Paulo Vidal Campregher

Comprehensive assessment of T-cell receptor β-chain diversity in αβ T cells

The adaptive immune system uses several strategies to generate a repertoire of T- and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. In alphabeta T cells, which primarily recognize peptide antigens presented by major histocompatibility complex molecules, most of this receptor diversity is contained within the third complementarity-determining region (CDR3) of the T-cell receptor (TCR) alpha and beta chains. Although it has been estimated that the adaptive immune system can generate up to 10(16) distinct alphabeta pairs, direct assessment of TCR CDR3 diversity has not proved amenable to standard capillary electrophoresis-based DNA sequencing. We developed a novel experimental and computational approach to measure TCR CDR3 diversity based on single-molecule DNA sequencing, and used this approach to determine the CDR3 sequence in millions of rearranged TCRbeta genes from T cells of 2 adults. We find that total TCRbeta receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO(+) antigen-experienced alphabeta T cells is at least 10-fold higher than previous estimates. These methods should prove valuable for assessment of alphabeta T-cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging.

Overlap and Effective Size of the Human CD8+ T Cell Receptor Repertoire

Deep sequencing of the T cell receptor repertoires of seven healthy adults reveals that the adaptive immune system is far less diverse than expected and the person-to-person overlap is thousands of times larger. Not So Diverse After All You never know what nasty microbe lurks around the corner. To guard against these potential foes, our immune cells produce a vast variety of antibody and T cell receptor (TCR) shapes ready to recognize these pathogens. This diversity is manufactured by gene rearrangement, with fragments from the so-called V, D, and J gene groups joined together to form an array of sequences, much as colored beads can be arranged on a string to make many combinations. With several choices for each of the V, D, and J fragments, supplemented with a few random nucleotides inserted at the junctions, an astronomical number of different arrangements can theoretically be created. Originally, this was thought to be a random process, but Robins et al., by high-throughput genome sequencing of seven people, show that the generation of immune diversity is actually selective, creating an unexpectedly small assortment of TCRs that is similar in different people. By analyzing blood samples containing millions of T cells from healthy donors and sequencing the TCRs from each donor’s set of naïve and memory T cells, the authors revealed that the sequences of the TCRβ subunits in each subject are not randomly distributed. Instead, certain D and J segments preferentially associated with each other. The number of nucleotides inserted at junction sites between the segments was also smaller than expected. As a result, the actual repertoire of each person’s T cells is a fraction of that predicted by a computer model assuming random rearrangement, and the overlap between donors’ T cells is several orders of magnitude greater. If the rearrangements were truly random, two unrelated adults would only be expected to share on average five TCRβ sequences, of a total 3 million, but the overlap turned out to be more than 10,000 sequences. The authors also overturn another assumption in the field. It had been thought that a random set of V-D-J combinations was generated and then, by deletion and selection in the thymus of cells carrying less useful combinations, only a subset matured, forming the final immune repertoire. Instead, the authors show that the T cell repertoire is limited and biased from the time of the original V-D-J rearrangement during cell development. Now that deep sequencing of these key immune regions in individual genomes is achievable, we will be able to compare TCR sequences and antibodies in healthy individuals with those of patients with autoimmune disorders, or of transplant recipients suffering from graft-versus-host disease. The results may help to understand these illnesses and to look for ways to modify patients’ T cell repertoires for treatment or prevention of autoimmunity or to enhance compatibility between transplant donors and recipients. Diversity in T lymphocyte antigen receptors is generated by somatic rearrangement of T cell receptor (TCR) genes and is concentrated within the third complementarity-determining region 3 (CDR3) of each chain of the TCR heterodimer. We sequenced the CDR3 regions from millions of rearranged TCR β chain genes in naïve and memory CD8+ T cells of seven adults. The CDR3 sequence repertoire realized in each individual is strongly biased toward specific Vβ-Jβ pair utilization, dominated by sequences containing few inserted nucleotides, and drawn from a defined subset comprising less than 0.1% of the estimated 5 × 1011 possible sequences. Surprisingly, the overlap in the naïve CD8+ CDR3 sequence repertoires of any two of the individuals is ~7000-fold larger than predicted and appears to be independent of the degree of human leukocyte antigen matching.

Evaluation of NIH consensus criteria for classification of late acute and chronic GVHD

Historically, graft-versus-host disease (GVHD) beyond 100 days after hematopoietic cell transplantation (HCT) was called chronic GVHD, even if the clinical manifestations were indistinguishable from acute GVHD. In 2005, the National Institutes of Health (NIH) sponsored a consensus conference that proposed new criteria for diagnosis and classification of chronic GVHD for clinical trials. According to the consensus criteria, clinical manifestations rather than time after transplantation should be used in clinical trials to distinguish chronic GVHD from late acute GVHD, which includes persistent, recurrent, or late-onset acute GVHD. We evaluated major outcomes according to the presence or absence of NIH criteria for chronic GVHD in a retrospective study of 740 patients diagnosed with historically defined chronic GVHD after allogeneic HCT between 1994 and 2000. The presence or absence of NIH criteria for chronic GVHD showed no statistically significant association with survival, risks of nonrelapse mortality or recurrent malignancy, or duration of systemic treatment. Antecedent late acute GVHD was associated with an increased risk of nonrelapse mortality and prolonged treatment among patients with NIH chronic GVHD. Our results support the consensus recommendation that, with appropriate stratification, clinical trials can include patients with late acute GVHD as well as those with NIH chronic GVHD.

Lung Function and Long-Term Complications after Allogeneic Hematopoietic Cell Transplant

It is unknown if diminished pulmonary function early after allogeneic hematopoietic transplant is associated with poor long-term outcomes. The objective of this study was to determine if posttransplant lung function is associated with 5-year nonrelapse mortality (NRM) and the development of chronic graft-versus-host disease (cGVHD). Retrospective analysis was done for 2158 patients who had routine pulmonary function testing 60-120 days after transplant between 1992 and 2004. Cox regression was used to assess the hazard ratio for 5-year NRM. A second analysis assessed the hazard ratio for the development of cGVHD. Lung function score was the primary exposure, and was calculated according to forced expiratory volume in 1 second (FEV(1)) and carbon monoxide diffusion capapcity (DLCO). Individual pulmonary function parameters were secondary exposures. The primary outcomes were 5-year NRM and the development of cGVHD. Most patients had normal lung function following transplant. A higher lung function score, signifying greater impairment, was associated with an increased risk of mortality (category 1 hazard ratio [HR] 1.47 [1.17-1.85]; category 2 HR 3.38 [2.53-4.53]; category 3 HR 7.80 [4.15-14.68]). A similar association was observed for all individual pulmonary function parameters. Low FEV(1) was associated with the subsequent development of cGVHD (FEV(1) 70%-79% HR 1.26 [1.01-1.57]; 60%-69% HR 1.48 [1.10-2.01]; <60% HR 2.02 [1.34-3.05]). Models using either lung function score or individual pulmonary function parameters performed about equally well as judged by the C-statistic. Impaired lung function at day 80 posttransplant was associated with a higher risk of NRM. A low FEV(1) following transplant was associated with developing cGVHD within 1 year.

Novel Prognostic Gene Mutations Identified in Chronic Lymphocytic Leukemia and Their Impact on Clinical Practice

Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy characterized by progressive accumulation of mature lymphocytes in the peripheral blood, bone marrow, liver, and lymphoid organs. Although most patients with CLL have an insidious clinical course, a subset of cases present with fast evolution and chemotherapy resistance, leading to high morbidity and mortality. Few clinically validated prognostic markers, such as TP53, are available for use in clinical practice to guide treatment decisions. Recently, several novel prognostically relevant molecular markers have been identified in CLL. We conducted a narrative literature review of the latest findings to evaluate the potential inclusion of these markers in the management of CLL cases.

Primary central nervous system lymphoma: what a neurologist/neurosurgeon should know?

Primary central nervous system lymphoma is a rare disease, with bad prognosis. Neurologists and neurosurgeons should be familiar with the diagnostic,and biologic features, as well as the initial management of patients. A correct approach to these patients is mandatory for a better outcome.

Survival and graft-versus-host disease in patients receiving peripheral stem cell compared to bone marrow transplantation from HLA-matched related donor: retrospective analysis of 334 consecutive patients

The objective of this study was to compare the major transplant outcomes between patients receiving hematopoietic stem cell transplantation (HSCT) from bone marrow (BM) or peripheral blood stem cells (PBSC).

Granulocyte whole exome sequencing and endothelial JAK2V617F in patients with JAK2V617F positive Budd‐Chiari Syndrome without myeloproliferative neoplasm

Budd-Chiari Syndrome (BCS) is characterized by hepatic venous outflow obstruction. JAK2-positive myeloproliferative neoplasms (JAK2 MPN) are one of the most frequent thrombotic conditions underlying a diagnosis of BCS (Smalberg et al, 2012). A subset of BCS patients harbour the JAK2 mutation in peripheral blood (PB) granulocytes without evidence of overt MPN (JAK2 MPN-neg). While the occurrence of other myeloid-associated mutations in genes are common in patients with JAK2 MPN, it is unknown if such mutations are present in patients with JAK2 MPN-neg. In addition, it remains to be determined if there are differences in JAK2 allele burden between JAK2 MPN and JAK2 MPN-neg. Recently, it has been demonstrated that a subset of patients with JAK2 MPN have endothelial cells (ECs) that are positive for JAK2(Teofili et al, 2011) and that these cells may possibly contribute to the prothrombotic state. However, the presence of JAK2-positive endothelial colony-forming cells (E-CFC) in JAK2 MPN-neg patients with BCS has not been evaluated. The objective of this study was to assess the presence of JAK2 in BM E-CFC from JAK2 MPN-neg patients with BCS and to evaluate the JAK2 allele burden and the presence of additional mutations. We selected patients diagnosed with BCS and the presence of JAK2 mutation in the absence of other thrombophilic conditions at Hospital Israelita Albert Einstein, Sao Paulo, Brazil, between March 2013 and June 2015. Patients were investigated for the presence of an associated MPN and diagnosed according to the 2001 World Health Organization criteria. This study was approved by the Institutional Review Board and conducted according to the Principles of the Declarations of Helsinki. Paired DNA (sorted CD66b-granulocytes/skin biopsy) was subjected to whole exome sequencing (WES) on the HiSeq 2000 platform (Illumina Inc., San Diego, CA) using the SureSelect library preparation kit (Agilent, Santa Clara, CA). Somatic variant calls were generated by combining the output of SomaticSniper (http://gmt.genome.wustl.edu/packages/somatic-sniper/), MuTect (https://www.broadinstitute.org/cancer/cga/mutec) and Pindel (www.sanger.ac.uk/ science/tools/pindel) plus additional in-house criteria (minimum coverage at both tumour/germline ≥8 reads; ratio of allele fraction tumour:germline >2). Bone marrow (BM) mononuclear cells were obtained by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Approximately 20 ml of BM was washed twice with phosphate-buffered saline and 2% fetal bovine serum and then suspended in Endocult liquid medium (STEMCELL Technologies Canada Inc., Vancouver, BC, Canada), as previously described (Wu et al, 2012), but extending the culture time by 7 days. ECs grown in culture were then evaluated by flow cytometry (ECs were considered CD146+, CD144+, CD34 , CD117 ) (Ozdogu et al, 2007). ECs were isolated by fluorescence-activated cell sorting (FACS) in a FACSAria (BD Biosciences, San Jose, CA). DNA was extracted from sorted ECs and tested for the presence of the JAK2 mutation by an allele-specific polymerase chain reaction (PCR). Data on JAK2 allele burden was extracted from WES results obtained from a cohort of 77 patients with JAK2-positive MPN (including 4 patients with JAK2 MPN-neg) (Santos et al, 2014) and from 110 patients previously reported by Nangalia et al (2013). We identified 32 cases of BCS, 10 (31 2%) of who were positive for the JAK2 mutation. Three of these patients passed away before inclusion in the study and 7 patients were included; their baseline features are summarized in Table I. Two patients were diagnosed with primary myelofibrosis (PMF) (Patients 1 and 7) during evaluation and the remaining 5 cases were JAK2 MPN-neg. Samples from Patients 1–6) were analysed by WES. In the 5 JAK2MPN-neg patients (Patients 2–6), the only known pathogenic somatic mutation found was JAK2. We found no additional myeloid-associated mutations in these patients. The median JAK2 variant allele frequency Table I. Demographic features of BCS patients and JAK2 alleleburden.

High body mass index among patients undergoing hematopoietic stem cell transplantation: results of a cross-sectional evaluation of nutritional status in a private hospital

BACKGROUND nutritional status before hematopoietic stem cell transplantation (HSCT) affects prognosis: better nourished patients have shorter time to engraftment, while malnutrition is associated with increase of mortality rates, complications, medical costs, poor quality of life and hospitalization stay. Furthermore, underweight patients have increased risk of death in the early post- HSCT period, and non-relapse mortality is greater for those who are extremely underweight, overweight and obese. Obesity is associated with treatment-related toxicity, higher incidence of grade II-IV acute graft-versus- host disease (GVHD), infections and mortality. The objective of this study was to investigate the nutritional status of patients undergoing HSCT between 2007-2013 in a private hospital, by calculating the body mass index (BMI), to verify the prevalence of any nutritional imbalances, especially obesity. METHODS in this retrospective study, based on medical records, we analyzed data from all patients with malignant and nonmalignant diseases who underwent HSCT from January 2007 to February 2014 in the Hematology- Oncology and Bone Marrow Transplantation Center at a large, tertiary referral center in Brazil. RESULTS a total of 257 cases were treated in the period and analyzed, of which 79% were aged up to 65 years old. Among these, 56% were overweight or obese. We observed a higher prevalence of obesity in elderly patients (P < 0.001). The mean BMI of the total sample was 26.4 kg/m2. BMI was significantly different between genders, with higher prevalence of overweight among men (P < 0.001). CONCLUSION differently from other studies, our investigation has shown low rates of underweight and more overweight and obesity rates in men and elderly patients undergoing HSCT.

Metastatic melanoma mimicking acute leukaemia.

A 71-year-old male who was immunosuppressed following liver transplantation underwent resection of a 3 1 mm-deep melanoma on his back. At the time of surgery, a positron emission tomography/computed tomography (PET-CT) scan and biopsy of the left sentinel axillary lymph nodes showed no evidence of metastasis. Three months later he presented with back pain. Another PET-CT was performed, which showed uptake in the left axilla and diffuse activity in the bone marrow. Blood counts were normal and lactic dehydrogenase was 20 times normal. A bone marrow aspirate (upper left) and staining with alpha naphthyl acetate esterase (upper right) were suggestive of acute monocytic leukaemia. Flow cytometry identified a population of cells that were CD45 , CD117+, CD61+, CD71+, CD56+ and were negative for myeloid markers. Bone marrow biopsy sections stained with haematoxylin and eosin (bottom left) and HMB45, recognizing a melanocyte antigen (bottom right), confirmed massive infiltration by melanoma. The axillary lymph nodes were also infiltrated. The tumour was tested for BRAF mutations and was found to harbour c.1801A > G resulting in K601E substitution and 1750C > T resulting in L584F substitution. Little is known about the clinical implication of these mutations. Despite a significant initial response to treatment with vemurafenib, the patient developed numerous brain metastases within 8 weeks and died 2 weeks later. We attribute the atypical pattern of metastasis to the transplantrelated immunosuppression.