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Follicular T-cell lymphoma (FTCL), one of the nodal T-cell lymphomas with T follicular helper (TFH) phenotype, is an uncommon disease. The diagnosis of FTCL is challenging on the distinction from the morphological mimics mostly exemplified by follicular lymphoma. Here, we described a case of FTCL that mimicked lymphocyte-rich classic Hodgkin lymphoma (LRCHL). A 47-year-old male presented with cervical lymphadenopathy. The biopsy specimen demonstrated nodular lymphoid proliferation, which included scattered CD30+ CD15- CD20- PAX5 weakly+ Hodgkin and Reed-Sternberg (HRS)-like cells and a rich distribution of CD3+ CD4+ PD1+ T-cells. Epstein Barr virus was not detected in HRS-like cells, but it was detected in a small proportion of the scattered lymphocytes. The large cells were also negative for programmed cell death ligand 1, which appeared to be coincidental as described in our previous report of LRCHL. However, flow cytometry showed a CD3- CD4+ T-cell population that constituted 37.4% of all gated lymphocytes. A PCR analysis showed a clonal T-cell receptor-gamma gene rearrangement, but not a clonal immunoglobulin heavy chain gene rearrangement, and showed RHOA G17V mutation. The constellation of these findings led us to revise the diagnosis to FTCL. This result indicated that our case belonged to a relatively indolent subgroup of nodal peripheral T-cell lymphoma of TFH phenotype, which affects patients ≤60 years old, recently proposed by our group. This case report expands our understanding of the morphologic spectrum of FTCL and its clinicopathologic significance.

Assessment of T-cell receptor γ gene (TRG) rearrangements is an importants consideration in the diagnostic workup of lymphoproliferative diseases. Although fragment analysis by PCR and capillary electrophoresis (CE) is the current standard of such assessment in clinical molecular diagnostic laboratories, it does not provide sequence information and is only semi-quantitative. Next-generation sequencing (NGS)-based assays are an attractive alternative to the conventional fragment size–based methods, given that they generate results with specific clonotype sequence information and allow for more accurate quantitation. The present study evaluated various test parameters and performance characteristics of a commercially available NGS-based TRG gene-rearrangement assay by testing 101 clinical samples previously characterized by fragment analysis. The NGS TRG assay showed an overall accuracy of 83% and an analytical specificity of 100%. The concordance rates were 88% to 95% in the V(γ)1–8, V(γ)10, and V(γ)11 gene families, but lower in the V(γ)9 gene family. This difference was mostly attributed to the incomplete polyclonal symmetry resulting from the two-tube CE assay versus the one-tube design of the NGS assay. The NGS assay also demonstrated strengths in distinguishing clonotypes of the same fragment size. This clinical validation demonstrated robust performance of the NGS-based TRG assay and identified potential pitfalls associated with CE assay design that are important for understanding the observed discrepancies with the CE-based assay.

BACKGROUND Lymphomatoid papulosis (LyP) type D (LyP D) and type E (LyP E) have recently been described in small series of cases or isolated case reports. AIM To further describe the clinical and histological features of LyP D and E based on a retrospective multicentre study. METHODS The clinical and histopathological features of 29 patients with an initial diagnosis of LyP D or LyP E were retrospectively assessed using standardized forms. RESULTS After exclusion of 5 cases, 24 patients (14 LyP D, 10 LyP E) were enrolled in the study. The median follow-up was 2.5 years (range 1 month to 13 years). LyP D was characterized by multiple recurrent self-regressing small papules that developed central erosion or necrosis, whereas LyP E presented as papulonodular lesions that rapidly evolved into necrotic eschar-like lesions > 10 mm in size. Epidermal changes were more frequent in LyP D, whereas dermal infiltrates were deeper in LyP E. Anaplastic cytology was rare and the DUSP22 rearrangement was never observed. Two patients (8%) had an associated cutaneous lymphoma. CONCLUSION LyP D and E have distinct clinical findings and may be associated with other cutaneous lymphomas.

γδ T cells are the unique T cell subgroup with their T cell receptors composed of γ chain and δ chain. Unlike αβ T cells, γδ T cells are non-MHC-restricted in recognizing tumor antigens, and therefore defined as innate immune cells. Activated γδ T cells can promote the anti-tumor function of adaptive immune cells. They are considered as a bridge between adaptive immunity and innate immunity. However, several other studies have shown that γδ T cells can also promote tumor progression by inhibiting anti-tumor response. Therefore, γδ T cells may have both anti-tumor and tumor-promoting effects. In order to clarify this contradiction, in this review, we summarized the functions of the main subsets of human γδ T cells in how they exhibit their respective anti-tumor or pro-tumor effects in cancer. Then, we reviewed recent γδ T cell-based anti-tumor immunotherapy. Finally, we summarized the existing problems and prospect of this immunotherapy.

γδ T cells function as sentinels in early host response to infections and malignancies. Although γδ T cells are regarded as innate immune cells and recognize antigens in a non-MHC restricted manner, they possess a huge diversity of complementary determinant region 3 (CDR3) of T cell receptor (TCR) generated by the rearrangement of germ-line gene V- (D) -J-C fragments. However, the detailed characteristics of the TCRγδ CDR3 repertoire remain unclear. A comprehensive analysis would answer fundamental questions about the diversity of the TCRγδ CDR3 repertoire and elucidate the mechanism underlying γδ T cell recognition of pathogens and tumor antigens. In this study, we used Immune Repertoire Sequencing (IR-SEQ) to analyze the diversity of TCRγδ CDR3 repertoires from 30 healthy donors. The results show that IR-SEQ had sufficient repeatability to analyze the TCRγδ CDR3 repertoire. The diversity of TCRγδ CDR3 repertoire is quite dispersed and individually different. The TCR δ chain (TRD) repertoire displayed more diversity and less sharing among individuals compared with TCR γ chain (TRG). To our knowledge, this is the first study to use IR-SEQ to characterize the repertoire of TCRγδ CDR3 in human peripheral blood γδ T cells by using IR-SEQ. Our findings provide a basic understanding of the diversity of TCRγδ repertoire in the physiological condition, which provides a clue to the underlying mechanism of γδ T cell recognition of pathogens and tumor antigens.

Growing adult T-cell leukemia/lymphoma (ATLL) cells in vitro is difficult. Here, we examined the effects of static electricity in the culture medium on the proliferation of ATLL cells. Six out of 10 ATLL cells did not proliferate in vitro and thus had to be cultured in a medium containing negatively charged polymers. In the presence of poly-γ-glutamic acid (PGA) or chondroitin sulfate (CDR), cell lines (HKOX3-PGA, HKOX3-CDR) were established from the same single ATLL case using interleukin (IL)-2, IL-4, and feeder cells expressing OX40L (OX40L(+)HK). Dextran sulfate inhibited growth in both HKOX3 cell lines. Both PGA and OX40L(+)HK were indispensable for HKOX3-PGA growth, but HKOX3-CDR could proliferate in the presence of CDR or OX40L(+)HK alone. Thus, the specific action of each negatively charged polymer promoted the growth of specific ATLL cells in vitro.

Autoimmune myelofibrosis is a rare, distinct clinicopathological entity that can occur in isolation (primary) or in association with systemic autoimmune disorders (secondary), such as systemic lupus erythematosus and Sjogren's syndrome. This disease is characterized by isolated or combined chronic cytopenias associated with autoimmune phenomena and bone-marrow fibrosis. Due to the rarity of this disease, patients are frequently misdiagnosed as having primary myelofibrosis, the most common form of bone-marrow fibrosis. Distinguishing between both disease entities is essential given the drastic therapeutic and prognostic differences between both disorders. We report a case of primary autoimmune myelofibrosis presenting with severe isolated anemia refractory to multiple lines of therapy. This patient was initially misdiagnosed as primary myelofibrosis. The absence of the characteristic features of primary myelofibrosis and the lack of a clonal abnormality on cytogenetic and molecular studies, particularly JAK2, CALR, and MPL mutation analyses, confirmed the absence of an aberrant neoplastic process. Furthermore, the presence of monoclonal T-cell receptor gamma gene rearrangements delineated the presence of an autoimmune disorder supporting our diagnosis of primary autoimmune myelofibrosis.

Chronic active Epstein-Barr virus (EBV) infection of T-cell and NK-cell type, systemic form (CAEBV-T/NK-S) is characterized by EBV T-cell and/or NK-cell proliferation with no changes suggesting malignancy. Therefore, when Hodgkin/Reed-Sternberg (HRS)-like cells are scattered in CAEBV-T/NK-S, it is more likely to be misdiagnosed as classic Hodgkin lymphoma. We encountered a case wherein the patient showed HRS-like cells with typical NK phenotype. Therefore, we further investigated 8 similar cases to provide clinicopathologic and genetic features and discuss their distinction from other related diseases. Clinically, all cases met the diagnostic criteria of CAEBV. Moreover, 4/8 patients had hemophagocytic lymphohistiocytosis. The median survival was 16 months (range, 5 to 35 mo). Pathologically, all lymph node samples had a remarkably similar morphology with scattered HRS-like cells surrounded by a mixture of small-sized lymphocytes, plasma cells, and macrophages that masqueraded classic Hodgkin lymphoma. Besides, erythrophagocytosis was detected in 4/11 samples. The HRS-like cells were positive for CD2, CD3p, CD30, CD56, GrB, and EBER-ISH, but negative for CD20, CD5, PAX-5, and LMP-1. The surrounding lymphocytes were mainly CD8 cytotoxic T cells, without obvious aberrant expression. In addition, all patients were polyclonal in the T-cell receptor γ rearrangement test. The harbored mutations were mainly in epigenetic modifiers, JAK-STAT signaling pathway, and apoptosis/cell cycle pathway, including SOCS1, DDX3X, and KMT2D, similar to other EBV-associated T/NK-cell lymphoproliferative disorders. Therefore, the evidence indicates that "HRS-like cells of NK phenotype" is a variant of CAEBV-T/NK-S. This study may raise awareness of such confounding CAEBV-T/N-S cases in clinical practice to avoid misdiagnosis and treatment delay.

The presence of circulating tumor cells (CTCs) serves as a prognostic marker and indicator of disease relapse, as well as a means of evaluating treatment efficacy in human and canine lymphoma patients. As an extension of our previous study for the construction of clinically useful GeneScan system, we utilized the GeneScan system for detecting CTCs in canine lymphoma patients. Samples from the primary lesion and peripheral blood mononuclear cells (PBMCs) were obtained from 32 dogs with lymphoma at initial diagnosis. All samples were subjected to polymerase chain reaction (PCR) for antigen receptor gene rearrangements (PARR) followed by GeneScan analysis. Common clonal rearrangements with identical amplified fragments were detected in both the primary lesion and PBMCs in 19 of the 32 dogs (59.4%). However, the detection rate of CTCs varied among the anatomical classification of lymphoma studied. GeneScan analysis following PARR would facilitate studies on determining the clinical significance of CTCs in canine lymphoma patients.

BACKGROUND Hydroa vacciniforme-like lymphoproliferative disorder (HVLPD) is a rare Epstein-Barr virus (EBV)-associated lymphoma that mainly affects children. OBJECTIVES To examine the similarities and differences in the clinical pathological features, EBV infection status, and gene rearrangements in adults and children patients with HVLPD. METHODS We compared the clinical manifestations, histopathology, immunophenotypical features, EBV infection status, and T-cell receptor gene rearrangements in the adult and children HVLPD groups. RESULTS Clinical manifestations differed between children and adults groups. The children were characterized by blisters and severe facial swelling, whereas the adults were characterized by mild facial swelling and papules. Mosquito bite was significantly related to morbidity in the children group. Histologically, the number of mast cells in the adult group was greater than in the children group (P < 0.05). There were no significant differences in EBV infection status or TCR-γ gene rearrangements between 2 groups. CONCLUSIONS There were differences in clinical pathology and prognosis between the 2 groups. A higher mast cell count and T-cell phenotype might be associated with a poor prognosis.

γδ T lymphocytes are a unique T cell population with important anti-inflammatory capabilities. Their role in acute lung injury, however, is poorly understood but may provide significant insight into lung-protective mechanisms occurring after injury. In a murine model of lung injury, wild-type C57BL/6 and TCRδ−/− mice were exposed to Escherichia coli LPS, followed by analysis of γδ T cell and macrophage subsets. In the absence of γδ T cells, TCRδ−/− mice developed increased inflammation and alveolar-capillary leak compared with wild-type C57BL/6 mice after LPS exposure that correlated with expansion of distinct macrophage populations. Classically activated M1 macrophages were increased in the lung of TCRδ−/− mice at d 1, 4, and 7 after LPS exposure that peaked at d 4 and persisted at d 7 compared with wild-type animals. In response to LPS, Vγ1 and Vγ7 γδ T cells were expanded in the lung and expressed IL-4. Coculture experiments showed decreased expression of TNF-α by resident alveolar macrophages in the presence of γδ T cells that was reversed in the presence of an anti-IL-4-blocking antibody. Treatment of mice with rIL4 resulted in reduced numbers of M1 macrophages, inflammation, and alveolar-capillary leak. Therefore, one mechanism by which Vγ1 and Vγ7 γδ T cells protect against LPS-induced lung injury is through IL-4 expression, which decreases TNF-α production by resident alveolar macrophages, thus reducing accumulation of M1 macrophages, inflammation, and alveolar-capillary leak.

Multiple distinct T-cell clones have been demonstrated in a subset of mycosis fungoides (MF), but have so far not been documented in folliculotropic MF, a clinical and histological variant of MF. We analyzed T-cell receptor (TCR) gene rearrangements in 8 patients with folliculotropic MF with multiple biopsies (range, 2-5) taken during the course of disease. Two patients had disease stage IA-IIA, 5 stage IIB-IVA, whereas data were not available for 1 patient. TCR β and γ gene rearrangements were analyzed according to the BIOMED-2 PCR protocol. Multiple clonal TCR gene rearrangements indicating more than 1 T-cell clone were found in 5 patients. Although the number of patients is small, the finding of multiple distinct T-cell clones in 5 out of 8 patients suggests that chronic T-cell stimulation contributes to the development of folliculotropic MF.

[Figure][1] A 51-year-old HIV-negative male from El Salvador presented with neurologic symptoms and computed tomography findings of an acute/subacute cerebral infarction. Physical examination identified indurated and hyperpigmented cutaneous lesions on the legs. A skin biopsy revealed an

In the present study, 21 cases of adult/late-onset EBV-associated lymphoproliferative disease (AELPD) with an uncertain malignant potential were investigated with regard to their histomorphology, immunophenotype, clonal rearrangement of the heavy chain (IgH) and T-cell receptor (TCR) genes and clinical course. The cases were histomorphologically reevaluated and assigned to one of three morphological groups: mononucleosis-like, Hodgkin-like, or polymorphous. In addition, cases with or without detectable necrosis were investigated for differences in clinical outcome. Overall survival was highest in the group with Hodgkin-like morphology (4/4 patients), followed by patients with mononucleosis-like phenotype (4/5 patients surviving). Cases with polymorphous morphology showed the poorest survival rates with 7/12 patients dead of disease (58%). 4/6 patients with histologically detectable necrosis died (66%), but only 4/15 patients without necrosis (27%). 11/21 cases with AELPD showed clonal rearrangement for IgH (n = 4), TCR (n = 5) or IgH + TCR (n = 2). 5/11 patients with clonal rearrangement died (45%), and this percentage was similar in all of the three subgroups. In conclusion, the present study shows that polymorphous morphology and detection of necrosis in AELPD are frequently linked to a fatal clinical course, whereas Hodgkin-like morphology seems to be associated with a more favourable prognosis. Clonal rearrangement of IgH or TCR is frequent in AELPD, but prognosis is unpredictable from this feature.

Mycosis fungoides is the most common primary cutaneous lymphoma; however, it remains a significant diagnostic challenge, in part because of the overlap with several inflammatory dermatoses. Despite advances in immunohistochemistry and molecular diagnostics, false-positive, false-negative, and indeterminate diagnoses are not uncommon. In most cases, the overall balance of morphologic, immunophenotypic, and genetic features must be considered carefully because there are few sensitive and specific clues to the diagnosis. Moreover, an appropriate clinical presentation is essential to the diagnosis and helps to favor or exclude inflammatory/reactive processes. Herein, we discuss 3 important inflammatory dermatoses that may closely simulate mycosis fungoides, and we review the use of ancillary studies in these challenging cases.

The SET-NUP214 (TAF1/CAN) fusion gene is a rare genetic event in T-cell acute lymphoblastic leukemia (T-ALL). Eleven (6%) of 196 T-ALL patients enrolled in the French Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL) 2003 and 2005 trials harbored a SET-NUP214 transcript. SET-NUP214-positive patients were predominantly (10 [91%] of 11) T-cell receptor (TCR)-negative and strikingly associated with TCRγδ lineage T-ALLs, as defined by expression of TCRγδ, TCRδ and/or TCRγ rearrangements but no complete TCRβ variable diversity joining rearrangement in surface CD3/TCR-negative cases. When compared with SET-NUP214-negative patients, SET-NUP214-positive patients showed a significantly higher rate of corticosteroid resistance (91% vs 44%; P = .003) and chemotherapy resistance (100% vs 44%; P = .0001). All SET-NUP214-positive patients but one achieved complete remission, and 9 were allografted. Despite the poor early-treatment sensitivity, the outcome of SET-NUP214-positive patients was similar to that of SET-NUP214-negative patients.

Objectives: To design and evaluate a next-generation sequencing (NGS)–based method for T-cell receptor γ (TCRG) gene-based T-cell clonality testing on the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA) platform. Methods: We analyzed a series of peripheral blood, bone marrow, and formalin-fixed paraffin-embedded tissue specimens with NGS vs traditional capillary electrophoresis methods. Results: Using a custom analysis algorithm that we developed, our NGS assay identified between 2,215 and 48,222 unique TCRG rearrangements in a series of 48 samples. We established criteria for assigning clonality based on parameters derived from both the relative and absolute frequencies of reads. In a comparison with standard capillary electrophoresis, 19 of 19 polyclonal samples and 24 of 27 samples that appeared clonal were in agreement. The three discrepant samples demonstrated some of the pitfalls of amplicon length–based testing. Dilution studies with T-lymphoid cell lines demonstrated that a known clonal sequence could be routinely identified when present in as few as 0.1% of total cells demonstrating suitability in residual disease testing. A series of samples was also analyzed on a second NGS platform and yielded very similar results with respect to the frequency and sequence of the clonal rearrangement. Conclusions: In this proof-of-concept study, we describe an NGS-based T-cell clonality assay that is suitable for routine clinical testing either alone or as an adjunct to traditional methods.

Primary central nervous system lymphoma (PCNSL) expressing T-cell markers is rare, among which nasal-type extranodal NK/T-cell lymphoma is an extremely rare subtype associated with Epstein-Barr virus (EBV) infection. Here we report the clinicopathologic features of a case of EBV-associated PCNSL showing a cytotoxic T-cell phenotype. The patient, a 73-year-old woman, presented with rapidly progressive mental deterioration. Brain MRI revealed multiple lesions with swelling in the bilateral cerebral hemispheres, which were hypointense on T1-weighted images, hyperintense on T2-weighted and fluid-attenuated inversion recovery images, and slightly hyperintense on diffusion-weighted images. Biopsy specimens from the temporal region showed many medium-sized anaplastic lymphocytic cells with perivascular and angio-invasive patterns in the cortex. Immunohistochemically, the cells were positive for CD3, CD8, T-cell-restricted intracellular antigen-1 (TIA-1), granzyme B and perforin, but negative for CD56 and CD20. In situ hybridization revealed EBV-encoded RNAs in the tumor cell nuclei. A rearrangement study showed T-cell receptor γ-chain gene rearrangement with a clonal appearance. The patient died 6 months after surgery, and a general autopsy revealed no lymphoma cells outside the brain. These cellular profiles are inconsistent with those of extranodal NK/T-cell lymphoma, and have not been previously described. This case appears to represent an unusual CNS manifestation of EBV-associated T-cell lymphoma.

Feline large granular lymphocyte lymphomas are rare but very aggressive tumors with a poor prognosis. In this study, a cell line from an abdominal effusion of a cat with large granular lymphoma was characterized. Immunophenotype staining was positive for CD3 and CD45R, and negative for CD4, CD8, CD56, CD79α, BLA.36 and NK1. A TCR γ gene rearrangement was detectable by PARR. Neither FeLV antigen nor exogenous FeLV provirus could be detected. A chromosomal instability associated with a centrosome hyperamplification could also be determined. The cell line is able to lyse target cells without antigen presentation or interaction with antigen presenting cells. Therefore, these cells were classified as genetically instable non-MHC-restricted cytotoxic T cells with large granular lymphocyte morphology.

BACKGROUND Refractory celiac disease (RCD) is a preneoplastic condition as many patients develop an enteropathy-type T-cell lymphoma, a mature T-cell receptor α-β lymphoma arising in the gut with an ominous outcome. Recently, research focused on a population of intraepithelial intestinal lymphocytes expressing the same lymphoma T-cell receptor variable region (V)γ, as shown by polymerase chain reaction (PCR) analysis and sequencing. Meanwhile, the Biomedicine and Health-2 Concerted Action has made available standardized, highly specific, and sensitive PCR assays not only for Vγ but also for Vβ. GOALS We verified whether analyzing both rearrangements in duodenal biopsies from RCD patients increases the diagnostic accuracy of this method. STUDY Duodenal biopsies were analyzed from 15 RCD patients, 21 negative controls, and 2 positive controls (enteropathy-type T-cell lymphoma complicating celiac disease). Multiplex clonality analyses were performed according to the Biomedicine and Health-2 protocols. PCR products were cloned and sequenced. RESULTS Monoclonal rearrangements were found in 5/15 samples from patients with RCD (both rearrangements in 2 cases, Vβ only in 2, and only 1 solitary Vγ clonality). Monoclonality was found in 4/8 of the RCD patients who subsequently died, whereas only 1/7 of the patients still alive presented a monoclonal rearrangement. Positive controls revealed both monoclonal rearrangements; rearrangements were not detected in 20 of 21 negative controls. Sequencing of the amplified fragments confirmed the results. CONCLUSIONS The combined analysis of both rearrangements allowed recognition of monoclonal populations in otherwise negative patients, with detection rates from 20% (Vγ only) to 33% (Vγ and Vβ), thus raising the likelihood of early identification of RCD patients at high risk of death.

We report 13 cases of anaplastic large cell lymphoma (ALCL) associated with breast implants. Patient age ranged from 39 to 68 years, and the interval from implant to ALCL was 4 to 29 years. All tumors were composed of large, pleomorphic cells that were CD30 and ALK1, and all 7 cases assessed had monoclonal T-cell receptor γ-chain rearrangements. Two patient subgroups were identified. Ten patients presented with effusion surrounded by fibrous capsule without a grossly identifiable tumor mass. Nine patients had stage I and 1 had stage II disease. Eight patients underwent implant removal and capsulectomy. Four patients received chemotherapy and 4 radiation therapy. All patients were alive without disease at last follow-up. A second subgroup of 3 patients had effusion and a distinct mass adjacent to the implant. One patient had stage I and 2 stage II disease. One patient had a 3-year history of lymphomatoid papulosis, and 1 patient had a 1-year history of CD30 T-cell lymphoma adjacent to the breast before the diagnosis of ALCL associated with breast implant. Two patients received chemotherapy and 1 radiation therapy. Two patients died 2 and 12 years after diagnosis, respectively. We conclude that the clinical behavior of ALCL associated with breast implants is heterogeneous. Patients who present with effusion without a distinct mass have an indolent disease course, similar to CD30 lymphoproliferative disorder of skin. In contrast, patients who present with a distinct mass may have advanced stage or possibly systemic disease and have a poorer prognosis.

Demonstration of T-cell receptor gene monoclonality often plays an important role in the diagnosis of T-cell lymphoma. When a test to detect monoclonality is performed on whole tissue sections, the presence of a reactive lymphocyte population may reduce sensitivity. This may be especially true for early or borderline cases of lymphoma. Microdissection techniques may be utilized to more readily identify a clonal population of lymphocytes. Subcutaneous panniculitis-like T-cell lymphoma represents a cutaneous lymphoid neoplasm whose clinical course may vary from an indolent, waxing and waning course to an aggressive course resulting in death. We report the first case of a microdissection technique used to facilitate diagnosing a case of subcutaneous panniculitis-like T-cell lympoma. Hoffman D, Chaffins M, Cankovic M, Maeda K, Meehan S. Manual microdissection technique in a case of subcutaneous panniculitis-like T-cell lymphoma: a case report and review.

Background: The diagnosis of early mycosis fungoides (MF) is a big challenge to dermatologists and dermatopathologists because it lacks specific clinicopathologic features. Methods: Fifty-two paraffin-embedded skin samples from 50 patients, including 31 with suspected MF, 10 with typical MF and 9 with benign inflammatory dermatosis (BID), were obtained from our archives. DNA was extracted both by traditional phenol-chloroform method and by the laser-capture microdissection (LCM)-proteinase K approach. The TVG/TJG, V2–5/V8–12/JGT1 and BIOMED-2-TCR-γ primers were used to assess TCR-γ monoclonal rearrangement as measured by polymerase chain reaction (PCR). Results: In the suspected MF group, clonal TCR-γ gene rearrangements were detected in 11/31 cases (35.5%) by phenol-chloroform DNA extraction and in 25/31 cases (80.7%) by LCM-proteinase K extraction (p 0.05) in the typical MF group, no TCR-γ monoclonal rearrangement was detected in the BID group. Conclusions: The strategy of multiple PCR/heteroduplex analysis for TCR-γ gene rearrangement combined with LCM increases the detection rate of clonal TCR-γ gene rearrangement in early MF cases and could provide strong evidence to confirm the diagnosis of early MF. Yang H, Xu C, Tang Y, Wan C, Liu W, Wang L. The significance of multiplex PCR/heteroduplex analysis-based TCR-γ gene rearrangement combined with laser-capture microdissection in the diagnosis of early mycosis fungoides.

Primary central nervous system (CNS) lymphomas are relatively rare with the most common subtype being diffuse large B-cell lymphoma. Primary CNS T-cell lymphomas (PCNSTL) account for 1 mutation, and none showed overlapping mutations. These included mutations in DNMT3A, KRAS, JAK3, STAT3, STAT5B, GNB1, and TET2 genes, genes implicated previously in other T-cell neoplasms. The outcome was heterogenous; 2 patients are alive without disease, 4 are alive with disease, and 6 died of disease. In conclusion, PCNSTLs are histologically and genomically heterogenous with frequent phenotypic aberrancy and a cytotoxic phenotype in most cases.

Extranodal natural killer/T-cell lymphoma, nasal type (NK/TCL) is more prevalent in Asia and in some areas of South and Central America, but it is rarely seen in the United States and Europe. In this study, a series of 122 cases of NK/TCL from Brazil was analyzed with respect to clinicopathologic features. Clinical characteristics and geographic distribution were evaluated in 97 cases of nasal/nasopharyngeal region and 23 cases in extranasal sites including 6 nodal cases. Clinical staging and follow-up information was available in a subset of 21 patients. All cases harbored Epstein-Barr virus (EBV), 95% and 85% expressed cytoplasmic CD3 and CD56, respectively, and all cases were positive for at least 1 marker for cytotoxic granules. The global distribution of EBV subtypes showed predominance of strain subtype A, 89%, and subtype B, 11%. No dual infections were detected. TCR-γ TCR-gene rearrangement was observed in 7 cases; all of them extranodal. Three of TCR-γ(+) cases showed EBV subtype A. Two TCR-γ(+)/CD56(+) cases showed EBV subtype B. Geographic distribution of NK/TCL showed higher frequency in the southeast and northeast regions of Brazil. Striking differences among geographic regions were seen with the vast majority of EBV subtype B (86%) occurring in the south and southeast regions.

γδ T cells represent a minor T-cell subset that is mainly distributed in mucosal surfaces. Two distinct lymphomas derived from these cells have been recognized: hepatosplenic γδ T-cell lymphoma (HSTL) and primary cutaneous γδ T-cell lymphoma (PCGD-TCL). However, whether other anatomic sites may also be involved and whether they represent a spectrum of the same disease are not well studied. The lack of T-cell receptor (TCR)β expression has been used to infer a γδ origin when other methods are not available. We studied 35 T-cell tumors suspected to be γδ TCL using monoclonal antibodies reactive with TCR δ or γ in paraffin sections. We were able to confirm γδ chain expression in 22 of 35 cases. We identified 8 PCGD-TCLs, 6 HSTLs, and 8 γδ TCLs without hepatosplenic or cutaneous involvement involving mainly extranodal sites. Two such cases were classified as enteropathy-associated T-cell lymphoma, type II. The other γδ TCL presented in the intestine, lung, tongue, orbit, and lymph node. In addition, we observed 13 cases with mainly extranodal involvement that lacked any TCR expression ("TCR silent"). In all cases, a natural killer cell origin was excluded. In conclusion, the lack of TCRβ expression does not always predict γδ-T-cell derivation, as TCR silent cases may be found. The recognition of γδ TCL presenting in extranodal sites other than skin and liver/spleen expands the clinical spectrum of these tumors. However, non-HSTL γδ TCL do not seem to represent a single entity. The relationship of these tumors with either HSTL or PCGD-TCL requires further study. Supported by Instituto de Salud Carlos III, Fondo de Investigación Sanitaria) PI080095 (AM) and PI050458 (TE). The Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT) SAF08-3630 (EC), Red Temática de Investigación Cooperativa del Cáncer (RTICC) RD06/0020/0039 (EC), AGH is a fellow supported by the Instituto de Salud Carlos III. This work was also supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute

Prostaglandin D2 (PGD2) exerts its effects through two distinct receptors: the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the D prostanoid (DP) receptor. Our previous study demonstrated that CRTH2 mediates contact hypersensitivity (CHS) in mice. However, the function of DP receptor remains to be fully established. In this study, we examine the pathophysiological roles of PGD2 using DP-deficient (DP(-/-)) and CRTH2/DP-deficient (CRTH2(-/-)/DP(-/-)) mice to elucidate receptor-mediated PGD2 action in CHS. We observed profound exacerbation of CHS in DP(-/-) mice. CRTH2(-/-)/DP(-/-) mice showed similar exacerbation, but to a lesser extent. These symptoms were accompanied by increased production of interferon-γ and IL-17. The increase in IL-17 producing γδ T cells was marked and presumably contributed to the enhanced CHS. DP deficiency promoted the in vivo migration of dendritic cells to regional lymph nodes. A DP agonist added to DCs in vitro was able to inhibit production of IL-12 and IL-1β. Interestingly, production of IL-10 in dendritic cells was elevated via the DP pathway, but it was lowered by the CRTH2 pathway. Collectively, PGD2 signals through CRTH2 to mediate CHS inflammation, and conversely, DP signals to exert inhibitory effects on CHS. Thus, we report opposing functions for PGD2 that depend on receptor usage in allergic reactions.

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs, substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγcnull (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples, 39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However, 33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients, but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction, and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function, and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies.

Adult T-cell leukemia/lymphoma (ATLL) is the neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). We performed oligo-array comparative genomic hybridization (CGH) against paired samples comprising peripheral blood (PB) and lymph node (LN) samples from 13 patients with acute ATLL. We found that the genome profiles of the PB frequently differed from those of the LN samples. The results showed that 9 of 13 cases investigated had a log2 ratio imbalance among chromosomes, and that chromosome imbalances were more frequent in LN samples. Detailed analysis revealed that the imbalances were likely caused by the presence of multiple subclones in the LN samples. Five of 13 cases showed homozygous loss regions in PB samples, which were not found in the LN samples, indicating that tumors in the PB were derived from LN subclones in most cases. Southern blot analysis of TCRγ showed that these multiple subclones originated from a common clone. We concluded that in many ATLL cases, multiple subclones in the LNs originate from a common clone, and that a selected subclone among the LN subclones appears in the PB.

We established a novel feline B-cell line, MS4, from the neoplastic pleural effusion of a cat with cutaneous B-cell lymphoma. Immunophenotype staining of the MS4 cells was positive for CD20, CD79α, and IgA and negative for CD3, CD4, CD5, CD8α, CD18, CD21, CD22, IgM, IgG, Ig light chain, and MHC class II. PCR analysis for immunoglobulin heavy chain gene rearrangements revealed a monoclonal rearrangement, whereas no clonal rearrangement of the T-cell receptor γ gene was detected. Southern blotting with an exogenous feline leukemia virus (FeLV) U3 probe revealed no integration of exogenous FeLV provirus. The MS4 cell line is the first FeLV-negative feline B-cell lymphoma cell line, and may be used to investigate the pathogenesis of spontaneously occurring feline lymphoma and the development of new therapies.

V(D)J recombination of Ig and TCR genes is strictly regulated by the accessibility of target gene chromatin in a lineage- and stage-specific manner. In the mouse TCRγ locus, rearrangement of the Vγ2 gene predominates over Vγ3 rearrangement in the adult thymus. This preferential rearrangement is likely due to the differential accessibility of the individual Vγ genes, because the levels of germ line transcription and histone acetylation of the Vγ genes are well correlated with the rearrangement frequency in adult thymocytes. However, factors responsible for the differential regulation of the Vγ gene rearrangement have been largely unknown. In this study, we demonstrated that Vγ2 rearrangement in the adult thymus was substantially reduced in mice deficient for the basic helix-loop-helix protein, E2A. The decreased rearrangement is likely caused by the reduced accessibility of Vγ2 chromatin, since germ line transcription and histone acetylation of the Vγ2 gene were reduced in an E2A dosage-dependent manner. We further showed that E2A bound around the Vγ2 gene in vivo and we identified two canonical E-box sites downstream of Vγ2, to which E2A can bind in vitro. Furthermore, these two E-box sites had the ability to activate transcription upon E2A over-expression. These data suggest that E2A directly binds to and increases accessibility of Vγ2 chromatin, thereby facilitating Vγ2 rearrangement in the adult thymus.

Mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, has various unspecific clinical and histological characteristics. Its early diagnosis is challenging. The application of T-cell receptor (TCR) gene clonal rearrangement to the diagnosis of MF has been widely studied. In this study, we used polymerase chain reaction (PCR) to investigate the diagnostic significance of detecting TCR-γ and -β gene clonal rearrangement in the early diagnosis of mycosis fungoides. PCR for TCR-γ and TCR-β gene rearrangement was performed on 19 patients with suspected early MF, 6 with typical MF, and 6 with chronic dermatitis. Of the 19 patients with suspected early MF, 13 had TCR-γ gene clonal rearrangement, whereas none had TCR-β gene clonal rearrangement. All patients with typical MF had TCR gene clonal rearrangement, in which 4 showed TCR-γ clonal rearrangement, 1 showed TCR-β gene clonal rearrangements, and 1 showed both. No patients with chronic dermatitis had TCR gene clonal rearrangement. These results indicate that TCR gene clonal rearrangement analysis is a useful tool in diagnosing early MF. TCR-γ gene is recommended to the routine analysis, whereas TCR-β gene has potential in combination toward intractable cases.

Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus-negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD.

Primary central nervous system (CNS) extranodal NK/T-cell lymphoma, nasal type (NKTCL), is an extremely rare tumor. To the best of our knowledge, only four cases have been described previously. Here, we report a case of primary CNS NKTCL in a 25-year-old immunocompetent Chinese male. The patient presented with worsening dizziness, headaches, and vomiting for approximately 2 weeks. Magnetic resonance imaging demonstrated three masses with solid components entirely in the parenchyma of the right hemisphere, and no sinonasal/nasopharyngeal lesions were found. The patient underwent a partial resection of the right temporal mass. Histological examination revealed that intermediate-sized, pleomorphic lymphocytes were arranged in an angiocentric distribution with large geographic necroses. The tumor cells expressed CD3ε, CD56, TIA-1, granzyme B, and Epstein-Barr virus-encoded RNAs. A rearrangement study showed T-cell receptor γ-chain gene rearrangement with monoclonal appearance. Postoperative chemotherapy and radiotherapy were also given, but the lymphoma failed to respond to therapy and the patient died 18 months later. Our observation and the four others found in the literature indicate that primary CNS NKTCL occurs predominantly in adult males. This is the youngest patient with primary CNS NKTCL reported.

Patients with rheumatoid arthritis, whether treated or not with immunosuppressive agents including methotrexate, have an increased risk of lymphoproliferative disorders. Termed "iatrogenic immunodeficiency-associated lymphoproliferative disorders" in the 2008 World Health Organization classification of lymphoid neoplasms, they include Hodgkin and non-Hodgkin lymphomas. Composite lymphomas are rare, particularly in skin, with none reported in immunodeficiency states. We report the case of a 67 year-old woman with a long history of rheumatoid arthritis, on methotrexate treatment, who developed multiple skin lesions exhibiting a malignant infiltrate displaying both B- and T-cell phenotypes and dual clonal gene rearrangement by polymerase chain reaction, consistent with a cutaneous composite lymphoma. The patient received chemotherapy including rituximab with partial response, but the T-cell component recurred. To the best of our knowledge, this is the first case report of a cutaneous composite lymphoma in a patient with an iatrogenic immunodeficiency representing a dual challenge, diagnostic for the pathologist and therapeutic for the hematologist.

Lupus erythematosus panniculitis (LEP) is an inflammatory disorder of the subcutaneous fat in patients with lupus erythematosus (LE). It is a rare variant of the disease, which occurs approximately in 1%-3% of patients with cutaneous LE. The purpose of this study was to investigate the clinical, histopathologic, immunophenotypical, and molecular profiles of LEP. We performed a retrospective study of 19 biopsy specimens from 17 patients with LEP. We reviewed their clinical data and reexamined the histopathology. Immunophenotyping and molecular studies were done using sections from paraffin-embedded formalin-fixed tissue. The most common clinical manifestation was a depressed patch on upper arm. Patients showed good response to variable treatment modalities, but, generally, relapse of panniculitis was noted when treatment was discontinued. Histopathologically, most specimens revealed lymphoplasmacytic lobular panniculitis with epidermal and dermal changes of LE, hyaline fat necrosis, and lymphoid follicles. Immunohistochemistry showed a mixture of T and B cells in dermis and subcutis with a slight preponderance of T cell. Although the polymerase chain reaction analysis of the T-cell receptor-gamma gene rearrangement showed a polyclonal smear in 89.5% of cases, a small portion of specimens demonstrated monoclonality. LEP is a chronic recurrent disease with characteristic features. Its diagnosis is often challenging, and a precise diagnosis is achievable only upon elaborate clinicopathologic correlation and integrated interpretation of all diagnostic criteria.

Background: Recently, Petrella et al. described four patients with an unusual CD8+ lymphoid proliferation arising on the ear. These cases do not correspond clearly to any recognized category of cutaneous T-cell lymphoma (CTCL) described in the World Health Organization (WHO)/European Organization for Research and Treatment of Cancer (EORTC) 2005 classification. Methods and Results: Three patients (all men; median age 64; range: 61-69) presented with plaques or small tumors localized on the ears. All lesions showed histopathologically a dense, diffuse infiltration of lymphocytes within the entire dermis without epidermotropism. Cytomorphology revealed predominance of medium-sized pleomorphic lymphocytes. Immunohistochemistry showed a cytotoxic phenotype (CD3 + /CD4 −/CD8 +). Polymerase chain reaction (PCR) analysis of the T-cell receptor (TCR)-gamma gene revealed a monoclonal rearrangement in two of three patients. Follow-up data of two patients were available; one is alive without skin or systemic manifestations of the disease after 28 months, whereas the other is alive with persistent skin disease after 7 months. Conclusions: Our observation confirms that some patients present with a peculiar lymphoid proliferation of small-medium pleomorphic cytotoxic lymphocytes located on the ear, probably representing a phenotypic variant of the cutaneous small/medium pleomorphic T-cell lymphoma (CSMPTCL). These cases should not be misinterpreted as a high-grade cytotoxic lymphoma.

Background A T-cell clone, thought to be the source of eosinophilopoietic cytokines, identified by clonal rearrangement of the T-cell receptor and by the presence of aberrant T-cell immunophenotype in peripheral blood defines lymphocytic variant of hypereosinophilic syndrome (L-HES).Design and Methods Peripheral blood samples from 42 patients who satisfied the diagnostic criteria for HES were studied for T-cell receptor clonal rearrangement by polymerase chain reaction according to BIOMED-2. The T-cell immunophenotype population was assessed in peripheral blood by flow cytometry. The FIP1L1-PDGFRA fusion gene was detected by nested polymerase chain reaction.Results Forty-two HES patients (18 males and 24 females) with a median age at diagnosis of 56 years (range 17–84) were examined in this study. Their median white blood cell count was 12.9×109/L (range 5.3–121), with an absolute eosinophil count of 4.5×109/L (range 1.5–99) and a median eosinophilic bone marrow infiltration of 30% (range 11–64). Among the 42 patients, clonal T-cell receptor rearrangements were detected in 18 patients (42.8%). Patients with T-cell receptor clonality included: T-cell receptor β in 15 patients (35%), T-cell receptor γ in 9 (21%) and T-cell receptor δ in 9 (21%) patients, respectively. Clonality was detected in all three T-cell receptor loci in 4 cases, in two loci in 7 patients and in one T-cell receptor locus in the remaining 7 patients. The FIP1L1-PDGFRA fusion transcript was absent in all but 2 patients with T-cell receptor clonality. Three patients out of 42 revealed an aberrant T-cell immunophenotype. In some patients, an abnormal CD4:CD8 ratio was demonstrated.Conclusions T-cell abnormalities are present at high frequencies in patients with HES.

We describe a case of human immunodeficiency virus-associated T-lymphoblastic leukemia/lymphoblastic lymphoma in a 43-year-old Italian man with a history of human immunodeficiency virus infection lasting 9 years. Immunoperoxidase stains showed that neoplastic cells were positive for CD3, TdT, CD45, CD10, CD1a, CD2, CD7, CD5, and CD43 (focal). The proliferation rate was approximately 70%, assessed by Ki-67/MIB-1 staining. Flow cytometry of the marrow aspirate revealed an intermediate/cortical T-lymphoblastic phenotype: negative for surface CD3 and positive for cytoplasmic CD3, CD1a, TdT, CD2, CD7, CD5, and CD8, with partial coexpression of dimCD4. Analysis of T-cell receptor gamma polymerase chain reaction products showed clonality. T-lymphoblastic leukemia/lymphoblastic lymphoma is a very rare occurrence in the clinical setting of human immunodeficiency virus infection. It is not listed in the World Health Organization classification of lymphomas associated with human immunodeficiency virus infection. Only 4 cases of human immunodeficiency virus-associated T-lymphoblastic leukemia/lymphoblastic lymphoma are reported in the current medical literature. (C) 2009 Elsevier Inc. All rights reserved.

Although the new World Health Organization-European Organization for Research and Treatment of Cancer classification focuses on providing uniformity in the diagnosis of cutaneous lymphomas, cutaneous peripheral T-cell lymphoma (PTL) remains a poorly defined subgroup. As follow-up to a study of systemic PTL complicated by a proliferation of B cells, we studied 16 cases of cutaneous PTL that contained morphologically atypical T cells associated with a significant infiltrate of B cells (about 20%-50%). A clonal T-cell receptor gamma chain gene rearrangement was present in all cases. In contrast, a clonal immunoglobulin heavy chain gene rearrangement was present in only 1 case. Clinical staging in 14 cases identified systemic involvement in 2. At last follow-up, both patients with systemic involvement had died of disease, and the majority of patients with primary cutaneous disease were alive (11/12). The presence of numerous atypical B cells and T cells caused diagnostic confusion in these cases. Comprehensive pathologic studies, coupled with clinical staging, are necessary for the accurate diagnosis of this unusual manifestation of cutaneous PTL.

Summary Epstein-Barr virus–positive B-cell lymphoproliferative disorders similar to those seen in posttransplantation patients have recently been described in immunocompetent, elderly Asian individuals. To our knowledge, this entity has not been described in the United States. The clinicopathologic features of 6 Epstein-Barr virus–positive B-cell lymphomas of the elderly from 5 patients 60 years and older were reviewed. Three specimens exhibited a polymorphous infiltrate with Reed-Sternberg–like cells. The remaining specimens resembled conventional diffuse large B-cell lymphoma. Most tumor cells were Epstein-Barr virus–positive by in situ hybridization and had a nongerminal center B-cell immunophenotype. In addition, 90 cases of diffuse large B-cell lymphoma in patients 60 years and older were screened for Epstein-Barr virus and were negative. All 5 patients were treated with combination chemotherapy; however, they had a poor outcome. Epstein-Barr virus–positive B-cell lymphomas of the elderly occur uncommonly at a United States tertiary medical center. Pathologic features that may aid in identification and trigger Epstein-Barr virus testing include a polymorphic infiltrate, plasmacytic morphology, Reed-Sternberg–like cells, angioinvasion, necrosis, and a nongerminal center B-cell immunophenotype. Further investigation is needed to determine if Epstein-Barr virus status plays an independent role in the prognosis of diffuse large B-cell lymphoma in non-Asian countries, which would also impact the need to screen all diffuse large B-cell lymphoma in older patients for Epstein-Barr virus.

We describe 9 patients with T-cell large granular lymphocyte leukemia (T-LGL) who also had a myelodysplastic syndrome (MDS). There were 6 men and 3 women with a median age of 60 years (range, 25-74 years). All patients had anemia at initial examination, 7 had neutropenia, and 5 had thrombocytopenia. The median absolute lymphocyte count was 1,300/microL (1.3 x 10(9)/L; range, 700-3,600/microL [0.7-3.6 x 10(9)/L]). Immunophenotypic analysis showed a CD8+ T-cell population, and molecular analysis showed monoclonal T-cell receptor gene rearrangement in every case. The MDS was classified as refractory cytopenia with multilineage dysplasia (RCMD, n = 5), refractory anemia (n = 2), RCMD with ringed sideroblasts (n = 1), and chronic myelomonocytic leukemia (n = 1). We compared the data for these patients with T-LGL/MDS with a group that had only T-LGL. The median hemoglobin level and absolute lymphocyte count were lower in patients with T-LGL/MDS (P < .05). The frequency of coexistent T-LGL and MDS at our institution suggests an etiologic relationship rather than simple coincidence.

The analysis of total plasma DNA and the monitoring of leukemic clone-specific immunoglobulin and/or T-cell receptor gene rearrangements for the evaluation of minimal residual disease (MRD) in the plasma may be useful tools for prognostic purposes or for early detection of subclinical disease recurrence in children with acute lymphoblastic leukemia (ALL). The aim of this paper is to establish reference ranges for total plasma DNA concentrations and to test the feasibility of MRD measurements employing plasma DNA from children with ALL by using real-time quantitative (RQ)-PCR. Despite wide inter-individual variation, the median concentrations of total plasma DNA for 12 healthy donors (57 ng/ml), 21 children with ALL after day 4 of treatment initiation (62 ng/ml) and 13 children with other malignancies (76 ng/ml) were similar. However, ALL patients had significantly higher concentrations at diagnosis (277 ng/ml) and on treatment day 3 (248 ng/ml) before returning to normal afterwards. Early plasma DNA MRD kinetics could be established for 15 ALL patients and showed good concordance with bone marrow MRD. Plasma DNA was higher in children with ALL at diagnosis but returned to normal within the first four treatment days. Despite low concentrations of DNA, it is feasible to measure MRD kinetics in plasma DNA during ALL induction therapy by adapted real-time PCR methodologies.