Clinical Application Of Peripheral Blood Flow Cytometry In The Diagnosis Of Cutaneous Lymphoid Tissue Tumors

Cutaneous lymphoid tissue tumors are a group of rare,heterogeneous diseases of the lymphohematopoietic system with skin involvement,the diagnosis are challenging.Mycosis fungoides and Sézary syndrome(MF/SS)are the most common primary cutaneous T-cell lymphoma,and peripheral blood involvement is an independent adverse prognostic factor.Erythroderma is a severe disease characterized by diffuse erythema,can be caused by a variety of causes.Erythrodermic mycosis fungoides and Sézary syndrome(EMF/SS)often mimic a variety of inflammatory erythroderma(IE),and the differential diagnosis is difficult,but the prognosis of EMF/SS is worse.Flow cytometry(FCM)is a rapid,objective and quantitative technique and allows simultaneous multi-parameter analysis of a single cell.However,the application of FCM in MF/SS is relatively rare.Therefore,in this study,peripheral blood FCM was analyzed of patients with MF/SS at different stages and patients with erythroderma of different etiologies,to investigate the value of FCM in the diagnosis of MF/SS,the evaluation of peripheral blood tumor load and the differential diagnosis between EMF/SS and patients with IE.Blastic plasmacytoid dendritic cell neoplasm(BPDCN)is a rare,aggressive hematopoietic malignancy,often presents skin lesions as the initial manifestations.E2-2 is an important transcription factor in BPDCN.Therefore,this study investigated the value of immumohistochemical staining against E2-2 and peripheral blood FCM in the diagnosis of BPDCN.Part 1 Peripheral blood flow cytometry in the diagnosis of mycosis fungoides/Sézary syndrome and its associated erythrodermaChapter 1 Peripheral blood flow cytometry in mycosis fungoides/Sézary syndromeObjective To investigate the value of peripheral blood FCM in MF/SS.Methods TCR-v? clonality and T lymphocytes immunophenotypes were analyzed by FCM in 78 peripheral blood samples from 69 patients with different stages of MF/SS diagnosed in the Institute of Dermatology,Chinese Academy of Medical Sciences from 2017 to 2020.Results Clonal TCR-v? expression was detected in 30 of 78 MF/SS samples,which accounted for 13.9%,54.8%,50%and 100%of the samples with early stage,tumor stage,stage ? MF and SS,respectively.And the proportion of clone cells were 6.5%,18.3%,12.2%and 74.9%,respectively.The species of the clonal TCR-v? were heterogeneity.Abnormal expression of at least one of the T lymphocyte markers were detected in 37(47.4%)MF/SS samples,mainly manifest as the reduced or lost expression of CD7,followed by the reduced expression of CD3 and CD4,and occasionally abnormal expression of CD2,CD5 and CD45.Clonal TCR-v? cells were consistent with CD4+CD7-CD26-immunophenotype cells.Tumor-cell counts in two tumor stage MF were greater than 1000/?l,and the diagnosis was revised from stage ?B to stage IV.Two SS patients showed the expression of CD 164.Conclusions Peripheral blood involvement can occur in patients with MF/SS at any stage.Peripheral blood immunophenotypes and TCR-v? clone analysis by flow cytometry is helpful for the diagnosis and accurate staging of MF/SS.Chapter 2 Peripheral blood flow cytometry in lymphoma-associated erythrodermaObjective To investigate the value of peripheral blood FCM in erythroderma.Methods peripheral blood FCM were analyzed of 33 patients with erythroderma(6 EMF,5 SS,22 IE)diagnosed in the institute from 2017 to 2020.Results Clonal TCR-v?expression was detected in 3 EMF and 5 SS patients,while all of the IE patients were negative.The specificity and positive predictive value of clonal TCR-v? in EMF/SS both were 100%.Abnormal expression of at least one of the T lymphocyte markers were detected in all of the EMF/SS samples,while merely 4 IE patients showed exclusively abnormal expression of CD7.The CD4/CD8 ratio,the count of cloned TCR-v? cells and the proportion of abnormal immunophenotype T lymphocytes in SS were significantly higher than those in IE and EMF(P<0.05).T lymphocytes function subtypes analysis showed that the tumor cells in EMF/SS were mainly TCM,while the IE patients were mainly infiltrated by TEM.In addition,CCR4 and CLA were higher in EMF/SS than in IE,CCR4 was higher in generalized eczema and drug rash patients than in healthy donors,and CXCR3 and CCR5 were higher in erythodermic psoriasis than in eczema and EMF/SS patients.Conclusions Peripheral blood FCM is helpful for the definitive diagnosis of SS,the differential diagnosis between EMF and IE,even for the differential diagnosis among IE patients caused by different etiology.Chapter 3 Flow cytometry combined with clinicopathological analysis in the diagnosis of transformed mycosis fungoidesObjective:To investigate the clinicopathological features and prognosis of transformed mycosis fungoides(TMF).Methods:Retrospectively analyzed the clinicopathologic features and prognosis of 24 patients with TMF diagnosed in the institute between 2014 and 2020,and the peripheral blood FCM data of 16 TMF patients.Results Of the 24 patients with TMF,9 were early stage and 15 were tumor stage.Histopathologically,large cells in 20 patients infiltrated in a diffuse pattern,and coalesced into gaint cells in 4 patients.Immunohistochemically,the proportion of large cells were greater than 75%in 9 of 18 patients showed positive expression of CD30.Peripheral blood FCM showed the proportion of patients with clonal TCR-v? expression and the proportion of cloned cells in TMF were higher than those without transformation,and the forward scatter of tumor cells was significantly increased.Follow-up results showed 5 tumor-stage patients died at an average of 3.3 years after transformation.Conclusions Large cell transformation can occur in patients with MF at any stage and often has a poor prognosis.Histopathology combined with peripheral blood FCM is helpful for the early diagnosis and accurate evaluation of TMF.Part 2 Flow cytometry combined with clinicopathological analysis in the diagnosis of blastic plasmacytoid dendritic cell neoplasmObjective:To investigate the diagnosis of blastic plasmacytoid dendritic cell neoplasm(BPDCN)with cutaneous involvement.Methods:Retrospectively analyzed the clinical,pathological and immunohistochemical characteristics of 28 patients with BPDCN diagnosed between 2010 and 2020.And analyzed of the peripheral blood flow cytometry of 6 patients.Results:The age at diagnosis presented a bimodal pattern.The lesions mainly manifest as disseminated(65%),sporadic(20%)or solitary(15%)plaque,nodule or tumor.Histopathologically,67.9%of lesions revealed tumor cells infiltrated in a diffuse and nodular pattern.Interstitial,lichenoid and perivascular pattern was observed in 14.3%,7.1%and 10.7%lesions,respectively.Angioinvasion and necrosis were absent.All tumor cells were lymphoblastic-like with polymorphic nuclei except one lesion revealed monomorphous immunoblastic-like cells.Immunohistochemically,CD4 and CD56 were negative in 17.9%and 7.1%of BPDCN patients,respectively.CD123 and TCL-1 were weakly positive in 2 patients with myeloid sarcoma,respectively.Simultaneous expression of the four markers were observed in 65.0%of lesions of BPDCN.Homogenous expression of E2-2 was observed in 96.4%BPDCN lesions,but negative in all myeloid sarcoma cases.Flow cytometry showed the proportion of tumor cells in peripheral blood ranged from 1.5%to 83.4%,with heterogeneous expression of CD56.Conclusions:The morphologic and phenotypic features of BPDCN are heterogeneous.E2-2 may serve as a useful and specific marker to the definitive diagnosis of BPDCN.Peripheral blood flow cytometry is helpful in assessing the tumor burden.