is associated with difficult early diagnosis, very low incidence, easy to- miss misdiagnosis, and possible transformation to ANKL. Hence, early screening for NK cell proliferative disease and prompt clinical intervention are crucial an important means to treat and improve the prognosis of this group of patients, but the international standards for its treatment are not uniform, and there are few separate reports on this disease in the literature. Hence, the diagnosis and treatment of this disease still needs to be further explored. In this study, we retrospectively analyzed the clinical and prognostic features of 18 patients diagnosed with CLPD-NK, reflecting the diagnosis and treatment of CLPD-NK in our center to present key experiences for clinical treatment.

Materials and methods

Case information

Eighteen patients with CLPD-NK admitted to our Hospital between September 2016 and September 2022, and definitely diagnosed clinically, pathologically or in the laboratory were enrolled. Patient data including general information, clinical symptoms, flow cytometry and pathological test results, treatments and follow-up information were obtained via an electronic case system and telephone follow-ups.

Diagnostic criteria

Most patients included in the study met the following criteria: 1) Increased abnormal NK cells in peripheral blood or bone marrow and mature cerebrospinal fluid NK cells. 2) Abnormal proliferating lymphocytes express the NK cell flow phenotype CD3-CD16 + CD56+. 3) Inert development of initial clinical symptoms, no systemic symptoms, organ infiltration; patients with severe clinical symptoms at the time of initial diagnosis, along with prolonged abnormal anemia and neutropenia history. 4) negative EBV expression; and 5) NK/T-cell lymphoma, ANKL, T-LGL were excluded (if there was disagreement regarding the patient's diagnosis, the comprehensive diagnosis was made when consensus was reached by discussion). The clinical differential diagnosis of CPLD-NK, T-LGL, ANKL, and NK/T lymphoma is shown in Table 1.

At present, there are no clear diagnostic criteria for ANKL at home and abroad, and its diagnosis is mainly based on the comprehensive consideration of clinical manifestations, cell morphology, genetics, and immunological characteristics of patients. Basic diagnostic criteria: 1) Acute presentation, accompanied by fever and systemic symptoms. 2) Systemic (multi-organ) proliferation of tumor lymphoid cells. 3) NK cell immunophenotype. 4) Lack of expression of TCR protein and/or lack of clonal rearrangement of TR gene Ideal diagnostic criteria. 5) EBER positive (90% positive). 6) Hemophagocytic lymphohistiocytosis.

T-LGLL is defined by WHO (2016) as a persistent (> 6 months) increase of large granular lymphocytes (LGL) in peripheral blood, usually 2 × 10⁹ /L-20 × 10⁹/L, with unknown etiology. In rare cases, if the diagnostic criteria for T-LGLL are met, the diagnosis is still valid even if the number of LGL is < 2 × 10⁹/L.

Flow cytometry

10-color flow cytometry was performed using the following markers: CD3, CD2, CD4, CD5, CD7, CD8, CD10, CD16, CD25, CD26, CD30, CD45RA, CD52, CD56, CD57, CD62L, TCRγδ, TCRαβ, PD-1, and CCR4 [7].

Treatment efficacy assessment criteria

Patients were evaluated for treatment efficacy after 4 months of treatment: complete remission (CR) was defined as hemoglobin (Hgb) >12 g/dL, no transfusion, absolute neutrophil count (ANC) > 1.5 × 10⁹/L, platelet count (PLT) > 150 × 10⁹/L, absolute lymphocyte count < 4 × 10⁹/L, and abnormal NK cell count < 0.5 × 10⁹/L. Partial remission (PR) was defined as Hgb > 8 g/dL, ANC > 0.5 × 10⁹/L, PLT > 50 × 10⁹/L, and no requirement of transfusion. Responses that did not achieve PR were considered treatment failures [5].

Treatment options

The treatment regimen for patients included chemotherapy (gemcitabine, cisplatin, pemetrexed, etoposide, and formostine), hormones (methylprednisolone and dexamethasone), small molecule drugs (selinexor), immunosuppressive agents (methotrexate, cyclophosphamide, lenalidomide, hydroxychloroquine). Some patients with observational follow-up were administered symptomatic treatment, including blood transfusions, antibiotic anti-infective therapy, and colony-stimulating factors to increase white blood cell level.

Data analysis

Statistical analyses were performed using IBM affiliation of SPSS 25.0, and the continuous data were expressed as medians and the categorical data were expressed as percentage. The Log-rank test is used to analyze the survival of two or more groups. Kaplan-Meier survival curves were plotted, with P < 0.05, indicating statistically significant differences.

Results

Clinical features

Among 18 patients (Table 2), 12 (67%) were females and 6 (13%) were males, with a median age of 52 years (44–75 years), all patients had abnormal NK cell proliferation in bone marrow fluid or peripheral blood without abnormalities in T and B cells, two patients (cases 6 and 9) had abnormal NK cells in the peripheral blood and also in the central cerebrospinal fluid. One patient (case 3) tested positive for Epstein–Barr virus (EBV) expression and was diagnosed with ‘EBV-positive CLPD-NK.’ Two patients (cases 4 and 7) had positive HBV expression. Two patients (cases 16 and 18) had abnormal autoimmune antibody indexes and combined immune-related diseases (Sjogren’s syndrome). One patient (case 5) had enlarged lymph nodes, and pathological examination of lymph node aspirations suggested lymphoid tissue hyperplasia.

Table 2. Basic characteristics of the 18 patients.

CASE	Age	Sex	First symptoms	EBV expression	source of abnormal NK cells	Associated Diseases	Treatment modality	Prognosis	Follow-up duration (months)
1	51	Female	○	-	BM	No	●	Survival	72
2	44	Female	○	-	BM	No	●	Survival	62
3	61	Female	○△	+	BM	HLH	▪	Death	2
4	48	Female	○△	-	BM PB	HBV (+)	●	Survival	53
5	59	Female	○△▽	-	BM	No	▾	Survival	23
6	47	Male	○□	-	BM PB CSF	No	▴	Survival	3.5
7	58	Female	○□	-	BM PB	HBV (+)	▾	Survival	8.5
8	52	Male	○△	-	BM	HLH	▪	Death	7
9	55	Female	○□	-	BM CSF	No	▪★	Survival	7
10	49	Male	○△▽	-	BM	No	●	Survival	9
11	60	Female	○	-	BM	No	▾	Survival	16
12	72	Female	○	-	BM	No	●	Survival	18
13	75	Female	○	-	BM	No	●	Survival	33
14	52	Female	○△✩	-	BM	No	●	Survival	35
15	46	Male	○✩	-	BM	HLH	▪	Death	2
16	52	Female	○△	-	BM	Sjogren’s syndrome	▾	Survival	10
17	59	Male	○	-	BM	No	●	Survival	89
18	55	Male	○	-	BM	Sjogren’s syndrome	●	Survival	24

Notes: ○Anemia, neutropenia, thrombocytopenia □Neurological symptoms ▽Lymph node enlargement ✩Hepatosplenomegaly △Infection ●Observational follow-up ▪Chemotherapy ▾Immune-related treatment ▴Glucocorticoids alone ★Small molecule drugs (selinexor). BM, bone marrow; HLH, Hemophagocytic lymphohistiocytosis; PB, peripheral blood; CSF, cerebrospinal fluid; HBV, hepatitis B virus.

Clinical symptoms during diagnosis included abnormal blood findings (100%, 18/18), infection (44%, 8/18), neurological symptoms (17%, 3/18), hepatosplenomegaly (11%, 2/18), and enlarged lymph nodes (5%, 1/18) (Figure 1).

Figure 1. Clinical symptoms of 18 cases of chronic NK-cell lymphoproliferative disorder.

Treatment and efficacy assessment

Three patients (cases 3, 8, 15) were treated with chemotherapy, one patient (case 7) was treated with glucocorticoids alone, four patients (cases 5, 7, 11, 16) were treated with immunomodulatory therapy (methotrexate, cyclophosphamide, lenalidomide, hydroxychloroquine), four patients without clinical symptoms (cases 1, 2, 4, 11) were followed up and observed, and five patients (cases 10, 12, 13, 14, 18) were administered symptomatic treatment (blood transfusion, subcutaneous injection of granulocyte colony-stimulating factor, antibiotic anti-infection treatment) and subsequently followed up. Case 9 developed elevated abnormal NK cells in the cerebrospinal fluid and symptoms of systemic invasion during the follow-up, and was considered to progress to ANKL. The patient was treated with the DDGP regimen (platinum, glucocorticoids, gemcitabine, pemetrexed) and then progressed, administered pemetrexed, formestine and glucocorticoids. The clinical symptoms were remitted; however, abnormal NK cells continued to decline and then rebounded. Bemadomide and celecoxib were added to consolidate the treatment. The disease is now stable and clinical symptoms are in remission (Figure 2).

Figure 2. Treatment in 18 cases of patients with chronic NK-cell lymphoproliferative disorder.

Prognosis

Up to November 24, 2022 follow up, 15 patients survived with a 5-year survival rate of 83%, Efficacy was evaluated after at least four months of treatment, two patients (cases 5, 7) achieved complete remission (CR), seven patients (cases 6, 9, 12, 13, 14, 16, and 17) achieved partial remission (PR), one patient (case 18) was still in active disease, three asymptomatic patients (cases 1, 2, and 4) were not reviewed, and three patients (cases 3, 8, 15) died (Figure 3).

Figure 3. Prognosis of 18 patients with chronic NK-cell lymphoproliferative disorder.

In this study, it was found that some patients (those who were treated with symptomatic treatment and observation but did not undergo special treatment) hardly underwent a review of peripheral flow patterns and bone marrow aspiration during follow-up, which was related to the patients’ willingness to be treated. However, it could not be excluded that abnormal NK cells persisted but did not show obvious clinical symptoms; therefore, complete remission could not be diagnosed when determining the degree of remission. Patient’s survival analysis is shown in Figure 4.

Figure 4. Survival curve of 18 patients with chronic NK-cell lymphoproliferative disorder.

Discussion

CLPD-NK is a rare inert hematopoietic disorder, which is misdiagnosed and underdiagnosed due to mild clinical symptoms in most patients. Moreover, some patients have a long-term history of anemia and neutropenia, which may lead to symptoms such as malaise and infection if not corrected in time and may even develop into another large granulocytic, ANKL. Therefore, accurate diagnosis and prompt intervention are crucial. The pathogenesis of CLPD-NK is believed to involve a combination of exogenous antigen stimulation (e.g. Cytomegalovirus, Hepatitis B Virus) and autoimmune dysfunction, which in turn abnormally activates transduction pathways such as JAK-STAT and eventually leads to the abnormal proliferation of NK cells [8]. Most of the patients in our study had a long history of anemia or neutropenia; some of them had combined viral infections and autoimmune diseases, and treatment with immune-related drugs was effective, which confirms that the disease is closely related to immune dysfunction [9, 10].

Although CLPD-NK has a slow progression and mild clinical symptoms, some patients can transform into ANKL, which is facilitated by NK cell proliferation and positive EBV expression caused by some mosquito bites [11]. ANKL is a class of highly aggressive malignant tumors, prone to invade the central nervous system, associated with positive EBV expression, with a poor clinical prognosis and a median survival time of 2 months for patients. There is no standard regimen for treatment and some patients are not sensitive to chemotherapy. Some retrospective studies suggested that autologous hematopoietic stem cell transplantation can improve the prognosis [12]. Case 9 produced abnormal NK cells centrally after a long-term abnormal peripheral blood lymphocyte ratio and developed a systemic multiorgan infiltration with a rapid rise in peripheral blood NK cells. Hence, we concluded that the patient progressed from CLPD-NK to ANKL. Related studies have suggested that NK cell abnormalities that occur only in tissues are more likely to provide laboratory evidence for the diagnosis of ANKL [13]. This suggests that the abnormal NK cells of CLPD-NK can not only occur in the peripheral blood and bone marrow, but can also break the blood–brain barrier and infiltrate the central nervous system, making such patients more susceptible to ANKL progression. In another patient with central symptoms, symptoms were relieved after timely diagnosis and treatment, and the abnormal NK cells in the center subsided completely. This suggests that patients with central symptoms need to undergo a lumbar puncture to rule out central invasion after an abnormal elevation of peripheral blood NK cells is detected, and necessary treatments, such as immunosuppressants, hormones, and chemotherapy, can be administered in time to avoid further aggravation of the disease. In addition, case 9 was treated with chemotherapeutic agents such as formostine and pemetrexed, which can break the blood–brain barrier to infiltrate the center. After disease progression, we added XPO1 inhibitors to increase the efficacy of other drugs, which is the first case in the current treatment of ANKL. These drugs which act on the center and small molecules have achieved good efficacy in the treatment of ANKL with central invasion, increasing the selectivity and innovation of the therapeutic options for ANKL, suggesting that this protocol has a potential therapeutic value.

In our study, the prognosis was significantly poor when CLPD-NK was combined with hemophagocytic syndrome. Case 3 was an elderly female with positive EBV expression and a long history of abnormal NK cell proliferation, perhaps the cause of long-term EBV infection whose development of cachexia eventually combined with hemophagocytic syndrome led to death from multiorgan failure. Case 8 and 15 presented with a combination of phagocytic syndromes but died of severe organ failure without the next targeted tests and related treatment. The hemophagocytic syndrome involves an elevation of inflammatory factors caused by activation of the immune system, which eventually activates the macrophage system abnormally, abnormal CLPD-NK autoimmune function, abnormal hematopoietic function [14]. Some studies suggest that CD56 ^dim/neg CD16 ^high NK cells can release cytokines, which may promote hemophagocytic syndrome [15]. All 3 patients in our study were diagnosed in the ICU, in poor general condition and life-threatening state, and in addition to the diagnosis of CLPD-NK, they had sepsis, respiratory failure, and other diseases that tend to trigger the release of systemic inflammatory factors. Hence, the production of abnormal NK cells and hemophagocytic syndrome may be a mutually- promoting relationship. ANKL is prone to hemophagocytic syndrome and has a poor prognosis, and such patients are not excluded from being converted to ANKL. Even if the condition of this group of patients improves with treatment for hemophagocytic syndrome, ANKL-related tests should be performed and if necessary, high-intensity chemotherapy regimens should be used to avoid further disease development.

For the treatment of CLPD-NK, most centers consider the same entity, T-LGL, due to the similarity of clinical symptoms and pathogenesis, and some studies have considered both as two aspects of the same disease and have performed retrospective studies together with T-LGL [16, 17]. In such studies, asymptomatic patients can be considered for follow-up observation, with recurrent history of anemia and neutropenia can be treated with immunosuppressive agents such as methotrexate (MTX), cyclophosphamide (CTX), and Cyclosporin A (CyA), and efficacy was assessed after 4 months of treatment. The overall response rates (ORR) of the three immunosuppressive agents were similar, ranging from 21% to 85% (median, 50%), whereas the complete remission rates were relatively low, approximately 21% for MTX, 33% for CTX, and 5% for CyA. In addition, the long-term efficacy of the three immunosuppressive agents differed between studies. A prospective study of 55 patients with LGLL by ECOG showed an ORR of 38% for first-line use of MTX and 64% for patients who were replaced wirelessly with CTX in first-line treatment, indicating that CTX treatment may be more efficient in patients with MTX-naïve LGLL [5]. Patients treated with immune-related therapy in our study (cases 5, 7, 9, 11, and 16) showed improvement in clinical symptoms after four months, therefore, this study concluded that immunosuppression remains the treatment of choice for CLPD-NK patients with recurrent blood abnormalities. However, which immunosuppression is preferred in the absence of contraindications to treatment still needs to be explored in a larger sample.

Unlike the TCR rearrangement technique, which routinely detects monoclonal T cell proliferation, CLPD-NK is characterized by the chronic expansion of NK cells with a restrictive killer immunoglobulin-like receptor (KIR) pattern [18, 19]; therefore, detecting abnormalities in the associated KIR is one of the features of the disease. However, this test is still difficult to perform owing to its complexity, and the diagnosis of CLPD-NK is still based on flow cytology. The majority of patients in this study had an abnormal NK cell phenotype, CD3^-/CD16⁺/CD56⁺, which can be distinguished from NK/T-cell lymphoma and T-LGL. EBV was usually expressed negatively without evidence of systemic organ invasion during initial diagnosis, with low ki-67 expression, which can be distinguished from another entity, ANKL. In contrast, the discovery of mutated genes such as STAT3 and TET2 (mutation frequencies of 27% and 34%, respectively) resulted in new advances in the diagnosis of CLPD-NK on flow [20, 21], although not all patients are detectable, it has also been noted that patients with SATA3 mutations are more likely to have granulocyte deficiency, while TET2 abnormalities are more likely to be accompanied by thrombocytopenia [22], and patients with STAT5B N642H mutations develop more aggressive disease [15]. At the same time, studies have shown that STAT3 mutations are common in CLPD-NK patients, predicting more pronounced clinical manifestations [23]. Mutations in these genes suggest that this could be a potential future therapeutic target and allow for more targeted symptomatic treatment [24]; however, this genetic testing program is not yet fully operational, and fewer treatment centers can support it.

Conclusions

In summary, CLPD-NK has mild clinical symptoms and most cases progress slowly. When long-term hematological abnormalities are detected, peripheral blood flow cytometry can be used to clarify the diagnosis, and asymptomatic patients can be followed up for a long time. When a combination of hemophagocytic syndrome, positive EBV expression, history of mosquito bites, and central NK cell abnormalities are suspected, therapies such as hormones and chemotherapy need to be applied promptly to achieve a better prognosis for the patient.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by National Natural Science Foundation of China: [grant no 81970184]; National Natural Science Foundation of China: [grant no 82170183]; National Natural Science Foundation of China: [grant no 82070210].