Multi-center analysis of the effect of T-cell acute lymphoblastic leukemia subtype and minimal residual disease on allogeneic stem cell transplantation outcomes.

This study aims to provide a detailed analysis of allogeneic stem cell transplantation (allo-SCT) outcomes in a large T-cell acute lymphoblastic leukemia (T-ALL) cohort with a specific emphasis on the effects of pre-transplant minimal residual disease (MRD) and disease subtype, including the aggressive early-thymic precursor (ETP) subtype. Data from 102 allo-SCT patients with a diagnosis of T-ALL from three centers were retrospectively analyzed. Patients were grouped into four T-ALL subtypes: ETP, early, cortical and mature. At 3 years, overall survival (OS), PFS, non-relapse mortality and cumulative incidence (CI) progression were 35, 33, 11 and 55%, respectively. Patients transplanted in first complete remission (CR1) had a 3-year OS of 62% versus those transplanted in CR2 or greater (24%) (hazards ratio 1.6, P=0.2). Patients with MRD positivity at the time of transplant had significantly higher rates of progression compared with those with MRD negativity (76 vs 34%, hazards ratio 2.8, P=0.006). There was no difference in OS, PFS or cumulative incidence (CI) progression between disease subtypes, including ETP (n=16). ETP patients transplanted in CR1 (n=10) had OS of 47%, comparable to other disease subtypes, suggesting that allo-SCT can overcome the poor prognosis associated with ETP. MRD status at transplant was highly predictive of disease relapse, suggesting novel therapies are necessary to improve transplant outcomes.

Bendamustine added to allogeneic conditioning improves long-term outcomes in patients with CLL.

Bendamustine has shown a favorable safety profile when included in chemotherapy regimens for several types of lymphoma, including CLL. This study investigated the long-term effect of adding bendamustine to a conditioning regimen on survival, rate of engraftment, immune recovery and GvHD after allogeneic stem cell transplantation (alloSCT) in CLL patients. These outcomes were compared with the fludarabine, cyclophosphamide and rituximab (FCR) conditioning regimen. We reviewed the data for 89 CLL patients treated on three trials at our institution. Twenty-six (29%) patients received bendamustine, fludarabine and rituximab (BFR) and 63 (71%) received FCR. Patient characteristics were similar in both groups. Ten (38%) BFR-treated patients vs only two (3%) FCR-treated patients did not experience severe neutropenia (P=<0.001). The 3-year overall survival estimates for the BFR and FCR groups were 82 and 51% (P=0.03), and the 3-year PFS estimates were 63% and 27% (P=0.001), respectively. The 2-year treatment-related mortality was 8 and 23% and the incidence of grade 3 or 4 GvHD was 4% and 10%, respectively. This study is the first to report that addition of bendamustine to alloSCT conditioning for CLL patients is associated with improved survival and lower mortality, myelosuppression, and GvHD.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Multi-color flow cytometric immunophenotyping for detection of minimal residual disease in AML: past, present and future.

Current chemotherapeutic regimens achieve CR in a large percentage of patients with AML. However, relapse after CR remains a significant problem. The presence of leukemic cells at levels too low to be detected by conventional microscopy, termed minimal residual disease (MRD), has been associated with an increased risk of relapse and shortened survival. Detection of MRD requires the use of highly sensitive ancillary techniques. Multi-color flow cytometric immunophenotyping is a sensitive method for quick and accurate detection of MRD. Use of this method in patient management may result in lower rates of relapse and improved survival, and is an effective means of assessing novel therapeutic agents. This method can be used in the vast majority of patients with AML, regardless of the immunophenotypic, cytogenetic and molecular genetic abnormalities present. Unfortunately, conflicting data regarding optimum methods of measurement and reporting, as well as the expertize required to interpret results have limited broad application of this technique. We provide a broad overview of this technique, including its advantages and limitations, and discuss the methods employed at our institution. We also review several possible areas of future investigation.

TCR-Vbeta flow cytometric analysis of peripheral blood for assessing clonality and disease burden in patients with T cell large granular lymphocyte leukaemia.

AIMS: T cell large granular lymphocytes (T-LGLs) are commonly increased in reactive conditions as well as T-LGL leukaemia. This differential diagnosis often requires a combined assessment of clonality and tumour burden. In this study we assessed the utility of flow cytometric (FC) analysis of T cell receptor beta chain variable region (TCR-Vbeta) expression by using 24 antibodies reactive to 70% of the TCR-Vbeta repertoire. METHODS: Analyses were performed on peripheral blood samples obtained from 20 patients with a confirmed diagnosis of T-LGL leukaemia and 18 patients without known T cell lymphoproliferative diseases. RESULTS: The results were compared with TCR gene rearrangement status assessed by PCR. By FC analysis, 19/20 T-LGL leukaemia cases were CD3+CD8+ and one case was CD3+CD4+. All the cases demonstrated at least one immunophenotypic aberration, with altered CD5 expression being most frequent. Abnormal Vbeta expression was detected by FC in 19 of 20 (95%) T-LGL leukaemia cases, but in none of the controls; this showed 100% concordance with TCR gene rearrangement studies. In addition to establishing clonality, FC Vbeta analysis enables calculation of absolute numbers of clonal T cells; this is important in monitoring tumour burden after treatment. CONCLUSIONS: It is concluded that FC Vbeta analysis is a fast, reliable and quantitative method that can simultaneously assess T-LGL leukaemia clonality and tumour burden.

Receptor expression in orbital inflammatory syndromes and implications for targeted therapy.

To investigate the expression of multiple therapeutic targets in tissue specimens from patients with orbital inflammatory syndromes, the clinical records of 16 patients treated for orbital inflammation between January 2003 and November 2005 for whom tissue blocks were available were reviewed retrospectively. Immunohistochemical staining was performed on archived specimens using commercially available monoclonal antibodies against CD3, CD20, CD22, CD23, CD25, and CD52 antigens. The histologic diagnoses were confirmed, and the immunohistochemical staining patterns were agreed upon by both collaborating pathologists (JLJ and PC-B). The study included 13 women and 3 men who ranged in age from 4 to 79 years (mean, 46 years). The histologic diagnoses were as follows: orbital pseudotumor in six patients; sarcoidosis, three; eosinophilic granuloma, one; necrobiotic xanthogranuloma, one; nonspecified granulomatous inflammation, one; Graves' ophthalmopathy, one; Wegener's granulomatosis, one; and reactive lymphoid hyperplasia, two. One orbital lymphoma specimen and one foreign body reaction specimen were used as controls. CD20 was strongly expressed in all specimens except three (Wegener's granulomatosis, eosinophilic granuloma, and nonspecified granulomatous inflammation specimens), and CD25 was strongly expressed in all specimens except the Wegener's granulomatosis specimen, in which this antigen was only moderately expressed. CD20 and CD25 were strongly or moderately expressed in most of the tested specimens of orbital inflammation. If our findings are confirmed in a larger study, rituximab, which targets CD20, and denileukin diftitox (ONTAK), which targets CD25, should be considered for future clinical trials for orbital inflammatory syndromes.

Unilocular retroperitoneal cyst of mesothelial origin presenting as a renal mass.

We report the first 2 cases, to our knowledge, of retroperitoneal cysts with features of mesothelial differentiation that clinically mimic renal masses. The first lesion occurred in a 71-year-old man who presented with flank pain. Ultrasound and magnetic resonance imaging studies showed a unilocular cystic structure arising from the upper pole of the left kidney. The second lesion was in a 44-year-old woman who presented with left flank pain. Imaging studies revealed an 8-cm hemorrhagic cyst at the lower pole of the left kidney. Histologic examination of the nephrectomy specimens in each case revealed a unilocular cyst with intracystic and pericystic hemorrhage. In each case, the cyst was lined by a single layer of cells with ample eosinophilic cytoplasm and benign nuclear features without mucinous or müllerian differentiation. Histochemical staining showed Alcian blue positivity on the cell surface, which was sensitive to hyaluronidase digestion. Intracytoplasmic mucin, however, was not detected. Immunostaining showed that the cyst lining cells were positive for keratin, vimentin, HBME-1, WT1, and thrombomodulin but negative for carcinoembryonic antigen, B72.3, Leu-M1, and BerEP4. The first case was positive for calretinin, whereas the second was negative. These findings support the mesothelial nature of the cysts.

Leiomyosarcomas of the oral cavity: an unusual topographic subset easily mistaken for nonmesenchymal tumours.

AIMS: Oral leiomyosarcoma is rare and poorly documented. We aimed to characterize these lesions clinicopathologically in order to facilitate their distinction from other spindle cell neoplasms in the oral cavity. METHODS AND RESULTS: Ten cases of oral leiomyosarcoma were retrieved and studied histologically and immunohistochemically. Clinical data were obtained from referring pathologists and prior literature concerning 46 comparable cases was reviewed. Nine out of 10 cases occurred in adults; 50% arose in the jaws and four showed bone involvement. Histological appearances were similar to leiomyosarcomas elsewhere. In addition to myogenic markers, two cases were also keratin-positive. Four patients developed local recurrence or metastatic disease and three died of tumour (median follow-up 37 months). CONCLUSIONS: Leiomyosarcoma is under-recognized in the mouth, often being mistaken for a spindle-celled epithelial neoplasm. Aside from an unusual but infrequent tendency to spread to lymph nodes and a location-specific differential diagnosis, its clinicopathological features are comparable to leiomyosarcomas at other locations.

Characteristic proliferations of reticular and dendritic cells in angioimmunoblastic lymphoma.

Angioimmunoblastic lymphoma (AIL) is a T-cell proliferation with a distinct clinical presentation that often poses a difficult diagnostic challenge. Angioimmunoblastic lymphoma is characterized by prominent vascular and stromal proliferations. Using a panel of antibodies, we investigated the nature of the stromal component in 15 cases of AIL as compared with 40 cases of nodal-based peripheral T-cell lymphoma (PTCL) of other types. As has been previously noted, extrafollicular proliferations of follicular dendritic cells (detected by CD21 and low molecular-weight nerve growth factor receptor staining) were highly associated with AIL and were only rarely seen in other lesions. Unexpectedly, large networks of desmin-positive reticulum cells also were noted in all cases of AIL evaluated. These cells with characteristic long cytoplasmic processes were present in much smaller numbers or only rarely in other types of peripheral T-cell lymphoma. This population of nodal stromal cells, a subset of the fibroblastic reticulum cells detected by vimentin immunostaining, may be responsible for the prominent reticulum deposition seen in AIL. No association of AIL with proliferations of other types of reticulum cells (e.g., interdigitating dendritic cells or histiocytes) was noted. These findings suggest that networks of follicular dendritic and desmin-positive reticulum cells are useful diagnostic features in angioimmunoblastic lymphoma and probably are related to the pathogenesis of this entity.

T cell receptor interaction with peptide/major histocompatibility complex (MHC) and superantigen/MHC ligands is dominated by antigen.

While recent evidence strongly suggests that the third complementarity determining regions (CDR3s) of T cell receptors (TCRs) directly contact antigenic peptides bound to major histocompatibility complex (MHC) molecules, the nature of other TCR contact(s) is less clear. Here we probe the extent to which different antigens can affect this interaction by comparing the responses of T cells bearing structurally related TCRs to cytochrome c peptides and staphylococcal enterotoxin A (SEA) presented by 13 mutant antigen-presenting cell (APC) lines. Each APC expresses a class II MHC molecule (I-Ek) with a single substitution of an amino acid residue predicted to be located on the MHC alpha helices and to point "up" towards the TCR. We find that very limited changes (even a single amino acid) in either a CDR3 loop of the TCR or in a contact residue of the antigenic peptide can have a profound effect on relatively distant TCR/MHC interactions. The extent of these effects can be as great as that observed between T cells bearing entirely different TCRs and recognizing different peptides. We also find that superantigen presentation entails a distinct mode of TCR/MHC interaction compared with peptide presentation. These data suggest that TCR/MHC contacts can be made in a variety of ways between the same TCR and MHC, with the final configuration apparently dominated by the antigen. These observations suggest a molecular basis for recent reports in which either peptide analogues or superantigens trigger distinct pathways of T cell activation.

Transfer of putative complementarity-determining region loops of T cell receptor V domains confers toxin reactivity but not peptide/MHC specificity.

We have used multiple-amino acid replacement mutagenesis to examine the roles of the TCR homologues of Ig complementarity-determining regions (CDR) and framework sequences in Ag-MHC and Staphylococcus aureus enterotoxin reactivity. In the three cases examined, transplantation of Ig CDR3 homologues between I-Ek-restricted TCR that recognize distinct peptides did not result in transfer of peptide reactivity. Thus the structural context of the CDR3 loops, e.g., both neighboring CDR and the V beta structure, must play a crucial, albeit supporting, role in ligand recognition. The extreme lability of this context was also shown by the fact that transplantation of the CDR1, -2, and -3 loops from the beta chain of 5C.C7 onto a V beta 1 framework failed to transfer MHC-peptide specificity even when the TCR-alpha chains were identical. In contrast, superantigen reactivity was readily transferred in several cases, with CDR2 transplants conferring strong staphylococcal enterotoxin B and A reactivity and CDR1 transplants yielding weak reactivities. This suggests that bacterial (and perhaps other) superantigens bind to many of the same regions of the TCR V beta that are believed to interact with MHC molecules. These regions of V beta may be ideal targets for superantigen binding precisely because they interact with MHC molecules and thus may be relatively conserved.

Molecular components of T-cell recognition.

We review recent data that increase our understanding of the ternary complex of the T cell receptor (TCR), antigenic peptides, and molecules of the major histocompatibility complex (MHC). Studies using synthetic peptide analogs for T-cell antigens have identified peptide residues that appear to interact with the MHC molecule and/or the TCR. The logical extension of these studies, using a complete replacement set of peptide analogues for a model peptide antigen, has more precisely defined the biochemical character of putative MHC and TCR contact residues, and indicated that the TCR is highly sensitive to subtle changes in peptide conformation. Insight into the binding site for peptide on the TCR has recently come from variant peptide immunization of TCR single-chain transgenic mice. These experiments indicate that residues encoded by the V(D)J junctions of both TCR chains contact peptide directly. TCR-MHC contacts have also been studied, using in vitro-mutagenized MHC molecules, particularly those altered at residues predicted to point "up," toward the TCR. These studies reveal that TCR-MHC contacts appear to be quite flexible, and vary between even closely related TCRs. A measure of the affinity of TCR for peptide/MHC complexes has come from competition experiments using soluble MHC complexed with specific peptides. This affinity, with a KD of 5 x 10(-5) M, is several orders of magnitude lower than that of most antibodies for their protein antigens and suggests that the sequence of events leading to T-cell activation begins with antigen-independent adhesion.