T-cell Receptor Gamma Gene Rearrangement Research Articles

Supratentorial metastatic enteropathy-associated T-cell lymphoma: A case report and literature review

Background:We are describing a rare case of supratentorial metastatic enteropathy-associated T-cell lymphoma (EATL). While these lesions are a rare complication of EATL, the implications are grave and they must be evaluated as a diagnostic possibility when a patient with known celiac disease presents with acute neurological deterioration. In addition, multidisciplinary care teams are recommended by the authors as critical to providing the most comprehensive patient care.Case Description:A 65-year-old female presented to the emergency room with uncontrolled abdominal pain, nausea, and vomiting. Initial abdominal computed tomography (CT) scan indicated a small bowel obstruction with a transition point at the jejunal area. Differential diagnosis included small bowel neoplasm, adhesions, or a reactive intestinal inflammatory process. Shortly after presentation, the patient's clinical condition worsened, requiring emergency small bowel resection. Histological analysis of the resected bowel segments demonstrated medium-sized infiltrating lymphocytes with characteristic pleomorphic nuclei and prominent nucleoli. Immunohistochemical stains revealed tumor cells positive for CD-3. Immunohistochemical analysis for Ki-67 showed a markedly increased proliferative index, with 90% of lymphocytes staining positive. Polymerase chain reaction analysis for T-cell receptor-gamma gene rearrangement was positive, demonstrating the presence of a clonal population of T-cells. The combined morphological and immunophenotypic features of this lesion were consistent with jejunal EATL.Five weeks post-diagnosis, she developed new onset neurological symptoms consisting of changes in her mental status and left facio-brachial weakness. Brain magnetic resonance imaging (MRI) demonstrated a single ill-defined, irregular, right fronto-parietal enhancing lesion surrounded by vasogenic edema. Surgical resection and histopathologic evaluation of the biopsied lesion confirmed the diagnosis of metastatic EATL involving the brain.Conclusion:Intracranial metastasis is a rare but grave complication of EATL and must be evaluated as a diagnostic possibility when a patient with known celiac disease presents with acute neurological deterioration. Although the prognosis of these patients is dismal, aggressive oncology management is mandatory.

Clonality and Phenotype in Spleens From Patients with Primary Immune Thrombocytopenia,

Abstract 3291Primary immune thrombocytopenia (PIT) results from both increased platelet destruction and insufficient platelet production. Although the development of autoantibodies against platelet glycoproteins remains central in the pathophysiology of PIT, several abnormalities in the immune modulation system have been identified (Blood 2008 Aug 15;112(4):1147; Hematol Oncol Clin North Am. 2009 Dec;23(6):1177; Hum Immunol. 2010 Jun;71(6):586);We analysed morphology, lymphocyte phenotype and heavy immunoglobulin (IgH) and TCR gamma (TCRg) gene rearrangements in spleens from 31 PIT patients to assess possible impact on diagnosis and course, in comparison with 19 spleens surgically removed for trauma.Age ranged from 15 to 68 (mean 41.61y) with M/F of 9/22 in PIT and 18 to 89 (mean: 66y) with M/F 11/8 in controls. All PIT patients experienced at least one line therapy before surgery and 4 underwent anti-CD20 immunotherapy.Immunohistochemistry for CD3, CD20, CD4, CD8, CD56, CD57, PD1, Tia1, Granzyme B, FoxP3, CD72 was made in all cases; both IgH and TCRg gene analysis were possible in 24/31 PIT and 17/19 controls, while 4/31 PIT and 2/19 controls were examined for either one (PIT: 2 TCR and 2 IgH, controls 1 TCR and 1 IgH) and 5 PIT and 1 control were excluded for unsatisfactory material. Ay histology all cases but 2 showed expanded white pulp (that was hypoplastic in the 2/4 cases that underwent Rituximab before surgery), moderate lymphocytic and modest granulocytic infiltrates in the red pulp; haemorragic lakes were present in control spleens. Immunohistochemistry showed similar stains in the 2 groups with normal distribution (white pulp: CD20+ B follicles, CD72-+ in the marginal zone, CD3+/CD4+ T cells, PD1/CD57+ T cells in the germinal centers; red pulp: regular CD20/CD3 and CD4/CD8 ratio, PD1/Tia1/Granzyme B expressed in roughly less than 20% CD3+ T cells; scattered CD57+/CD56- T cells. For molecular results see table 1 below. Monoclonality was defined if 1 or 2 peaks of amplified polymerase chain reaction products were obtained, oligoclonality with 3 to 5 peaks and polyclonality with a Gaussian peak distribution.Table 1IGHWDCtrlIGHWDCtrlPoly2015Poly-Oligo2116Oligo10Mono32Mono32TCRWDCtrlTCRWDCtrlPoly75Poly-Oligo128Oligo53Mono129Mono129The results showed no statistically significant differences between PIT and controls as for morphology, phenotype and clonality. Since a decrease in regulatory T cells (T regs) is reported in PIT among other immune-impairments (Eur J Haematol. 2007 Oct;79(4):310; Zhonghua Nei Ke Za Zhi. 2010 Mar;49(3):213;Blood. 2009 Mar 12;113(11):256) we immunostained all cases with T reg-related nuclear molecule Foxp3: in control cases few cells in the white and red pulp were observed, while in PIT spleens fewer Foxp3 positive cells could only be seen in the red pulp: although results are slightly different, the low amount of positive cells in both groups decreases the reliable reproducibility of such observation. Moreover, the use of glucocorticoids by all patients before splenectomy, could have further reduced (Eur J Haematol. 2007 Oct;79(4):310) T regs levels.Overlapping molecular results were also obtained in the two groups in agreement with previous reports (Hematology. 2009 Aug;14(4):237; Platelets. 2009 Mar;20(2):135; Int J Hematol. 2003 Dec;78(5):461; Blood. 2002 Aug 15;100(4):1388). The attempt to translate the molecular findings into immunomorphologic differences between monoclonal and non monoclonal cases failed since neither amount/distribution of B and T cells nor T cell subtypes showed evident differences. On the whole, our results show that neither lymphoid phenotype nor IgH or TCRg clonality can be used as specific features of refractory PIT or guide treatment choice. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Serous effusion cytology of extranodal natural killer/T-cell lymphoma

Extranodal natural killer/T-cell lymphoma, nasal type (ENKTCL-N), is a rare form of lymphoma that typically occurs at extranodal sites. It is one of the most common extranodal lymphomas in China. Literature on effusions and cytological findings relating to ENKTCL-N is limited. We studied five consecutive cases of ENKTCL-N effusions collected over a 3-year period. The cytomorphological, immunocytochemical and molecular biological features were evaluated with literature review. The purpose of this study is to discuss how to diagnose ENKTCL-N cytologically in effusions. Smears and cell block sections were reviewed for each case. Immunocytochemistry was performed on 4-μm paraffin sections. Antibodies used were as follows: cCD3 (intracytoplasmic CD3), CD45RO, surface CD3, CD20, CD79a, CD56, TIA-1, granzyme B, CD30, CD99, TdT and Ki-67. In situ hybridization for EBER1/2 (EBER-ISH) and T-cell receptor γ (TCRγ) gene rearrangement were performed for all cases. Large to medium-sized tumour cells with pleomorphic nuclei and coarse chromatin were found in a necrotic background in all cases. The cytoplasm of the tumour cells was scant to moderately abundant with occasional cytoplasmic projections; in Giemsa-stained smears, fine granules were present in some tumour cells. Mitotic figures were frequent. The tumour cells were all positive for CD56, granzyme B, TIA-1 and cCD3, and were negative for surface CD3, CD20 or CD79a, CD99 and TdT. The MIB index was 50-80%. Epstein-Barr virus-encoded RNA (EBER) hybridizing signals were detected for most neoplastic cells. The T-cell receptor gamma gene rearrangement analysis showed germ-line configuration, except for one case. Effusion cytology may be appropriate for establishing the diagnosis of ENKTCL-N, particularly for patients in whom tissue biopsy is not possible.

Atraumatic Splenic Rupture: An Unusual Manifestation of Acute HIV Infection

A 27-year-old white male, who had sex with other men, presented to the emergency department with 3 days of left shoulder and abdominal pain. He reported no history of trauma to the abdomen. On abdominal imaging, he was found to have hemoperitoneum from a ruptured spleen; he underwent splenectomy. Causes of atraumatic splenic rupture can be divided into six main categories: infectious, neoplastic, inflammatory, congenital or structural, iatrogenic, and idiopathic. Work-up of the atraumatic splenic rupture revealed that his HIV antibody was newly positive. He had a documented negative HIV antibody 3 weeks prior to the current admission. CD4 cell count, obtained after splenectomy, was 904 cells per microliter and the HIV-1 plasma RNA level was 4657 copies per milliliter. Spleen pathology demonstrated an enlarged spleen with increase in the number of small to intermediate size lymphoid cells in the red pulp, and reactive follicular lymphoid hyperplasia, with numerous secondary lymphoid follicles and reactive germinal centers in the white pulp. T-cell receptor (TCR) gene rearrangement studies demonstrated a positive TCR beta gene rearrangement, without a TCR gamma gene rearrangement, consistent with a clonal CD8(+) T-cell population. The case gives rare insight into what happens in the spleen during acute HIV infection and encourages HIV testing in those presenting with atraumatic splenic rupture. Counseling patients with acute HIV to avoid potential trauma should also be considered.

Phenotype and TCR-gamma gene rearrangements in a Malaysian cohort of T-cell leukaemia/lymphoma cases

T cells undergo a series of complex phenotypic changes before achieving maturation. Discrete stages of T-cell differentiation are simplified to four stages (pro-, pre-, cortical and mature-T cell) and used in the classification of T-cell leukaemia. HLA-DR has been reported to be expressed in immature T-cell acute lymphoblastic leukemia (ALL) and also confer a poorer treatment outcome. Simultaneously, the genotype goes through distinct pattern changes due to rearrangement of T-cell receptor (TCR) genes. TCR gene rearrangement is important in the diagnosis of clonality and used as markers to detect minimal residual disease in lymphoproliferative disorders. We identified a subset within Pro-T and Pre-T cell cases distinguished by the expression of HLA-DR. These subgroups appeared to be more immature as rearrangement of the TCR-gamma gene was either at germline or involved only the first constant region (C1) unlike a more rearranged pattern in the HLA-DR-subgroups. We also observed a higher incidence of mediastinal mass (67%) in the HLA-DR-subgroup in the Pre-T stage. These characteristics may be useful as markers to further refine staging of T-cell ALL and determine prognosis.

T Cell Receptor Gamma and Delta Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in South India and Quantitation of Minimal Residual Disease

Abstract Background: T cell-Acute Lymphoblastic Leukemia (T-ALL) arises by clonal proliferation of lymphoid precursors arrested at particular stage of differentiation. The incidence of T-ALL in India is 37-43% of ALL. In this study, TCR gamma (TCRG) and TCR delta (TCRD) gene rearrangements were detected in diagnostic samples of ALL. Those clonal rearrangements detected at diagnosis are used as clonal markers for the quantitation of minimal residual disease (MRD) in follow up samples.Patients and Methods: BM/PB from 54 T-ALL patients (34 pediatrics and 20 adults) at diagnosis, treated by MCP 841 protocol were studied. Median age of the patients was 13. The frequency of clonal TCRG and TCRD gene rearrangements were studied by Polymerase Chain Reaction (PCR) coupled with Heteroduplex analysis (HD). Allele Specific Oligos (ASO) was designed by sequencing the junctional region sequence of clonal TCRG and TCRD gene rearrangements. MRD was studied in 4 patients with follow up samples. Diagnostic DNA with almost 100% tumor involvement as standard was serially diluted (50ng to 5ng) in 500ng control DNA to give a final concentration of one leukemic cell in 101 (1 in 101) cells to one leuekemic cell in 105 (1 in 105) cells. The serially diluted diagnostic standard in triplicates was amplified with TaqMan probe for respective TCRG/TCRD gene rearrangements in Real-time PCR along with 500ng of follow up DNA samples at different time points (Unknown) in triplicates. Analysis was done to calculate the amount of leukemic cells in follow up samples. Results: Using PCR-HD analysis, TCRG gene rearrangements were detected in 37 of 54 cases (68.5%) and TCRD gene rearrangements in 16 of 54 cases (29.6%). VgI-Jg1.3/2.3 was more commonly rearranged in 29 cases (53.7%) of T-ALL; VgII-Jg1.3/2.3 in 14 cases (26%). Both VgIII-Jg1.3/2.3 and VgIV-Jg1.3/2.3 rearrangements were detected in 4 cases respectively and VgI-Jg1.1/2.1 was detected in 3 cases. Vd1-Jd1 rearrangement was detected in 9 cases (16.6%); Vd2-Dd3 in 5 cases and Dd2-Dd3 in 4 cases of T-ALL. Both TCRG and TCRD gene rearrangements were detected in 8 cases (14.8%). The junctional region in TCRG rearrangements ranged from 1nucleotide to 11nucleotide (average 7.6 nt) and in TCRD rearrangements ranged from 14 nucleotide to 42 nucleotide (average 27 nt). In patient 1, the initial leukeimia load of 1 (before treatment) was reduced to 3 leukemic cell in 104 cells at the lost follow up. In patient 2, the initial leukemia load was reduced to 3.7 in 104 cells. In Patient 3, the initial leukemia load 1 was reduced to 6 in 102 cells at end of M2 and disease relapsed at M5. In patient 4, the initial leukemia load was reduced to 1.6 leukemic cell in 104 cells. Conclusion: Real-time PCR experiments reached a reproducible sensitivity of detecting one leukemic cell in 104 normal cells. Real-time PCR analysis showed that Patient 3 was not all responded to treatment and it was detectable by Real-time PCR at RI1 stage itself though the disease was clinically evident only at M5 stage of treatment. Thus, monitoring the MRD using Real-time PCR will help to quantitate the accurate amount of residual leukemic load, predate relapse and assess the response to treatment.

T Cell Receptor Gamma and Delta Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in South India and Quantitation of Minimal Residual Disease

T cell-Acute Lymphoblastic Leukemia (T-ALL) arises by clonal proliferation of lymphoid precursors arrested at particular stage of differentiation. The incidence of T-ALL in India is 37-43% of ALL. In this study, TCR gamma (TCRG) and TCR delta (TCRD) gene rearrangements were detected in diagnostic samples of ALL. Those clonal rearrangements detected at diagnosis are used as clonal markers for the quantitation of minimal residual disease (MRD) in follow up samples.Patients and Methods: BM/PB from 54 T-ALL patients (34 pediatrics and 20 adults) at diagnosis, treated by MCP 841 protocol were studied. Median age of the patients was 13. The frequency of clonal TCRG and TCRD gene rearrangements were studied by Polymerase Chain Reaction (PCR) coupled with Heteroduplex analysis (HD). Allele Specific Oligos (ASO) was designed by sequencing the junctional region sequence of clonal TCRG and TCRD gene rearrangements. MRD was studied in 4 patients with follow up samples. Diagnostic DNA with almost 100% tumor involvement as standard was serially diluted (50ng to 5ng) in 500ng control DNA to give a final concentration of one leukemic cell in 101 (1 in 101) cells to one leuekemic cell in 105 (1 in 105) cells. The serially diluted diagnostic standard in triplicates was amplified with TaqMan probe for respective TCRG/TCRD gene rearrangements in Real-time PCR along with 500ng of follow up DNA samples at different time points (Unknown) in triplicates. Analysis was done to calculate the amount of leukemic cells in follow up samples. Using PCR-HD analysis, TCRG gene rearrangements were detected in 37 of 54 cases (68.5%) and TCRD gene rearrangements in 16 of 54 cases (29.6%). VgI-Jg1.3/2.3 was more commonly rearranged in 29 cases (53.7%) of T-ALL; VgII-Jg1.3/2.3 in 14 cases (26%). Both VgIII-Jg1.3/2.3 and VgIV-Jg1.3/2.3 rearrangements were detected in 4 cases respectively and VgI-Jg1.1/2.1 was detected in 3 cases. Vd1-Jd1 rearrangement was detected in 9 cases (16.6%); Vd2-Dd3 in 5 cases and Dd2-Dd3 in 4 cases of T-ALL. Both TCRG and TCRD gene rearrangements were detected in 8 cases (14.8%). The junctional region in TCRG rearrangements ranged from 1nucleotide to 11nucleotide (average 7.6 nt) and in TCRD rearrangements ranged from 14 nucleotide to 42 nucleotide (average 27 nt). In patient 1, the initial leukeimia load of 1 (before treatment) was reduced to 3 leukemic cell in 104 cells at the lost follow up. In patient 2, the initial leukemia load was reduced to 3.7 in 104 cells. In Patient 3, the initial leukemia load 1 was reduced to 6 in 102 cells at end of M2 and disease relapsed at M5. In patient 4, the initial leukemia load was reduced to 1.6 leukemic cell in 104 cells. Real-time PCR experiments reached a reproducible sensitivity of detecting one leukemic cell in 104 normal cells. Real-time PCR analysis showed that Patient 3 was not all responded to treatment and it was detectable by Real-time PCR at RI1 stage itself though the disease was clinically evident only at M5 stage of treatment. Thus, monitoring the MRD using Real-time PCR will help to quantitate the accurate amount of residual leukemic load, predate relapse and assess the response to treatment.

Atypical fibroxanthoma with lymphomatoid reaction

Atypical fibroxanthoma (AFX) represents an uncommon skin tumor typically occurring on sun-damaged skin of the elderly. Histopathologic variants include spindled, clear cell, osteoid, osteoclastic, chondroid, pigmented, granular cell and myxoid lesions. To date, an atypical lymphoid infiltrate, including CD30-positive large cells mimicking lymphomatoid papulosis, has not been described in association with AFX. The clinical and histopathological characteristics of two AFX cases inciting an atypical lymphoid infiltrate, along with immunohistochemical profiles and T-cell receptor gamma (TCRγ) gene rearrangement results, were reviewed. Lesions in both cases occurred as solitary nodules in elderly patients. Microscopically, both lesions showed a cellular proliferation composed of pleomorphic spindle cells, associated with a prominent intralesional atypical lymphoid infiltrate. The spindle cells expressed CD10 but lacked the expression of S-100, cytokeratins and muscle markers, thereby confirming the diagnosis of AFX. CD30 highlighted a significant subset of large mononuclear cells in the lymphoid infiltrate of one case. TCRγ gene rearrangement analyses were negative for both cases. An atypical lymphoid infiltrate, including the one resembling lymphomatoid papulosis, associated with AFX has not been previously described. It is important to recognize the reactive nature of the infiltrate to avoid a misdiagnosis of lymphoma.

Secondary ALK Negative Anaplastic Large Cell Lymphoma in a Patient With Lymphomatoid Papulosis of 40 Years Duration

A 49 year-old man presented to our clinic. He had a history of lymphomatoid papulosis since childhood. At age 44, regional lymph node manifestation of anaplastic lymphoma kinase (ALK) anaplastic large cell lymphoma (ALCL) developed. Chemotherapy resulted in complete remission of the lymphadenopathy. Four years later, systemic relapse was detected which was refractory to therapy. Histology and immunohistochemistry showed congruent characteristics of multiple skin and lymph node biopsies: diffuse mixed infiltrate with large, anaplastic CD30 cells. Immunophenotype and microscopic morphology suggested a common origin of the different manifestations-however, this could not be proven due to lack of T-cell receptor (TCR) gamma gene rearrangement in most of the samples. The diagnosis of ALK-negative systemic ALCL with cutaneous symptoms was set up at the second flare up, however, the possibility of primary cutaneous ALCL was not excluded steadily. Lymphomatoid papulosis, primary cutaneous ALCL, and systemic ALK ALCL are 3 different entities but the separation of them cannot be solved without distinctive diagnostic tools.

Hemophagocytic lymphohistiocytosis associated with myelodysplastic syndromes

Hemophagocytic lymphohistiocytosis/hemophagocytic syndrome (HLH/HPS) is a reactive disorder of the mononuclear phagocytic system, characterized by generalized histiocytic proliferation with marked hemophagocytosis in the bone marrow [1]. It comprises two different categories: a primary (genetic) and a secondary (acquired) form. Malignant neoplasm-associated HPS (MAHS) is categorized as a secondary HLH. MAHS is mainly associated with lymphoma and rarely with other carcinomas [2]. To our knowledge, HLH associated with myelodysplastic syndromes (MDS) has not been described, although MDS with hemophagocytosis has been observed. Here, we describe a case of HLH associated with MDS, which presented with abundant CD8 T cells in the bone marrow and elevated plasma-soluble interleukin 2 receptor (sIL-2R). A 60-year-old Japanese man was admitted with pancytopenia, epitaxis, fever and general fatigue. A hematological examination showed a hemoglobin concentration of 6.5 g/dL, a platelet count of 14,000/lL and a leukocyte count of 3,200/lL with 42.8% atypical lymphocytes (Fig. 1a). Blood biochemistry showed increased levels of lactate dehydrogenase (LDH) (331 IU/L), ferritin (649 ng/mL) and sIL-2R (4,054 U/mL). NK cytolytic activity was measured by the standard 51-chromium (Cr) release assay. NK cytolytic activity was not reduced at both the E/T ratios of 10:1 (13.1%, normal range 8.9–29.5%) and 20:1 (22.5%, normal range 17.1–48.7%). Serological studies for hepatitis B, hepatitis C and HTLV-1 were negative. IgM antibodies to parvovirus were negative. Serological test for EBV revealed that anti-viral capsid antigen IgM was negative in the presence of anti-viral capsid antigen IgG (1609) and anti-EBV nuclear antigen antibody (409). The number of EBV DNA copies in serum was under 2.0 9 10 copies/mL (normal \200 copies/mL). Computed tomography (CT) revealed hepato-splenomegaly, ascites and bilateral pleural effusion. Bone marrow aspirate showed dyserythropoietic changes (abnormally lobulated nuclei) (Fig. 1c) and dysplastic megakaryocytes (micromegakaryocytes) (Fig. 1d). Erythrocytic precursors were 10.4% and bone marrow myeloblasts were 2.0% (Fig. 1e). Chromosome analysis showed severe complex karyotype: 54–57XY, ?1[2],?3[2],?4[4],?6[4],?8[4],add(9)(p22)[2],?11[3],add (15)(p11.2)[4],add(16)(q24)[4],add(19)(p13.1)[4],add(20) (p13)[4],?21[2],2–5mar[cp4]/46,XY[8]. A diagnosis of refractory anemia with excess blasts-1 (WHO classification) was made. In addition, bone marrow examination revealed increased numbers of atypical lymphocytes (Fig. 1b) and multiple sites of active hemophagocytosis (Fig. 2a–c). Atypical lymphocytes in bone marrow were positive for CD2, CD3 (surface, cytoplasmic), CD5, CD7, CD8, HLA-DR, TCR-ab, and negative for CD4. No monoclonal rearrangement of TCR beta, gamma and delta chain genes was detected by Southern blotting. These findings suggested that the atypical lymphocytes were reactive CD8 T cells. Combining fever, hemophagocytosis, splenomegaly, increased levels of ferritin and sIL-2R, a diagnosis of HLH was established. Although steroid therapy was administered for HLH, pancytopenia was not improved. Two months after the initial diagnosis, the levels of LDH and ferritin were further elevated (LDH 1,474 U/mL, ferritin 4,347 ng/mL). Bone marrow examination T. Tsuji (&) H. Yamasaki H. Tsuda Division of Hematology and Oncology, Kumamoto City Hospital, Kotoh 1-1-60, Kumamoto 862-8505, Japan e-mail: tsuji.takahiro@city.kumamoto.lg.jp

TCRγ-Chain Gene Rearrangement by GeneScan: Incidence and Significance of Clonal Heterogeneity in Sézary Syndrome

GeneScan (GS) analysis is a highly sensitive method for the early detection of cutaneous T-cell lymphoma (CTCL) and allows the identification of clonal heterogeneity, defined as the coexistence of two or more different T-cell clones in multiple samples from the same patient. We analyzed by GS the incidence and the significance of long-lived oligoclonal expansions in multiple skin and blood samples from 24 Sézary syndrome (SS) patients, and tried to correlate them with the clinical outcome. A skin clonal heterogeneity with additional reproducible TCRgamma-gene rearrangements (TCRgamma-GRs) was detected at diagnosis in 19/24 patients, 13 of whom had a constant prevalence of pathological TCRgamma-GRs in both skin and blood (dominant clonal pattern). During follow-up, an increase in oligoclones that were present at diagnosis or the appearance of new oligoclones was observed in 10 patients; all of them achieved a clinical response to treatment with extracorporeal photochemotherapy (ECP). The TCRgamma pattern (homogeneity or heterogeneity) in the skin at diagnosis showed a relevant prognostic value, and patients with an oligoclonal pattern had a significantly longer survival than those with a homogeneous pattern. In conclusion, multiple-sample approach GS analysis allows the identification of clonal heterogeneity and could also help in identifying SS patients with a potential higher response to therapy.

Primary cutaneous marginal zone B-cell lymphoma with amyloid deposition: report of two cases with review of literature

Amyloid deposition is rare. If there was a great amount of amyloid depositions in the skin tissue, it would be considered to be amyloid deposition disease at first, and then primary cutaneous marginal zone B-cell lymphoma (PCMZL). This study was to analyze the diagnosis and differential diagnosis of two cases of PCMZL with amyloid deposition. Clinicopathologic characteristics and follow-up of two cases of PCMZL were analyzed. Immunohistochemical staining was performed by EnVision method using antibodies LCA, CD19, CD20, CD79a, CD3, CD7, MUM1, kappa, lambda, Ki-67. IgH and TCRgamma gene rearrangement was detected by polymerase chain reactive (PCR). Case 1, a 71-year-old Chinese male, had a subcutaneous mass on the right elbow that was initially diagnosed with "amyloidosis" in 2004. Three years after the initial diagnosis, he developed recurrences on the right para-auxillary that was still diagnosed with "probably amyloidosis". Four years after the first diagnosis, the patient presented a lesion on the right para-auxillary with a diameter of 2 cm and a lesion on the temporal-parietal dural with a size of 6.0 cmx3.0 cmx3.0 cm. Case 2, a 68-year-old Chinese male, had a subcutaneous mass next to back of the left ear with a size of 9.0 cmx5.0 cm, and he underwent a operation one year previously because of subcutaneous mass in the same site. Microscopically, the tumors of both cases were located in dermis and subcutaneous, tumor cells were medium size with a nodular or diffuse distribution, and some of tumor cells were plasmacytoid/plasma cells. Morphologically, the temporal-parietal dural lesion was similar to subcutaneous lesion and infiltrated into cranial (case 1). Juxtaposed the tumor cells of two cases, there were the large amyloid deposits of amorphous hyaline material and concentrically laminated hyaline spherules in case 1, while cord-like amyloid deposits in case 2. Reactive lymphoid follicles with germinal centers and foreign body giant cells in the stroma were found surrounding the amyloid deposits. Congo red staining showed positive of amyloid deposition in tumor tissues of both cases. Immunohistochemical staining revealed that LCA, CD19, CD20, CD79a and MUM1 expressions were positive in tumor cells, and Ki-67 expression was about 8%-10%. IgL restricted expression as kappa positive while lambda negative was found in both cases. PCR results showed monoclone gene rearrangement of IgH gene in both cases. Our findings suggest that amyloid deposition rarely present in both primary and metastatic tumors in PCMZL, and its diagnosis should be considered to avoid misdiagnosis. The patients with PCMZL should undergo regular examinations and chemotherapy as well as a long-term follow-up since it is apt to recur or relapse.

Re: A Lifestyle Intervention Study in Patients with Diabetes or Impaired Glucose Tolerance: Translation of a Research Intervention into Practice

AIMS: Differentiation between actinic reticuloid and cutaneous T cell lymphoma can be extremely difficult. Demonstration of clonal T cell receptor (TCR) gene rearrangements has been suggested as a potential diagnostic criterion, but the results obtained thus far have been conflicting. This study investigated whether TCR gamma gene rearrangement analysis, using polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) and immunohistochemistry, can serve as a diagnostic criterion. METHODS: PCR/DGGE was performed on skin, peripheral blood mononuclear cells, and/or lymph nodes of seven patients with actinic reticuloid, 11 patients with Sézary syndrome, and 15 patients with a benign form of erythroderma. The results of PCR/DGGE and Southern blot analysis of TCR beta gene rearrangements were compared. In addition, CD4:CD8 ratios in skin and peripheral blood samples were investigated. RESULTS: Clonal T cell populations were detected in 19 of 21 samples obtained from patients with Sézary syndrome but were not detected in any of the 12 samples from patients with actinic reticuloid. Clonal T cells were detected in the peripheral blood of only one of 15 patients with a benign form of erythroderma. PCR/DGGE and Southern blot analysis gave concordant results in 28 of 29 samples. Immunophenotypic analysis demonstrated increased proportions of CD8+ T cells in skin (seven of seven cases) and peripheral blood (four of seven cases) of patients with actinic reticuloid. CONCLUSION: The results of this study demonstrate that gene rearrangement analysis, in combination with immunohistochemistry, may be an important adjunct in differentiating between actinic reticuloid and cutaneous T cell lymphoma. In patients suspected of having actinic reticuloid, application of both techniques is recommended.

CD4/CD8 double negative pagetoid reticulosis: a case report and literature review

Pagetoid reticulosis is an indolent primary cutaneous T-cell lymphoma. It typically presents as a solitary and slowly growing patch or plaque on the extremity, histologically characterized by an acanthotic epidermis infiltrated with atypical lymphocytes. Here, we present histological, immunophenotypical and molecular findings of a 29-year-old Jamaican man with bilateral wrist plaques. Histology showed marked acanthosis, hyperkeratosis and an intraepidermal infiltration consisting of large atypical lymphocytes. Immunohistochemical stains showed CD3 and CD5 positive T cells with significant loss of CD7, double negative CD4 and CD8 and strong positive CD30. Molecular analysis showed a monoclonal T-cell receptor (TCR) gamma gene rearrangement. Review of the literature confirms that the immunophenotype of pagetoid reticulosis is variable with decreasing frequency of CD8+ cytotoxic/suppressor T cell, CD4+ helper T cell and least commonly CD4/CD8 double negative phenotypes. Although CD4/CD8 double negative phenotype appears to be associated with higher proliferation index, it does not appear to confer prognostic significance.

Primary T-cell Lymphoma of the Thyroid Associated with Hashimoto's Thyroiditis, Histologically Mimicking MALT-Lymphoma

Most of thyroid lymphomas are B-lineage, and T-cell lymphomas are rare. Here, we report a case of primary thyroid T-cell lymphoma associated with Hashimoto's thyroiditis. A 48-yr-old woman presented with incidentally found neck mass. Histologically, the resected right lobe of the thyroid was replaced by monomorphic small atypical lymphoid cells with lymphoepithelial lesion-like change, most of which were immunoreactive for CD3, CD8, βF-1, and TIA-1. Peripheral T-cell lymphoma, unspecified, was finally diagnosed after molecular study for TCR-γ gene rearrangement. This is the second case of cytotoxic T-cell lymphoma reported in the thyroid gland so far. Unique association between thyroid follicles and neoplastic lymphocytes may be characteristic feature of this type of T-cell lymphoma.

Clinicopathologic Characteristics and Outcome of CD4+/CD56+ Blastic Plasmacytoid Dendritic Cell Neoplasm: A Single Institutional Experience.

Abstract 1625Poster Board I-651 BackgroundCD4+/CD56+ Blastic Plasmacytoid Dendritic Cell Neoplasm (BPCDC, also called hematodermic or blastic NK neoplasm) is a rare aggressive hematolymphoid malignancy with initial skin presentation and concurrent/subsequent marrow and systemic dissemination. BPCDC could pose a diagnostic challenge since it is a rare entity with varied clinical presentation, sharing overlap clinicopathologic features with other hematopoietic neoplasms such as acute leukemia of myelomonocytic differentiation given the high similarity of their ontogeny. Prognosis of this entity is poor and in contrast to AML there is no standard therapy established. MethodsEleven cases with a diagnosis of CD4+/CD56+ BPCD (WHO 2008 criteria) were retrieved from Moffitt Cancer Center's electronic clinical and pathology databases. Clinical notes and pathology material including skin biopsies, bone marrow aspirates and biopsies were reviewed. Immunophenotypic analyses were performed on fresh samples using flow cytometry and on paraffin-embedded samples using immunohistochemistry (IHC). Molecular analyses of TCR-alpha and TCR-gamma gene rearrangement by PCR and cytogenetic analysis were performed on available fresh skin punch biopsy and bone marrow aspirate samples to exclude T/NK cell lymphoma. In situ hybridization for detection of EBV-encoded RNA (EBER) was performed. Clinical presentation, treatment regimens, response to therapy and clinical course were analyzed. ResultsAll eleven patients were males, with age ranging from 17 to 85 years (median 68). Ten patients initially presented with cutaneous nodules (10/11) and one patient with a tonsillar mass (1/11), with or without marrow/lymph node involvement. Histopathologically, all skin biopsies demonstrated dense dermal infiltrates of primitive blasts without epidermotropism. Blasts expressed CD4 and CD56 (11/11), and HLA-DR (6/6), lacking expression of CD3, CD5, CD8. Blasts typically lacked CD11c, CD13, CD33, and CD57 (9/11). Molecular analysis showed germline configuration of TCRb and TCRg. In situ hybridization for EBV (EBER) was negative (3/3). One case showed complex cytogenetic karyotype (1/3). Two patients were diagnosed concurrently with transformed MDS/RAEB-1 and AMML/MPD followed by a CD4+/CD56+ BPCD. The majority (7/11) of patients were initially treated with antracycline-based induction chemotherapy regimen (CHOP n=4, CNOP n=1, hyperCVAD n=2). Results of induction therapy (Chemotherapy n=6, Radiation therapy n=1, Surgical excision n=1) were available for 8/11 patients. Overall response rate (ORR) was 73% (8/11). Equal number of patients (4/8) achieved the first complete remission (CR1) and partial remission (PR1) Median duration of CR1 was 5 (range 5-10) months. The second line therapy (Chemotherapy n=6, steroids n=1) was administered in 7/11 patients. Only 1 patient with limited disease to skin achieved CR2. Two younger patients (17 and 36yrs) underwent consolidation with autologous stem cell transplantation in CR1 and PR1, respectively. A patient transplanted in CR1 was alive with no evidence of disease at 11 months of follow-up. Overall median survival of entire cohort of patients was 8 (range 1-35) months. ConclusionsCD4+/CD56+ BPCD exhibits overlapping clinicopathologic features with peripheral T cell lymphoma, NK cell lymphoma, and acute myelomonocytic/monocytic leukemia. Diagnosis requires accurate histopathology, immunophenotyping by flow cytometry, tissue immunohistochemistry, as well as molecular studies. Clinically, this disease has an aggressive course in a majority of patients with a relatively short survival. Initial responses to induction antracycline-based chemotherapy regimens are of a very short duration. Patients with disease limited to skin and younger patients who can undergo consolidation with hematopoietic stem cell transplantation might have more favorable outcome. DisclosuresNo relevant conflicts of interest to declare.

Detection of Clonal T Cells in the Circulation of Patients With Nephrogenic Systemic Fibrosis

Nephrogenic systemic fibrosis is a sclerodermalike disease in patients with acute or chronic renal insuffiency related to administration of gadolinium-containing contrast agents. Previous studies have demonstrated clonal T-cell populations in the blood of patients with systemic sclerosis, suggesting that these cells may be involved in the pathogenesis of the disease. Facing the clinical similarities of both diseases, we hypothesized that clonal expansion of T cells could be present in nephrogenic systemic fibrosis as well. Findings from polymerase chain reaction and high-resolution capillary electrophoresis for T-cell receptor gamma gene rearrangement analysis showed that all 6 prospectively evaluated patients (100%) with nephrogenic systemic fibrosis had detectable clonal T cells in their peripheral blood. In contrast, only 4 of the 15 control patients (27%) with chronic renal failure and none of the 12 healthy individuals analyzed in this study had evidence for T-cell clonality using the same type of examination. Clonal T-cell-positive patients with systemic sclerosis have previously been reported to better respond to extracorporeal photopheresis. However, this was not the case in 2 of our patients with nephrogenic systemic fibrosis. Conclusion As in systemic sclerosis, clonally expanded T-cell populations could play a critical role in the pathogenesis of nephrogenic systemic fibrosis, probably as an in vivo-activated inflammatory response to gadolinium exposure.

Intraepithelial CD8-positive T lymphocytes predict survival for patients with serous stage III ovarian carcinomas: relevance of clonal selection of T lymphocytes

Background:The aim of this study was to investigate the prognostic effect of tumour-infiltrating lymphocytes (TILs) in serous stage III ovarian carcinoma to determine TIL clonality and to correlate this to Her2/neu expression.Methods:Formalin-fixed and paraffin-embedded ovarian carcinomas were examined for CD20-, CD3-, CD4- and CD8-positive lymphocytes (n=100), and for Her2/neu-positive tumour cells (n=55/100) by immunohistochemistry. Clonality analysis was carried out by T-cell receptor γ (TCRγ) gene rearrangements (n=93/100). Statistical analyses included experimental and clinico-pathological variables, as well as disease-free (DFS) and overall (OS) survival.Results:CD20-positive B lymphocytes were present in 57.7% (stromal)/33.0% (intraepithelial) and CD3-positive T lymphocytes in 99.0% (stromal)/90.2% (intraepithelial) of ovarian carcinomas. Intraepithelial CD3-positive T lymphocytes were correlated with improved DFS in optimally debulked patients (P=0.0402). Intraepithelial CD8-positive T lymphocytes were correlated with improved OS in all optimally debulked patients (P=0.0201) and in those undergoing paclitaxel/carboplatin therapy (P=0.0092). Finally, rarified and clonal TCRγ gene rearrangements were detected in 37 out of 93 (39.8%) and 15 out of 93 (16.1%) cases, respectively. This was marginally associated with improved DFS (P=0.0873). Despite a significant correlation of HER2/neu status and intraepithelial CD8-positive lymphocytes (P=0.0264), this was non-directional (R=−0.257; P=0.0626).Conclusion:Improved survival of ovarian cancer patients is related to the infiltration, clonal selection and intraepithelial persistence of T lymphocytes.

Diversity of T-cell receptor gene rearrangements in South Indian patients with common acute lymphoblastic leukemia.

Precursor B-Acute Lymphoblastic Leukemia (precursor B-ALL) occurs due to the uncontrolled proliferation of B-lymphoid precursors arrested at a particular stage of B-cell development. Precursor-B-ALL is classified mainly into pro-B-ALL, common-ALL and pre-B-ALL. The Common Acute Lymphoblastic Antigen CD10 is the marker for common-ALL. This study was aimed to examine the diversity of T-cell receptor Gamma (TCRG) and T-cell receptor Delta (TCRD) gene rearrangements in South Indian Common-ALL patients. Clonality of TCRG and TCRD was studied in 52 cases (pediatric=41 and adolescents and young adults=11) of common-ALL. TCRG and TCRD gene rearrangements were amplified by PCR and the clonality was assessed by Heteroduplex analysis of amplified products. In pediatric common-ALL, clonal TCRG and TCRD gene rearrangements were detected in 19 (46.3%) and 18 (43.9%) cases respectively. In adolescents and young adults (AYA), TCRG was rearranged in 8 (72.7%) cases and TCRD was rearranged in 4 (36.3%) cases. In the present study of common-ALL, the frequency of a TCRG rearrangement VII-J1.3/2.3 was significantly high in AYA compared to pediatric (36.3% vs 4.8%; p<0.025). Thus, VII-J1.3/2.3 was highly diverse in AYA compared to pediatric. That shows the difference in biology of the disease between pediatric and AYA in South Indian population. The reason for the high frequency of VII-J1.3/2.3 in AYA of common-ALL in South Indian population in connection with unknown infectious agents or environmental carcinogens needs to be evaluated further.

Subcutaneous Panniculitis-Like T-Cell Lymphoma With Overlapping Clinicopathologic Features of Lupus Erythematosus: Coexistence of 2 Entities?

We observed 5 patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL) who were unusual, in that they also exhibited features of lupus erythematosus (LE). This observation is in keeping with a recent study that reported an increased rate of autoimmune disease, including systemic lupus erythematosus (SLE), among patients with SPTCL. In all cases, attributes indicating SPTCL included an infiltrate of lymphocytes with pleomorphic nuclei involving subcutaneous lobules exhibiting a cytotoxic T-cell (CD3/CD8/betaF1) immunophenotype. Additionally, a high proliferation rate and a monoclonal T-cell receptor-gamma gene rearrangement were observed in most cases. The manifestations of LE in these patients included a spectrum of clinical and histopathological abnormalities. Clinical manifestations of LE included, in some patients, morphologic evidence of lupus erythematosus panniculitis (LEP) with subcutaneous nodules that healed with lipoatrophy on the face. In addition, all the patients exhibited serologic and/or extracutaneous end-organ abnormalities seen in patients with SLE, with 2 patients having sufficient findings to meet American College of Rheumatology criteria for SLE. Histopathological evidence of LE included vacuolar change at the dermal-epidermal interface in 3 patients, 2 of whom also showed interstitial deposition of mucin in the reticular dermis. One of these patients also had findings of LEP in the subcutaneous lobules with clusters of CD20 B cells partially arranged within germinal centers. In 2 patients in which neither the epidermis nor dermis was available for review, histopathological features of LE included, in one patient, a few small clusters of CD123 plasmacytoid dendritic cells within the adipose tissue and, in the other patient, a positive direct immunofluorescence test (lupus band) on clinically uninvolved and lesional skin. Our study shows that some patients show overlap between SPTCL and LE. We suspect that these patients may suffer from both diseases concomitantly. Furthermore, patients with LE, particularly LEP, should be monitored for evolution into SPTCL with biopsy of any subcutaneous lesion that is not typical of LEP. Additionally, screening for cutaneous LE and SLE could be considered in patients with SPTCL.