Concurrent development of small lymphocytic lymphoma and lung cancer: A report of two cases and a review of the literature

Small lymphocytic lymphoma (SLL) is a rare disease subtype which has the same morphological and immunophenotypic features as chronic lymphocytic leukemia (CLL) but does not demonstrate lymphocytosis and grows mainly in the lymph nodes and spleen. As with CLL, SLL patients tend to present with immune abnormalities, and are associated with an increased risk for developing second primary malignancies. We report here two cases of SLL who developed lung cancer concurrently. The biological and clinical features of these two patients were very similar to each other; they both developed SLL with trisomy 12 and lacked lymphocytosis or cytopenia. SLL cells involved nodal areas adjacent to lung adenocarcinoma which expressed PD-L1. One patient received immunochemotherapy including nivolumab and ipilimumab against lung cancer, and notably, transient deterioration of SLL occurred after the second cycle of immunochemotherapy along with the development of immune related adverse events. Immunohistochemical analysis of the SLL samples of the patient revealed that the tumor cells were positive for CTLA-4, suggesting that ipilimumab might have potentially induced the activation of SLL cells by blocking the inhibitory signal mediated by CTLA-4. These clinical findings indicate the potential biological relationship between SLL and lung cancer. According to these observations, we would like to draw attention to the possibility of deterioration of SLL when immune checkpoint inhibitors are used for the treatment of malignancies developed in SLL patients.

section_content: Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy that is characterized by monoclonal B-cell lymphocytosis in the blood and lymphoid organs. Small lymphocytic lymphoma (SLL) is a rare disease subtype which has the same morphological and immunophenotypic features as CLL but does not demonstrate lymphocytosis and grows mainly in the lymph nodes and spleen. 1 Specific chromosomal abnormalities including 13q, 11q, and 17p deletions and trisomy 12 are often detected in CLL/SLL, and are associated with distinctive clinical presentation and outcomes. 

The clinical course of the patients with CLL/SLL is highly heterogeneous. Although patients typically have an indolent disease and a sometimes do not even require treatment throughout their lifetime, others exhibit an aggressive clinical course and dismal outcome. The mutational status of the immunoglobulin heavy-chain variable region gene (IGHV) of CLL/SLL is also predictive of prognosis, and those without IGHV mutations tend to have poor prognosis. 2 t has been clinically recognized that CLL/SLL patients have an increased incidence of developing second primary malignancies. Although disease-and treatment-related immunosuppression is assumed to be an underlying cause of predisposition to malignancies, the precise mechanisms for developing other neoplasms have not been clarified. We report here the clinical course of two patients who concurrently developed SLL and lung cancer. Clinical and biological similarities in these two patients raise the possibility that there may be a causal relationship between the pathogenesis of these two malignancies.
section_name: INTRODUCTION

section_content: Kensuke Nakao, 1) Momoko Nishikori, 1) Masakazu Fujimoto, 2) Hiroshi Arima, 1) Hironori Haga, 2) Akifumi Takaori-Kondo 1) Small lymphocytic lymphoma (SLL) is a rare disease subtype which has the same morphological and immunophenotypic features as chronic lymphocytic leukemia (CLL) but does not demonstrate lymphocytosis and grows mainly in the lymph nodes and spleen. As with CLL, SLL patients tend to present with immune abnormalities, and are associated with an increased risk for developing second primary malignancies. We report here two cases of SLL who developed lung cancer concurrently. The biological and clinical features of these two patients were very similar to each other; they both developed SLL with trisomy 12 and lacked lymphocytosis or cytopenia. SLL cells involved nodal areas adjacent to lung adenocarcinoma which expressed PD-L1. One patient received immunochemotherapy including nivolumab and ipilimumab against lung cancer, and notably, transient deterioration of SLL occurred after the second cycle of immunochemotherapy along with the development of immune related adverse events. Immunohistochemical analysis of the SLL samples of the patient revealed that the tumor cells were positive for CTLA-4, suggesting that ipilimumab might have potentially induced the activation of SLL cells by blocking the inhibitory signal mediated by CTLA-4. These clinical findings indicate the potential biological relationship between SLL and lung cancer. According to these observations, we would like to draw attention to the possibility of deterioration of SLL when immune checkpoint inhibitors are used for the treatment of malignancies developed in SLL patients.
section_name: Concurrent development of small lymphocytic lymphoma and lung cancer: A report of two cases and a review of the literature

section_content: A 67-year-old male, current smoker, presented with slowly growing cervical tumors. A computed tomography (CT) scan revealed enlarged lymph nodes in the bilateral cervical, axillary, supraclavicular, and paraaortic regions, and a mass lesion in the apex of the left lung (Figure 1A ). An 18-F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/CT scan demonstrated abnormal FDG uptake in these tumors. Blood examination revealed hemoglobin of 14. 6 g/dL, platelet count of 12. 3×10 4 /μL, and white blood cell count of 5,970 /μL with 47% neutrophils, 40% lymphocytes, 8% monocytes, 4% eosinophils, and 1% basophils. His serum lactate dehydrogenase (LD) was 210 U/L, IgA 210 mg/dL, IgG 1,327 mg/dL, IgM 54 mg/dL, soluble interleukin-2 receptor (sIL-2R) 1,450 U/mL (normal range, 121-613 U/mL), Pro-GRP 35. 4 pg/mL (normal range, < 81. 0 pg/mL), CEA 6. 8 ng/mL (normal range, < 5. 0 ng/mL), NSE 11. 42 ng/ mL (normal range, <12. 0 ng/mL), SCC 0. 7 ng/mL (normal range, < 1. 5 ng/mL), and CYFRA 1. 9 ng/mL (normal range, < 2. 2 ng/mL). Bone marrow examination revealed no involvement of lymphoma cells. An excisional biopsy of the left axillary node led to a diagnosis of SLL. 

The possibility of concurrent lung cancer in the left lung apex was indicated by the PET-CT scan, but it was considered difficult to precisely evaluate its distribution. Since the systemic lymph nodes grew larger in the following 4 months (Figure 1B ), we decided to treat SLL first following consultation with a thoracic physician. The patient was treated with 4 cycles of BR (bendamustine and rituximab), and a PET/CT scan after the completion of chemotherapy showed regression of all tumors except the left lung apex lesion (Figure 1C ). A bronchoscopic biopsy of the residual tumor led to the diagnosis of acinar-predominant lung adenocarcinoma. The tumor was positive for PD-L1 expression in immunohistochemistry and was found to carry an EGFR L858R gene mutation by Oncomine Dx Target Test Multi-CDx system. 3 A detailed chest CT scan demonstrated the presence of visceral pleural invasion. The patient has been treated with osimertinib and is alive with lung cancer for two years.
section_name: CASE PRESENTATION Case 1

section_content: A 73-year-old male, former smoker, presented with persisting productive cough. A chest CT scan demonstrated mediastinal lymphadenopathy and a mass lesion in his right lung (Figure 2A, B ). Blood examination revealed hemoglobin of 16. 5 g/dL, platelet count of 16. 7×10 4 /μL, and white blood cell count of 9,320 /μL with 80. 4% neutrophils, 12. 6% lymphocytes, 5. 5% monocytes, 1. 1% eosinophils, and 0. 4% basophils. Serum LD was 228 U/L, IgA 153 mg/dL, IgG 1,022 mg/dL, IgM 30 mg/dL, sIL-2R 683 U/mL, Pro-GRP 83. 1 pg/mL, CEA 9. 1 ng/mL, and CYFRA 10. 6 ng/mL. Bone marrow examination showed no involvement of lymphoma cells. 

Histological analysis of bronchoscopic biopsy samples of both the lung tumor and a mediastinal lymph node revealed lung adenocarcinoma. Only a few clusters of carcinoma cells were identified in these biopsy specimens, and histologic subclassification was not possible. Although no obvious lymphocyte clusters were found in these samples, flow cytometric analysis of the lymph node detected a CD20, CD5, CD23, IgM, and Igκ-positive monoclonal B-cell popu- lation accounting for 60% of the nucleated cells, and concurrent development of SLL was suggested. A cranial magnetic resonance imaging (MRI) scan revealed a brain metastasis of lung cancer, and immunochemotherapy consisting of carboplatin, pemetrexed, nivolumab, and ipilimumab was initiated for the treatment of lung cancer. 

On day 9 of the second cycle of chemotherapy, the patient was hospitalized for rapidly progressive hypoxia and thrombocytopenia with fever. A CT scan revealed further enlargement of systemic lymph nodes, although the lung tumors were regressing (Figure 2C, D ), and an axillary lymph node biopsy was performed for the evaluation of his illness. Surprisingly, the biopsied lymph node demonstrated diffuse infiltration of small monotonous B cells with CD20, CD5, and CD23 expression and the absence of cyclin D1 expression, and the patient was diagnosed with progression of SLL. His fever, hypoxia, and thrombocytopenia gradually resolved after the cessation of immunochemotherapy, and they were considered to be compatible with immunerelated adverse events (irAEs) caused by immune checkpoint inhibitors. Notably, systemic lymphadenopathy also regressed along with the resolution of other symptoms. Chemotherapy was resumed without immune checkpoint inhibitors, but the patient died of progression of lung cancer six months later. Figure 3 shows the summary of his clinical course.
section_name: Case 2

section_content: Histological analysis of the axillary lymph nodes of both Case 1 and Case 2 demonstrated disrupted architecture and diffuse proliferation of small lymphocytes with CD20, CD5, and CD23 expression, which led to the diagnosis of SLL (Figure 4 ). The Ki-67 labelling index of the tumor cells was 10% in Case 1 and 30% in Case 2. Both tumor cells were positive for IgM-κ in flow cytometric analysis. In Case 2, SLL seemed to be deteriorated along with the occurrence of irAEs during the immune checkpoint inhibitor treatment. We detected CTLA-4 but no obvious PD-1 expression in SLL cells in immunohistochemical analysis (Figure 4N, O), indicating that SLL cells might be activated by blockade of inhibitory CTLA-4 signaling following ipilimumab. 

Although G-banded chromosomal analysis of SLL cells demonstrated normal karyotypes, trisomy 12 was detected by interphase fluorescence in situ hybridization (FISH) in both patients (Figure 4G, M ). Informed consent was obtained from the patients for further genetic analysis, and the IGHV gene segment usage and somatic hypermutation status of SLL cells were examined. Semi-nested polymerase-chain reaction (PCR) amplification of monoclonal immunoglobulin gene rearrangements 4 and Sanger sequencing of the PCR products were performed using the following 3 primers:
section_name: HISTOLOGICAL AND GENETIC ANALYSIS OF CLL AND LUNG CANCER

section_content: were used for the first round PCR, FR2A and VLJH were used for the second round PCR, and VLJH was used for Sanger sequencing. The results demonstrated the usage of VH3-9 and VH1-2 in Case 1 and Case 2 SLL, respectively, both of which are reported as frequently used IGHV segments in CLL/SLL. 5 IGHV was not mutated in Case 1, but was mutated in Case 2. 

In both cases, biopsied lung cancer tissues were histologically evaluated to be adenocarcinoma with PD-L1 expression. The histological and genetic features of SLL and lung cancer in each case are summarized in Table 1.
section_name: T G A G G A G A C G G T G A C C ; V L J H , GTGACCAGGGTNCCTTGGCCCCAG. FR2A and LJH

section_content: CLL/SLL patients have an increased risk for comorbid malignancies, 6, 7 a phenomena thought to be associated with their predisposition to immunodeficiency due to the disease itself and/or cumulative immunosuppressive treatment. We reported two patients who developed SLL and lung adenocarcinoma concurrently and demonstrated very similar clinical features between the patients. In both cases, SLL was positive for CD5, CD23, and IgM-κ expression, as well as chromosomal abnormality of trisomy 12. SLL was not accompanied with lymphocytosis or cytopenia, but involved in lymph nodes adjacent to lung cancer, which led to the hypothesis that there might be a causal relationship between these two malignancies. 

In the analysis of second primary malignancies among CLL patients from the Surveillance, Epidemiology, and End Results (SEER) database between 1973 and 2015, 6,487 second primary malignancies were diagnosed with a 20% higher risk compared to the general population. 7 Moreover, patients with lung and various other cancers were shown to have inferior overall survival when developed with a background of CLL. 8 Trisomy 12 is one of the most common cytogenetic abnormalities in CLL/SLL, and is more frequently found in patients with SLL than CLL (28-36% vs. 13-16%). 9 Notably, as high as 9% of CLL/SLL patients with trisomy 12 demonstrate complications of other hematologic and nonhematologic malignancies. 9 t is also known that more than 20% of CLL patients carry a stereotyped B-cell receptor (BCR), 5 and CLL patients with trisomy 12 have a significantly higher incidence of carrying stereotyped BCR than those without (44. 1% vs 27%, p = 0. 01). 10 These findings suggest that persistent BCR activation by certain endogenous antigens plays a role in the pathogenesis of CLL, especially in those with trisomy 12. Although there is only limited information on the antigens bound to CLL/SLL BCRs, reported candidates include cytoskeletal proteins, such as vimentin, filamin B, and cofilin-1, and phosphorylcholine-containing antigens, such as Streptococcus pneumoniae polysaccharides and oxidized low-density lipoprotein. 11 Since these molecules compose motifs exposed on apoptotic cells and bacteria, it is speculated that CLL/SLL cells are derived from B cells that play roles in the elimination of apoptotic cells and/or pathogenic bacteria. 12 IGHV analysis of SLL in Case 2 demonstrated the usage of mutated IGHV1-2, which was previously reported to have an affinity for filamin B. 11 As filamin B is reported to be highly expressed in invasive cancer cells and contributes to their invasiveness, 13 it is possible that BCR expressed on SLL cells in Case 2 recognized the antigen presented by lung cancer. 

Moreover, transient SLL progression was observed in Case 2 along with the occurrence of irAEs during immunochemotherapy, and immunohistochemical examination detected CTLA-4 expression in SLL. In T-cell lymphomas, CTLA-4 and PD-1 are known to potentially act as tumor suppressors, and downregulation or blockade of these molecules may induce lymphoma progression in some situations. 14 TLA-4 and PD-1 are also reported to be occasionally expressed in CLL/SLL and Richter transformation; [15] [16] [17] [18] [19] [20] however, the functional roles they play in CLL are not well clarified. A previous report demonstrated that downregulation of CTLA-4 induced proliferation and survival of CLL cells in vitro. 21 Therefore, it can be speculated that ipilimumab might have augmented the immune response of SLL cells against lung cancer in Case 2, which led to the transient SLL progression. 

Although further investigation is necessary, our observations suggest the possible causal relationship between SLL and lung cancer. We would like to draw attention to the potential risk of deterioration of CLL/SLL when immune checkpoint inhibitors are used for the treatment of second primary malignancies in CLL/SLL patients.
section_name: DISCUSSION

section_name: CONFLICT OF INTEREST

section_content: The authors declare no potential conflict of interest in this work.
section_name: CONFLICT OF INTEREST