Immunoglobulin and T cell receptor gene rearrangements in human lymphoma and leukemia

DNA samples from blood leukocytes or tumor biopsies of 45 patients with phenotypic B or T cell neoplasms were analyzed for rearrangements of the immunoglobulin (Ig) or T cell receptor (TCR) genes by Southern blot hybridization analysis. Rearrangements of the Ig heavy chain joining region genes (JH) were present in DNA from each of 28 B cell lymphomas and leukemias; 14 of 21 of these tumors also had rearrangements of the Ig kappa light chain joining (JK) or deleting element (KDel) genes. Conversely, 16 of 17 T cell lymphomas and leukemias had rearranged TCR beta chain genes. One B cell and one T cell tumor had rearrangements of both Ig and TCR genes. There was a strong correlation between the rearrangements of specific genes and the immunophenotype of the tumor: JH rearrangement without TCR beta chain rearrangement occurred only in B cell tumors; TCR beta chain rearrangement with or without JH rearrangement occurred only in T cell tumors, with one exception; and JK and KDel rearrangements were found only in B cell tumors. Thus, rearrangements of the Ig heavy and light chain genes and the TCR beta chain genes were found to be highly sensitive markers of monoclonal human lymphomas and lymphoid leukemias, with the type of gene rearrangements well correlated with the cell lineage of these neoplasms.     

Radiogenic leukemia revisited

Radiation-induced leukemia is considered to be similar to the de novo disease. However, following an analysis of clinical and hematological findings in leukemia occurring in irradiated cervical cancer patients, adult Japanese atomic-bomb survivors, and spondylitics treated with x- ray, striking differences were noted. Acute leukemias in cervical cancer patients and Japanese survivors were similar in type to acute de novo leukemias in adults. Cell types among spondylitics were very dissimilar; rare forms, eg, acute erythromyelocytic leukemia (AEL) and acute megakaryocytic leukemia, were increased. Pancytopenia occurred in 25 of 35 cases and erythromyelodysplastic disorders were noted in seven of 35 acute cases. The leukemias and myelodysplastic disorders closely resembled those occurring in patients treated with alkylating agents. This similarity suggests a common pathogenesis involving marrow stem cell injury and extra-medullary mediators of hematopoiesis. Investigation of early acute leukemias and myelodysplastic disorders with newer techniques may provide valuable insights into the pathogenesis of leukemia in humans.     

Precision of genotyping of Epstein-Barr virus by polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C, and EBER): predominance of type A virus associated with Hodgkin's disease [published erratum appears in Blood 1993 Oct 1;82(7):2268]

To precisely determine the genotype of Epstein-Barr virus (EBV) in Hodgkin's disease (HD), we simultaneously analyzed three divergent gene loci (EBNA-2, EBNA-3C, and EBER) that distinguish type A and B viruses. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of the polymerase chain reaction (PCR)-amplified products or the mobility shifts in single-strand conformation polymorphism analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. We analyzed 15 EBV-infected cell lines and found a good correlation between EBNA-2 and EBNA-3C typing results. In contrast, approximately 33% of the cell lines analyzed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B, and 31% with dual viral sequences. Thus, type A virus is predominant in HD. Based on the histology, the frequencies of EBV positivity were 83%, 71%, and 33% for mixed cellularity, nodular sclerosis, and lymphocyte predominance, respectively. The detection of high frequency of both type A and B sequences in HD may provide a lead in investigating the role of dual viral infection in EBV pathogenesis.     

Influence of resection margin on survival in hepatic resections for colorectal liver metastases

Background:

Traditionally a 1-cm margin has been accepted as the gold standard for resection of colorectal liver metastases. Evidence is emerging that a lesser margin may provide equally acceptable outcomes, but a critical margin, below which recurrence is higher and survival poorer, has not been universally agreed. In a recent publication, we reported peri-operative morbidity and clear margin as the two independent prognostic factors. The aim of the current study was to further analyse the effect of the width of the surgical margin on patient survival to determine whether a margin of 1 mm is adequate.

Methods:

Two hundred and sixty-one consecutive primary liver resections for colorectal metastases were analysed from 1992 to 2007. The resection margins were assessed by microscopic examination of paraffin sections. The initial analysis was performed on five groups according to the resection margins: involved margin, 0–1 mm, >1–<4 mm, 4–<10 mm and ≥ 10 mm. Subsequent analysis was based on two groups: margin <1 mm and >1 mm.

Results:

With a median follow-up of 4.7 years, the overall 5-year patient and disease-free survival were 38% and 22%, respectively. There was no significant difference in patient- or disease-free survival between the three groups with resection margins >1 mm. When a comparison was made between patients with resection margins ≤1 mm and patients with resection margins >1 mm, there was a significant 5-year patient survival difference of 25% versus 43% (P < 0.04). However, the disease-free survival difference did not reach statistical significance (P= 0.14).

Conclusions:

In this cohort of patients, we have demonstrated that a resection margin of greater than 1 mm is associated with significantly improved 5-year overall survival, compared with involved margins or margins less than or equal to 1 mm. The possible beneficial effect of greater margins beyond 1 mm could not be demonstrated.     

Factors in the liquid portion of stored blood inhibit the proliferative response in mixed lymphocyte cultures

The transfusion of blood may suppress the immune responses of patients with renal transplants and with malignant disorders. To study the in vitro suppressive effects of banked blood, 4 units of blood were stored in CPDA-1 and ADSOL at 4 degrees C for 14 days. Lymphocytes and plasma or ADSOL supernatants were harvested on Days 0, 4, 7, 10, and 14. Subpopulations of lymphocytes were enumerated by flow cytometry. Recalcified and heat-treated plasma and supernatants from the units of blood were added to mixed lymphocyte cultures (MLC) composed of cells from normal individuals. No significant changes were noted in the proportions of T or B cells from blood stored under these conditions. A 60 +/− 3 percent inhibition in the proliferative response was observed when plasma from CPDA-1 units was added to MLCs (p less than 0.02). Supernatants from ADSOL units demonstrated a 29 +/− 4 percent inhibition (p less than 0.10) of the proliferative response, and this inhibition of response was observed on all 14 days of the study. When appropriate concentrations of dextrose or adenine were added to other MLCs, adenine (at the concentration found in ADSOL) caused a significant inhibition of the proliferative response. This inhibition was not, however, as marked as that observed with recalcified, heat- treated plasma from CPDA-1 units. We conclude that adenine plus some additional factor(s) found in the liquid portion of stored blood inhibits the proliferative response of normal lymphocytes. It is possible that these factors contribute to the immune suppression observed in vivo in some patients who receive blood transfusions.     

Lymphocyte predominance Hodgkin's disease: lineage and clonality determination using a single-cell assay

Lymphocyte predominance Hodgkin's disease (LPHD) is a clinically indolent condition. Although there is evidence that the putative neoplastic cell in this disease, the "L&H" cell, is of B-cell lineage, there is conflicting data concerning the clonality of these cells. Our study was aimed at clarifying the issue of lineage and clonality of the L&H cells of LPHD using a single-cell assay. Four cases of LPHD were studied. To circumvent the difficulties of obtaining fresh tissue and to be able to study representative cases, a new method was developed to obtain single-cell suspensions of L&H cells from archival formalin- fixed paraffin-embedded tissue. Single L&H cells were identified by morphology and immunostaining for epithelial membrane antigen, isolated using a micropipette, and subjected to polymerase chain reaction (PCR) amplification of the complematarity determining region 3 (CDR3) of the Ig heavy chain (IgH) gene, which is B-cell clone-specific. The PCR products were size-fractionated by polyacrylamide gel electrophoresis and representative products were directly sequenced. Single T cells and small B cells were also isolated from the tissues and used as negative and positive controls, respectively. In all four cases of LPHD, the IgH CDR3 of single L&H cells could be amplified. Within each case, the IgH CDR3 of single L&H cells was found to be of different length or of different sequence. Therefore, our results provide strong evidence for the B-cell origin of the L&H cells and the polyclonal nature of LPHD.     

von Willebrand disease in the RIIIS/J mouse is caused by a defect outside of the von Willebrand factor gene [published erratum appears in Blood 1995 Sep 15;86(6):2461]

An animal model for human type I von Willebrand disease (vWD) has been previously described in the inbred mouse strain RIIIS/J. Murine vWD is characterized by a prolonged bleeding time, normal von Willebrand factor (vWF) multimer distribution, autosomal dominant inheritance, and proportionately decreased plasma vWF antigen, ristocetin cofactor, and factor VIII (FVIII) activities. To study the molecular genetics of murine vWD, a portion of the vWF gene surrounding exon 28 was cloned, sequenced, and used to develop two informative DNA sequence polymorphisms for rapid genotyping by DNA polymerase chain reaction. RIIIS/J mice were crossed with PWK/Ph mice, an inbred line of Mus musculus musculus, and the F1 progeny backcrossed to the parental PWK/Ph strain. vWF antigen levels in F1 mice were not significantly different from the parental RIIIS/J strain but were markedly decreased compared with the parental PWK/Ph mice. Genetic linkage analysis of 104 backcross progeny showed no correlation between vWF antigen level and vWF genotype. These data indicate that murine vWD is caused by a defect at a novel genetic locus, distinct from the murine vWF gene. The distribution of vWF antigen levels among backcross progeny suggests the presence of one major dominant vWD gene in the RIIIS/J mouse with possible modifying contributions from one or more additional minor loci. These observations may provide new insights into the molecular basis and variable expressivity of human vWD.     

Deoxycoformycin-induced immunosuppression in patients with hairy cell leukemia

Immune function in patients with hairy cell leukemia (HCL) was examined serially during treatment with alternating monthly cycles of recombinant interferon alpha-2a and 2'-deoxycoformycin (dCF). At presentation, most patients had normal numbers of T lymphocytes and their cells had normal proliferative responses to mitogens [phytohemagglutinin (PHA) and concanavalin A (Con A)] and alloantigens. Patients had severe monocytopenia, decreased delayed-type hypersensitivity (DTH) reactions, and decreased peripheral blood natural killer (NK) activity. Treatment caused a profound decrease in all lymphocyte subpopulations. T cells were more affected than B cells or NK cells. Numbers of CD4+ and CD8+ lymphocytes decreased to levels less than 200 cells/microliters in all patients during treatment. This decrease in T cell number was associated with a marked decrease in proliferative responsiveness to PHA, Con A, and alloantigens. These abnormalities persisted throughout the 14 months of treatment and have continued for up to 6 months beyond discontinuation of treatment. NK cell activity increased during treatment, but cycled depending on the phase of treatment; highest activities were observed after interferon (IFN)-alpha and lower levels of activity were observed after dCF. DTH responses generally did not improve during therapy. Levels of IgM, IgG, IgA, and IgD did not change during treatment, but IgE levels rose in most patients. All immunosuppressive effects were attributable to dCF since patients receiving IFN-alpha 2a alone did not exhibit these same immunosuppressive effects, and patients receiving dCF alone after IFN failure exhibited similar abnormalities. Despite this severe immunosuppression from dCF, life-threatening opportunistic infections have not been observed in our patient population. Six patients developed localized Herpes zoster infection among 21 patients who had received dCF. Pending the results of long-term follow-up, we recommend that dCF be reserved for patients who have failed splenectomy and IFN therapy.     

Spontaneous regression of a monoclonal proliferation of large granular lymphocytes associated with reversal of anemia and neutropenia

A 43-year-old male with a phenotypically homogeneous, expanded subset of T cells presented in 1981 with anemia and neutropenia. The surface antigen phenotype of 99% of the peripheral blood lymphocytes was T3+, T8+, T4-, and they were morphologically large granular lymphocytes (LGL). The same cells comprised 37% of the marrow nucleated cells. Eight months after he presented, the peripheral blood T8+, LGL diminished spontaneously, and the anemia and neutropenia completely resolved. The patient remains hematologically normal as of October 1984. To determine if the T8+, LGL represented a clonal expansion, DNA from peripheral blood lymphocytes collected and cryopreserved when the patient was neutropenic and anemic, and when he was hematologically normal, was analyzed for clonal T-cell antigen receptor gene rearrangements. Using Southern blot analysis, a clonal DNA rearrangement was demonstrated, and this clone diminished but was still demonstrable in peripheral blood lymphocytes collected in 1984. The above observations implicate the expanded T8+, LGL in the pathogenesis of the neutropenia and anemia, yet the exact mechanism remains to be elucidated.     

Ablating hedgehog signaling in tenocytes during development impairs biomechanics and matrix organization of the adult murine patellar tendon enthesis

Restoring the native structure of the tendon enthesis, where collagen fibers of the midsubstance are integrated within a fibrocartilaginous structure, is problematic following injury. As current surgical methods fail to restore this region adequately, engineers, biologists, and clinicians are working to understand how this structure forms as a prerequisite to improving repair outcomes. We recently reported on the role of Indian hedgehog (Ihh), a novel enthesis marker, in regulating early postnatal enthesis formation. Here, we investigate how inactivating the Hh pathway in tendon cells affects adult (12‐week) murine patellar tendon (PT) enthesis mechanics, fibrocartilage morphology, and collagen fiber organization. We show that ablating Hh signaling resulted in greater than 100% increased failure insertion strain (0.10 v. 0.05 mm/mm, p<0.01) as well as sub‐failure biomechanical deficiencies. Although collagen fiber orientation appears overtly normal in the midsubstance, ablating Hh signaling reduces mineralized fibrocartilage by 32%, leading to less collagen embedded within mineralized tissue. Ablating Hh signaling also caused collagen fibers to coalesce at the insertion, which may explain in part the increased strains. These results indicate that Ihh signaling plays a critical role in the mineralization process of fibrocartilaginous entheses and may be a novel therapeutic to promote tendon‐to‐bone healing. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:1142–1151, 2015.