Chronic lymphocytic leukemia-associated paraneoplastic pemphigus: potential cause and therapeutic strategies

Paraneoplastic pemphigus (PNP) is a severe autoimmune syndrome commonly triggered by neoplasms. The prognosis of CLL-associated PNP is dismal due to its refractory course and secondary infection and no standard treatment was recommended. We retrospectively reported six CLL with PNP cases from 842 cases of CLL including diagnosis, treatment and prognosis. The median time between the initial of CLL to PNP was 36 months while the median overall survival from the diagnosis of PNP was 26 months. And three cases died of lung infection while 5 developed pulmonary symptoms. And 5 cases received fludarabine-based chemotherapy before developing PNP, which suggesting fludarabine was one of potential causes of PNP. For the treatment, five patients were rescued by combined regimens including rituximab, methylprednisolone, immunoglobulin, fresh frozen plasma and the last received ibrutinib combined with short-term prednisone. Fludarabine-based regimen may be one of the potential causes of PNP. The combined regimen might shed a new light, while ibrutinib is a promising drug for CLL with PNP, but needs much more evidence. PNP should be carefully treated to guide early diagnosis and intervention for a better prognosis.


Materials and methods
We retrospectively analyzed 842 CLL patients presented in our hospital from 2007 to 2018. The diagnosis of CLL was based on iwCLL criteria including clinical features, blood and bone marrow examination including morphology, immunology, cytogenetics and molecular biology. Specific clonal B lymphocytes expressing CD5, CD19, light chain, CD23 and CD79b were detected by flow cytometry in periphery blood [positive for light chain restriction (either kappa or lambda), CD5, CD23, CD79b, and surface immunoglobulin expression, and low levels of CD20] 10 . Chronic lymphocytic leukemia can be diagnosed if Matutes score reaches 4 points or more. Investigations to diagnose CLL-associated PNP should consist of checking for systemic complications to identify CLL, skin biopsies for histopathological and immunofluorescence studies, and serum immunological studies such as lichenoid or acantholytic changes in pathology, supportive immunofluorescence findings, particular intercellular and basement membrane binding 11 . All patients showed increased lymphocytes in periphery blood with typical morphology and immunophenotype of CLL scored with 4-5 tested by flow cytometry. Immunohistochemistry of Cyclin D1 or fluorescence in situ hybridization (FISH) of t (11;14) was considered routinely in cases with atypical immunophenotype. PNP was diagnosed based on the skin biopsy and immunoprecipitation 11 . All of them were diagnosed as CLL previously and developed PNP during the treatment or follow-up of CLL. The baseline characteristics of the 6 patients with PNP were shown in Table 1. informed consent. Informed consent was obtained from all individual participants included in the study.

Results
According to our data, a total of 0.71% incidence of PNP in CLL population could be drawn due to 6 PNP cases in 842 CLL patients. Besides, 4 of the 6 patients in our study were male. All the 6 patients developed PNP after the treatment of CLL and half of them were diagnosed PNP during the partial remission of CLL while the rest of them manifested as disease progression in CLL. The median time from the initial of CLL to the diagnosis of PNP was 36 months (range from 12 to 139 ms). The median overall survival from the diagnosis of PNP to the recent fellow-up or the death date was 26 months (range from 14 to 33 ms). Half of the 6 patients died while 2 cases were alive and 1 lost follow-up in Jun 2015.
In our study, 4 of the 6 patients presented as Binet C stage with decreased platelet count or hemoglobin, among which only patient 1 and 2 received treatment at the time of diagnosis of CLL. Only case 2 (dead) existed the deletion of 17p and ATM while case 4 (dead) and case 5(alive) existed the mutation of immunoglobulin heavychain variable region (IGHV) gene, and case 1 presented as IGHV1-69*01 indicating poor outcome. Besides, 5 cases of them received fludarabine-based regimen before the development of PNP except case 6.
Expressions of the IgG and C3 level in periphery blood were shown at a mild reduction level in nearly half of our PNP cases. Lactate dehydrogenase (LDH) was found to be elevated in 4 of the 6 patients, and its relevance was worthy to be distinguished with PNP complicated with CLL. β 2 -Microglobulin (β 2 -MG) in 2 of the 6 patients were found obviously increased while 1 case wasn't tested. Serum CA-125 was elevated in 3 patients. Albumin was fall below normal level in patient 1 and patient 2 but kept in the normal rang in others. EBV DNA was negative in 5 of the 6 patients, which might indicate EBV took less effect on the development of CLL-associated PNP. Interestingly, 5 of the 6 cases developed various degrees of pulmonary infection after the diagnosis of PNP.  Table 2). According to the Fig. 1A-C, we found cracks and blisters could be easily seen in sliced side of the epidermis. Acantholism released cells and mixed inflammatory cell infiltrated to cause blisters. It was proved to be moss-like dermatitis changes, especial vice-tumor pemphigus. And IgG and C3 were deposited linearly in the basement membrane (      www.nature.com/scientificreports/ cycles(every 28 days a cycle) because of her progressive increase in lymphocyte counts and lymphadenopathy. Her CLL was controlled to PR according to iwCLL while her skin lesions repeatedly appeared, disappeared and partially aggravated in August 2018. She accepted skin biopsy in dermatologic department owing to red spots, blisters and itching while Edema appeared in her eyes and nose. PNP was diagnosed according to anti-Dsg3 positive in serum and histological changes ( Table 2). During her hospitalization in August 2018, she received HDMP (1 g/m 2 , d1-3) and immunoglobin transfusion [0.4 g/(kg d), d1-5] based on the diagnosis of PNP. This patient intermittently receives an immunoglobulin infusion (5-10 g each time every month) when there is a significant drop in IgG. The symptoms of itching and erythema have been moderately improved and her CLL disease state assessment reaches Cru (without bone marrow evaluation) in May 2019. The patient 6 was a 49-year-old male complained of lymphocytosis and lymphadenectasis in Dec 2016 without treatment history. Dexamethasone (10 mg qd × 7ds) was given to the patient as pretreatment in June 2017 to control his dyspnea caused by increased lymphocytes (37 × 10 9 /L) and enlarged lymph nodes. During the pretreatment, the patient developed massive canker sores which was progressive existence and refractory to antibiotics and antifungal drugs while his dyspnea developed intermittently. The pathology of the oral mucosa lesion and serum examination were proved to be PNP (Table 2). With the combination therapy of HDMP [MP 1 g/(m 2 d), d1-5] and IVIG[0.4 g/(kg d), d1-5], the oral lesion recovered gradually. The 6 cycles of R-COP[RTX 375 mg/m 2 d0, CTX 750 mg/m 2 d1, VDS 3 mg/m 2 , pred 60 mg/(m 2 d) d1-5] were given to the patient to treat CLL with a PR response while oral lesion presented intermittently. The patient changed to take ibrutinib (420 mg/d) combining with dosage-adjusted prednisone acetate from June 2018 to now to control his CLL disease at a Cru (without bone marrow evaluation) stage and recovered from his PNP.
Totally, 5 cases of them received fludarabine-based regimen before the development of PNP except case 6. Although high doses of glucocorticoid were given to all 6 patients at the time of PNP suffered, the outcomes were presented in various way. Patient 2 died of the refractory PNP and the secondary pulmonary infection, patient 3 died of his CLL disease progression, pulmonary infection and heart failure, and patient 4 died of severe complex infections especially pulmonary infection and bleeding, the case 5 and 6 are still alive at the recent follow-up.

Discussion
Paraneoplastic pemphigus, as a rare and high mortality autoimmune blistering disease accompanied by both benign and malignant neoplasms, was first described by Anhalt et al. in 1990. The relatively frequently reported associated hematological malignancies are B-cell lymphoma, Castleman's disease, CLL and Waldenström's macroglobulinemia. Autoimmune diseases especially hemocytopenia are frequently associated with CLL. Patients with CLL/SLL have a 5-10% risk of developing autoimmune complications. It can develop at any stage in the process of CLL 12 . PNP most often occurs in cancer patients aged from 45 to 70 years old, while it can also appear in children and adolescents. Different from other types of pemphigus, PNP can invade not only epithelia in gastrointestinal but also respiratory tract, resulting in florid oral mucosal lesions including extensive polymorphous cutaneous eruption and lung involvement. Kaplan et al. 13 pointed out that the most severe lesions during PNP occur 2-3 years after NHL diagnosis. Except for the first set of diagnostic criteria of Anhalt et al. 2,13 , there are no clear diagnostic criteria for PNP. The criteria they summarized 2,14 included characteristic clinical manifestations and histopathology, detection of tissue binding, circulating autoantibodies detected by direct immunofluorescence, indirect immunofluorescence (IIF), and immunoprecipitation studies. Kaplan et al. have revealed that the typical manifestation of painful inflammation in oral mucosa, polymorphous skin eruptions including lichenoid or acantholytic changes in pathology, positive immunofluorescence findings especially the combination of cells and basement membrane, combined with column coexists with serum antibodies of transitional epithelium or lymphoproliferative diseases especially the presence of anti-dsg, desaminoprotin I and II, envoplakin, periplakin, bullous pemphigus antigen 1 and Plectin antibody 11 . The appearance of skin lesions is diverse and has five changes: pemphigoid-like, bullous pemphigoid-like, erythema-like, graft-versus-host disease, lichen planus-like 15 . Bronchiolitis obliterans, as a serious life-threatening complication of PNP, is still unknown in the occurrence frequency and pathological mechanisms, although it was reported in several researches at a lower proportion 16,17 and was irreversible with aggressive therapy 4,18,19 .
PNP was thought to be a severe autoimmune reaction resulting in the deposition of auto-antibodies in epithelium. It was identified as a distinct disease from the following five aspects: (i) painful mucosal erosions and polymorphous skin eruption in neoplasia, (ii) histological changes (acantholysis, keratinocyte necrosis, interface dermatitis); (iii) DIF showing abnormal deposition of IgG and complement in intercellular substance and basement membrane zone, (iv) IIF with the same deposition as for DIF, in skin or mucosa including simple, columnar, and transitional epithelium, (v) demonstration of serum antibodies through immunoprecipitation of a complex of four keratinocyte proteins (250, 230, 210, 190 kd) 13 . The developing concept of paraneoplastic autoimmune multi-organ syndromes has been proposed due to its clinical individuation and deposition of autoantibodies could not only be found existed in the skin and mucous membranes but also in multiple internal organs 20 . Skin lesions can be involved in up to 25% of patients with CLL while the more common secondary lesions are presented as purpura, pruritus, urticaria, erythroderma, cutaneous vasculitis, pyoderma gangrenosum, sweet's syndrome and so on. Above all, erythema multiforme is proved to be a poor prognosis of the disease 21 . Oral mucosa ulceration as the characteristic manifestation should be paid much more attention on to distinguish the life-threatened PNP from the other skin lesions complications of CLL. The possible antibodymediated pathologic mechanisms in PNP associated neoplasias are listed as follows: Tumor-induced production of autoantibodies targeting epithelial proteins; Cross-reactivity of neoplasm and epithelial antigens; Elevated IL-6 leading to abnormal B-cell differentiation and auto-immunoglobulin production; Epitope spreading (interface dermatitis induced by the neoplasia exposes epidermal epitopes with autoantibody production against multiple epidermal proteins) 3,22 . The stubborn oral ulcerations used to present drug-resistant to conventional treatments.
Scientific RepoRtS | (2020) 10:16357 | https://doi.org/10.1038/s41598-020-73131-y www.nature.com/scientificreports/ There is no standard regimen for PNP owing to its rarity and severity, diversity of clinical manifestations and tumor heterogeneity. Besides, PNP management involves a complex and multidisciplinary approach aiming to treat the associated hematologic neoplasia and the mucocutaneous lesions. High-dose corticosteroids including prednisolone are still recommended as the first line therapy 23,24 . Corticosteroids merely improve the dermal lesion, while mucosal involvement is not generally improved by steroids 25 , which is consistent with our patients' symptoms. It is reported that other drugs, including cyclophosphamide, intravenous immunoglobulin (IVIG), Rituximab 23 associated with corticosteroids show good efficacy and safety in specific patients 22 . IVIG is a good choice to counteract and decrease circulating pathogenic auto-antibodies, and to modulate cytokines, complements and lymphocytes 26 . Recalcitrant mucous involvement can be treated with a combination of adjuvant immunosuppressive agents, immunobiologics and corticosteroids. Considering the relapsed/refractory CLL, fresh frozen plasma was added to enhancing the action of rituximab due to its role in a source of complement 27 . In our research, 4 of the 6 cases were male even if female gender showed much more tendency to autoimmune diseases. Of note, two-thirds of them were diagnosed as Binet C and Rai III or IV. The development of CLL-associated PNP showed less links with CLL-IPI risk even if low risk CLL-IPI patient could develop PNP. Besides, we found two-thirds developed PNP during their progression of CLL and 5 of the 6 developed PNP after receiving fludarabine-based chemotherapy even if many reports revealed no relationship between PNP and the primary malignant neoplasm in sequence of PNP or neoplasm regardless of progression or remission of CLL. Except the case 6, all cases had skin lesions at different degree while case 1 and case 2 suffered more seriously skin lesions in pathology. Moreover, half of the 6 cases proved to experience repeated oral painful mucosal erosions. Fludarabine use is associated with the occurrence of PNP, suggesting the altered immunity led by fludarabine use may triggered PNP development, just like fludarabine associated autoimmune hemolytic anemia (AIHA). Functional deficits in CD4 + CD25 + FOXP3 + Treg cells after fludarabine treatment are one of the risk factors for autoimmune diseases in CLL patients. Interleukin-2, a cytokine that promotes Treg survival and function, could be beneficial for patients to recover their Tregs in specific population including autoimmune diseases 28,29 . However, whether IL-2 can be suggested as an option for PNP needs much more exploration while our patient 4 was treated with FFP and rituximab which may contribute much more to PNP. Furthermore, we found the primary CLL showed no obvious correlation with control of PNP in the patient 2 which was consistent in many reported cases 21,30 , while the patient 6 diagnosed with Rai I recovered much more quickly from PNP than others. Besides, the development of PNP during the treatments including FC and HDMP with the control of CLL equally remind us that the indolent PNP seems have no relationship with the stage and state of the primary neoplasm 31 .
PNP is often reported resistant to conventional immunosuppressive treatments and constantly leads to death caused by infectious complications and respiratory failure due to its abnormal deposition of auto-antibodies and complements in tracheas. The development of bronchiolitis obliterans was thought to be a severe high-mortality complication of PNP. Similarly, 5 of 6 cases developed various degree of pulmonary infection after the diagnosis of PNP. The common cause of the three dead cases was pulmonary infection. Their diagnosis of lung infection were identified by CT scans and sputum bacteriology examination. Pulmonary symptoms included cough, sputum, fever, auscultation of wet rales. It is a pity that our patients didn't receive pneumocystis jiroveci pneumonia (PJP) prophylaxis before their treatment regimens including fludarabine. Pulmonary PNP may be existed with lung infection while pathological examination of bronchoalveolar lavage fluid is the most valuable evidence. In our research, 4 of 6 patients benefited from treatments after they received chemoimmunotherapeutic regimen combined of rituximab and HDMP and FFP. Long-term and insistent application of rituximab will produce curative effect. Meanwhile, addition of FFP might offer sufficient complements especially in synergistic effect with rituximab by enhancing antibody depended cell-mediated cytotoxicity during the course of diseases. Besides, 2 of the 6 patients received ibrutinib therapy and the last one took the drug to control his CLL and PNP. Bruton's tyrosine kinase is a kind of Tec family kinase with a well-defined role in the B cell receptor and Fcγ receptor signaling pathways. Ibrutinib, as a classical BTK inhibitor, makes it a uniquely attractive target for a safe and efficacious treatment of autoimmune diseases especially in CLL patients. It is reported by Ito et al. in 2018 that the combination of ibrutinib and rituximab was effective against CLL/SLL-associated PNP, while PNP lesions did not improve with ibrutinib monotherapy 32 . And the last patient in our research took ibrutinib to control his CLL and PNP.
In summary, the above 6 patients of CLL companied with PNP showed favorable outcome than the other cases reported 4,31 , which might be benefit from the combination of FFP, rituximab, standard dose methylprednisolone and ibrutinib according to our speculation. conclusion Above all, CLL accompanied with PNP as a rare and fetal disease, is resistant to treatment resulting in a highly mortality rate over 75%. Patients with Binet C and previous treatment with fludarabine based chemotherapy are more likely to develop PNP. Prognosis of PNP is heterogeneous while some patients progressed rapidly. And the main cause of death in PNP is pulmonary infection. A surgical cure can be a better choice to treat an operable PNP. In most cases, symptoms in PNP can be well managed by nonsurgical regimens including standard doses of glucocorticoid therapy [prednisone 0.5-1.0 mg/(kg d)], cyclosporin, cyclophosphamide, azathioprine, mycophenolate mofetil and so on. Besides, rituximab at a dose of 375 mg/m 2 qw × 4w can be used in lymphoma or CLL while it can be used at 1 g once and repeated in 2w in rheumatologic diseases. The rest cycles and dosages can be administered according to clinical response and the rest population of CD20 positive B cells. If the patient showed the poor response to the conventional therapy like high doses of prednisone or the application of rituximab, he might be treated with IVIG combined with rituximab. IVIG can be used at 2 g/kg per month. Except the fact of reducing production of pathogenic autoantibodies, the addition of IVIG doesn't add additional immunosuppression like other immunosuppressants. Alemtuzumab has been reported sporadically effective