Microsatellite instability is associated with the histological features of the tumor in nonfamilial colorectal cancer.

Replication errors (RERs) at microsatellite loci were examined in 46 specimens of nonfamilial colorectal cancer. Somatic microsatellite alterations in at least two genetic loci, D11S904, D13S175, D2S123, and D10S197, consistent with a RER(+) phenotype were found in four cases (8.7%). Six additional cases (13%) showed alterations at a single locus. Mucinous differentiation was observed in 3 of 4 (75%) adenocarcinomas with a RER(+) phenotype and only in 19% (8 of 42) of RER(-) adenocarcinomas (P < 0.05). A distinct cap of inflammatory cells at the advancing edge of the tumor and Crohn's-like reaction in peritumoral stroma were histologically identified in 50 and 25% of RER(+) and in 5 and 0% of RER(-) tumors, respectively (P < 0.05). Also, the plexiform pattern of growth of carcinoma turned out to be significantly associated with the RER(+) phenotype (P < 0.05). Mucinous differentiation and stromal inflammatory reactions are frequent features of hereditary nonpolyposis colorectal cancer in which germ-line mutations of mismatch repair genes cause genetic instability. Our results indicate that a link exists between such histological features and somatic genetic instability consistent with a RER(+) phenotype also in nonfamilial colorectal cancer.

Characterization of EBV-positive lymphoblastoid cell lines obtained from HIV seropositive patients with or without lymphomas.

Epstein-Barr-virus- (EBV-) positive lymphoblastoid cell lines (LCLs) spontaneously arising in vitro were obtained from the peripheral blood of six HIV-seropositive patients and from the peripheral blood and the bone marrow of one patient (LAM) with AIDS and lymphoma. The LCLs from HIV-seropositive patients had phenotypic, cytogenetic, and biological characteristics indistinguishable from those of normal LCLs obtained by infecting B cells with EBV in vitro. The LCLs from LAM patient comprised composite cell populations. Cloning analysis and cell fractionation procedures showed that, beside normal EBV-infected cells, these lines contained a malignant subset population characterized by c-myc rearrangement, abnormal karyotype, and a surface phenotype similar to that of Burkitt's lymphoma cells. Analyses of Ig heavy chain and c-myc oncogene loci showed that these malignant cells were the progeny of a single precursor. Nevertheless, these cells had heterogeneous EBV-fused termini, a finding which indicates that EBV infection followed c-myc rearrangement.

Identical utilization of T-cell receptor gene regions in B-lymphoid blast crisis of chronic myeloid leukemia and B-precursor acute lymphoblastic leukemia.

The configuration of the T-cell receptor (TCR) beta, gamma and delta chain genes was analyzed in 16 cases of B-lymphoid blastic crisis of chronic myeloid leukemia (BC-CML) for a better definition of the biological aspects of this cellular population, in comparison with the molecular features of B-precursor acute lymphoblastic leukemia (ALL). All cases displayed B-phenotypic features, were Ph'-positive and had a rearranged configuration of the breakpoint cluster region (bcr) and of the immunoglobulin heavy chain gene region (JH). The TCR beta chain gene was rearranged in four cases (25%), all of which displayed a monoallelic rearrangement involving the J beta 2 region. The TCR gamma chain gene was rearranged in 13 cases (81%); 13 rearranged alleles utilized the J1/2 regions, while the remaining five utilized JP1. The V regions of the group I were mostly involved. The TCR delta chain gene was rearranged or deleted in 15 cases (94%); the 10 rearranged chromosomes displayed exclusively two patterns referable to partial recombinations, a V2-(D)-D3 and a (D)-D3 type. These two configurations are predominant in B-precursor ALL (75% of rearranged chromosomes) and almost absent in T-ALL. Taken together, these results document the close similarities between the genotypic features of B-lymphoid BC-CML and B-precursor ALL, not only in terms of the incidence of rearrangement but more relevantly with regard to the choice of regions involved in the recombinations. This aspect is particularly evident at the TCR delta locus level.

Molecular characterization of Ph' + hybrid acute leukemia.

The configuration of the immunoglobulin heavy chain (IgH), T-cell receptor (TcR) beta and gamma chain regions, and the major breakpoint cluster region (M-bcr) genes were analysed in four cases of Ph' + acute leukemia (AL). Monoclonal rearrangements of the IgH region were detected in three cases exhibiting two phenotypically distinct cell populations (i.e. one lymphoid and one myeloid. In one of these cases, identical genetic events were observed by molecular analysis of FACS separated blasts. Multi-lineage rearrangements involving also the TcR gamma gene were observed in a biphenotypic AL showing co-expression of markers. The lack of rearrangements within the M-bcr gene, together with demonstration in one case of the Ph' + AL specific p190 protein product, pointed against the occurrence of chronic myeloid leukemias presenting in blastic transformation. Our results imply that such cases are to be considered as true AL and should therefore be included in the definition of hybrid AL.

Molecular evaluation of clonal remission in hairy cell leukemia patients treated with 2-chlorodeoxyadenosine.

Several reports have documented complete and long-lasting remissions in hairy cell leukemia (HCL) patients after a single one-week course of 0.1 mg/kg/d of 2-Chlorodeoxyadenosine (2-CdA) administered by intravenous infusion. In these studies the evaluation of the clinical response was based on physical examination, blood cell counts, as well as cytochemical and immunological detection of residual leukemic cells. Since HCL is a chronic lymphoproliferative disorder characterized by the expansion of monoclonal B cells, analysis of the configuration of the immunoglobulin gene regions represents a valuable tool towards a more accurate monitoring of treatment response. We hereby describe the molecular assessment of the neoplastic clone in six HCL patients treated with 2-CdA; in five of them a disappearance of the rearranged bands could be documented in bone marrow cells aspirated between 2 and 21 months after a single course of continuous infusion 2-CdA. The last HCL case analyzed, revealed molecular persistence of the neoplastic clone. In all cases but one, the molecular evaluation was in agreement with the definition of clinical response based on conventional analysis. This study demonstrates that HCL patients treated with 2-CdA show a high incidence of molecularly-defined complete remissions, the likelihood of which is much greater than for patients treated with Interferon alpha.

Immunoglobulin light chain restriction and clonal rearrangement in nodular paragranuloma.

B-cell clonality was demonstrated in a typical nodular paragranuloma case (NP) by both immunoglobulin (Ig) surface analysis and Ig genes rearrangement studies. On frozen sections, immunostaining for Ig light chain expression revealed a clear-cut predominance of Ig lambda-expressing cells, recognizable as both small lymphocytes and lympho-histiocytic (L&H) cells. Accordingly, molecular analysis of the Ig genes showed a monoclonal rearrangement of the lambda chain gene, although no specific pattern of heavy chain gene rearrangement could be detected by JH analysis. The C lambda rearranged band was identified with two different restriction enzymes, excluding the hypothesis of a genomic polymorphism. Furthermore, the C kappa gene was almost completely deleted, indicating that the developmental hierarchy of Ig genes rearrangement has been respected. The molecular pattern of the C lambda hybridizing band was consistent with monoallelic rearrangement of almost the entire DNA sample, indicating that clonal proliferation was not limited to L&H cells, but also involved surrounding lymphocytes. This finding is in keeping with the immunohistochemical evidence of a lambda light chain restriction on both L&H cells and small lymphocytes, pointing to a close relationship between these two cell types. Our results as a whole suggest that L&H cells and B lymphocytes share a common origin and may both be involved in clonal proliferation in NP.

Intestinal T-cell lymphoma with massive tissue and blood eosinophilia mediated by IL-5.

A case of enteropathy associated T-cell lymphoma (EATCL) in a 62-year-old female with a previous history of coeliac disease, complicated during the clinical course by massive blood and tissue eosinophilia is described. The patient's serum contained a factor capable of stimulating the in vitro growth of eosinophilic colonies (CFU-Eo), that was absent in the serum of normal donors. We suggest that such factor was Interleukin-5 (IL-5), as indicated by the presence in the monoclonal tumor T cells of IL-5 encoding mRNA, usually absent in the normal enterocytes of the jejunum.

Cellular and Molecular Characterization of Two Cases of Castleman's Disease, Plasma Cell Variant.

We report the cellular and molecular characterization of two cases of Castleman's disease, plasma cell variant, that differed in their clinical presentation and course. Patient 1 had Castleman's disease in association with Kaposis's sarcoma unrelated to human immunodeficiency virus (HIV) infection and died while he was receiving an aggressive chemotherapeutic regimen for Kaposi's sarcoma. Patient 2 had an isolated retroperitoneal lymphoid mass with an adjacent enlarged limph nodes and his symptoms disappeared completely following the surgical removal of both. Pathologic and immunohistochemical analyses in both cases, revealed that there was a massive infiltration of polyclonal plasma cells in the interfollicular areas of the lymph nodes. Immunoglobulin gene rearrangement studies confirmed the polyclonal nature of B-lineage cells in the involved lymph nodes. Southern blot experiments failed to demonstrate the presence of EBV genome copies in the same lymph nodes. These paradigmatic cases lend further support to the notion that Castleman's disease is an extremely heterogeneous entity.

Cytogenetic rearrangement of C-MYC oncogene occurs prior to infection with Epstein-Barr virus in the monoclonal malignant B cells from an AIDS patient.

Two cell lines were originated from the peripheral blood (PB-LAM) and bone-marrow (BM-LAM) of a patient with Burkitt-type acute lymphoblastic leukemia and AIDS. 26 and 7 clones were isolated from PB-LAM and BM-LAM respectively by limiting dilution. All of these had surface IgM lambda and the CD10 marker with low to absent CD23, CD30, CD39 and surface adhesion molecules. Furthermore, they shared the same chromosomal abnormalities (trisomy 7 and t(8;14) translocation) and the same rearrangements of immunoglobulin L and H chain and of c-myc gene loci. These features are those most frequently found in Burkitt's lymphoma (BL) cells and were different from those of the parental cell lines, which, besides cells identical to those of the malignant clones, also contained normal lymphoblastoid cells. Therefore, the cloning procedure used selected for the growth of cells with malignant features. EBV latent antigens were detected in all clones by Western blotting and their pattern of expression resembled that usually observed in BL cells. All the clones were positive for the EBV genome by Southern blotting and had monomorphic EBV-fused termini as determined by using cDNA probes specific for sequences at either end of the viral genome. However, the clones derived from PB-LAM had EBV fused termini of a different size from that of the clones derived from BM-LAM. The presence of different EBV-fused termini in otherwise monoclonal malignant cells indicate that EBV infection was possibly a late event in lymphomagenesis following rearrangement of the c-myc and the Ig gene loci.

Immunoglobulin DNA Analysis as a Marker of Clonality in the Follow-up of Patients with Hairy Cell Leukemia Treated with Alpha-Interferon.

In fourteen patients with hairy cell leukemia (HCL) the configuration of the immunoglobulin (Ig) heavy chain genes was used as a marker of clonality, to monitor the response of the neoplastic population to treatment with alpha-interferon (a-IFN). In agreement with the morphological, hematological and immunological data, twelve of them showed, after a variable length of therapy, a complete disappearance of rearranged bands in peripheral blood cells. In one patient, who was treated less intensively, the molecularly-defined neoplastic population was still present on two consecutive determinations, whilst in the last patient persistence of disease was repeatedly documented despite prolonged A-IFN treatment. Three further cases were analyzed sequentially: in two, no rearranged bands could be found at repeated determinations; the third, who was in complete remission whilst on 3 × 10(6) U of α-IFN every other day, showed recurrence of disease nine months later when on a maintenance protocol with 3 × 10(6) U/weekly. Nine bone marrow specimens were also analyzed following treatment with α-IFN. In four a monoclonally rearranged band could still be detected, while in another four, reversal of fibrosis and hemopoietic recovery wits coupled with the absence of a molecularly recognizable neoplastic clone. In the last (case, persistence of disease paralleled the findings in the peripheral blood cells. These data indicate that α-IFPJ is capable of producing a specific cytolytic effect on the leukemic population in HCL, which in some cases may lead to complete clonal remissions. Analysis at the DNA level may represent a valuable tool towards monitoring the clinical course of HCL patients and for optimal individual therapeutic scheduling.

[Pleural effusion in a case of non-Hodgkin's lymphoma: diagnostic use of molecular analysis]. / Versamento pleurico in un linfoma non-Hodgkin: utilizzazione diagnostica delle analisi molecolari.

We describe a case of advanced non-Hodgkin lymphoma which lacked palpable superficial lymph nodes and in which conventional method did not allow a conclusive diagnosis. Recognition of lymphoproliferative disease was made by analysis at the DNA level of the configuration of the immunoglobulin and T-cell receptor gene regions.

Distinct patterns of novel gene mutations in poor-prognostic stereotyped subsets of chronic lymphocytic leukemia: the case of SF3B1 and subset #2.

Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subset #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%, lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically oriented therapy.

Hybrid lymphoid blast crisis of chronic myeloid leukemia with both immunoglobulin and T-cell receptor gene rearrangements.

A case of Ph1+ chronic myeloid leukemia in blast crisis (CML-BC) is reported, in which the periodic acid Schiff and myeloperoxidase negative blasts displayed high terminal deoxynucleotidyl activity and coexpressed both B- (CD19, CD10, and CD24) and T- (CD7) lymphoid markers. In line with the immunophenotype, DNA analysis revealed a rearranged configuration of both the immunoglobulin and T-cell receptor (beta, gamma, and delta) genes. In spite of this dual B/T phenotype and genotype, the negativity of CyCD3 favors the suggestion that the target of the neoplastic event is an early B cell, with a cross lineage involvement of the putative common recombinase. However, taking into account that a normal counterpart of a biphenotypic B/T ALL has been recognized, it could be hypothesized that the leukemic transformation may have involved an oligopotent B/T lymphoid precursor. This case confirms the lineage heterogeneity of CML-BC and suggests that DNA analyses coupled to extensive immunophenotyping may allow further insight for a more precise recognition of both normal and leukemic ontogenesis.

Nonrandom TRG gamma variable gene rearrangement in normal human T cells and T cell leukemias.

To estimate the extent of the TRG gamma variable (V) gene repertoire used in human T cell ontogeny, we have analyzed the variety of V gamma gene rearrangements in a large series of T and non-T acute and chronic leukemias. A limited heterogeneity of rearranged fragments was observed: only 13 types of differently rearranged fragments, four of which occurred only once, were found among 80 rearranged chromosomes. Furthermore, in the leukemic population as a whole, the frequency distribution of the most common types of rearranged V gamma gene-containing fragments appeared to be nonrandom (p less than 0.01). Of interest is the clear preference for functional vs. nonfunctional V gamma genes (nonfunctional genes being those which carry frameshifts or nonsense mutations but which presumably can still rearrange due to their conserved signal sequences). We discuss the possibilities that this preference may result either from selection of the TRG gamma product at some stage during T cell development or, alternatively, from an intrinsic, antigen-independent polarity in V gamma gene activation.

Cyclic neutropenia and severe hypogammaglobulinemia in a patient with excess of CD8-positive T lymphocytes: response to G-CSF therapy.

Cyclic neutropenia is a rare hematological disorder characterized by periodical severe granulocytopenia. A stem cell defect and/or immunological abnormalities are considered to play a role in this disease. Here we describe the case of an adult woman who was diagnosed as having both cyclic neutropenia and severe hypogammaglobulinemia. Her clinical history revealed that one or both abnormalities had been present since childhood. Normal in vitro growth of the patient's bone marrow CFU-GM was observed, while immunological analysis revealed the presence of a persistent excess of activated (HLA-DR+) CD8+ T lymphocytes in both bone marrow and peripheral blood. These T lymphocytes have been shown to be polyclonal by DNA analysis, and their role in determining the clinical picture of our patient remain uncertain since they could not be shown to produce inhibitors of in vitro CFU-GM growth. Intermittent low doses of human recombinant G-CSF were able to improve neutropenia and completely prevent infectious symptoms, thus confirming the efficacy of this cytokine in cyclic neutropenia patients.

Immunoglobulin and Myc gene structure in lymphnode biopsies from HIV-positive patients with LAS and lymphoma.

The pattern of immunoglobulin (Ig) gene rearrangement and the genomic structure of the Myc locus were investigated in the DNA of 20 lymphnode biopsies of patients suffering from lymphoadenopathy-associated syndrome (LAS) and from 3 patients with Burkitt's lymphoma. Although polyclonality was the prevalent pattern of Ig gene rearrangement observed in LAS, in 30% of the cases discrete bands of Ig heavy chain gene rearrangement were identifiable due to the presence of monoclonal or oligoclonal cell populations. However, structural alterations of the Myc gene were not detected in any cases. As expected, in all three Burkitt's lymphomas studied, the lymphnode DNA displayed a clonal pattern of Ig heavy chain gene rearrangement. The Myc was altered in two cases, which presented a truncation of the gene beginning within a very short region of the first intron. By contrast, the breakpoint positions on chromosome 14 mapped in different regions of the Ig loci, which in both cases involved the switch (SH) area. Data confirm the relatively common occurrence of oligoclonal expansions within B cells in LAS and the frequent involvement of the Myc oncogene in the process of lymphomagenesis in individuals positive for human immunodeficiency virus (HIV).

Analysis of the turin umbilical cord blood bank registry.

BACKGROUND: The polymorphic nature of the HLA system reduces a patient's probability of finding an HLA-compatible unrelated bone marrow (BM) donor, even though more than 6 million individuals are enrolled in international registries. Recently, umbilical cord blood (UCB) has been successfully employed as a source of HPCs. The use of such cells reduces the risk of GVHD and allows transplants with one or two HLA mismatches. UCB represents an expensive resource: therefore, it is necessary to carefully manage the UCB unit inventory. STUDY DESIGN AND METHODS: The current study analyzed the genetic heterogeneity of HLA-A, -B, and -DR gene frequencies between pools of UCB and unrelated-donor BM in the Piedmont (an administrative region of Italy). An Italian hematology patient's probability of finding complete or partial matches as a function of donor pool size was determined by considering subsamples randomly selected from the local unrelated BM donors. RESULTS: The HLA gene frequencies in UCB and unrelated-donor BM pools were not significantly different. The search simulation, based on actual HLA phenotypes, showed that the percentage of Italian patients matched with an HPC unit increases remarkably if 1 or 2 mismatches are accepted, reaching a proportion of 90 percent with an inventory of only about 500 units, while the increment is not so remarkable if the number of UCB units is greater. CONCLUSION: To optimize economic resources and to be internationally competitive, UCB banks should aim to increase the genetic heterogeneity of their units rather than increasing the UCB inventory, acquire efficient quality control systems, and acquire and preserve UCB units with a greater number of nucleated cells.

Long-term results of alpha interferon as initial therapy and splenectomy as consolidation therapy in patients with hairy cell leukemia. Final report from the Italian Cooperative Group for HCL.

PURPOSE: In 1987 the Italian Cooperative Group for the Study of hairy cell leukemia (HCL) started a prospective trial with the following three major aims: 1) to confirm the effectiveness of alpha-IFN as first-line treatment; 2) to assess the usefulness of splenectomy as consolidation treatment in patients achieving a satisfactory partial remission (PR) with alpha-IFN, and 3) to explore whether splenectomy in patients achieving complete remission (CR) with alpha-IFN can reduce the risk of relapse after discontinuation of the drug. PATIENTS AND METHODS: One-hundred seventy-seven patients with histologically-confirmed HCL were registered in the HCL88-A trial between December 1987 and January 1992. Inclusion criteria included no previous treatment and age less than 66 years. All patients received total doses of 3 MU of alpha-IFN daily for 12 months except for those who achieved early CR and would stop treatment after 6 or 9 months. Patients could be treated with different alpha-IFNs. At the time of the present analysis, 166 patients (93.8%) were fully evaluable. RESULTS: Treatment of HCL patients with alpha-IFN at the onset of the disease resulted in 28 CR (16.9%), 103 PR (62.0%), and 27 Minor Remissions (MR) (16.3%). Patients treated with different alpha-IFNs achieved similar results: the overall response rate (CR + PR + MR) was 92.7%, 97.2%, and 95.3% for patients treated with r-alpha-2a, r-alpha 2b, and alpha-N1, respectively. The presence of a leukemic phase and a poor performance status were associated with a statistically significant lower response rate. Patients who were randomly assigned and underwent splenectomy after achieving a PR had a better but not significant 4-year progression-free survival than cases randomized for observation (53% vs. 22%, p = 0.116). Overall, 5 patients died after study entry, with an actuarial 5-year survival rate of 96% for the entire group of 166 patients. After a mean follow-up time of 38 months, only one second malignancy has been recorded. CONCLUSIONS: Initial therapy with alpha-IFN, regardless of the type of alpha-IFN used, induces satisfactory responses in the majority of patients with HCL, but in most instances discontinuation of treatment results in recurrence of disease. In most cases alpha-IFN improves the performance status of patients and favors a satisfactory bone marrow recovery and thus could still play a role in the initial management of the disease. Although splenectomy following alpha-IFN could prolong the progression free survival, its use should be restricted to selected cases.