Chimerism refers to the relative proportion of donor and recipient DNA after hematopoietic stem cell transplantation (HSCT) and its quantitative follow-up is of great clinical utility in this setting. PCR of short tandem repeats (STR-PCR) constitutes the gold standard method for chimerism quantification, although more sensitive PCR techniques (such as qPCR) have recently arisen. We compared the sensitivity and the quantification capacity of both techniques in patient samples and artificial mixtures and demonstrated adequate performance of both methods, with higher sensitivity of qPCR and better quantification skills of STR-PCR. By qPCR, we then prospectively followed up 57 patients that were in complete chimerism (CC) by STR-PCR. Twenty-seven patients (59%) showed 0.1–1% recipient DNA in the bone marrow. Only 4 patients presented 0.1–1% recipient DNA in peripheral blood (PB), and one of them relapsed. Finally, by qPCR, we retrospectively studied the last sample that showed CC by STR-PCR prior to relapse in 8 relapsed patients. At a median of 59 days prior to relapse, six patients presented mixed chimerism by qPCR in PB. Since both approaches have complementary characteristics, we conclude that different techniques should be applied in different clinical settings and therefore propose a methodological algorithm for chimerism follow-up after HSCT.

A successful Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variant, B.1.1.7, has recently been reported in the UK, causing global alarm. Most likely, the new variant emerged in a persistently infected patient, justifying a special focus on these cases. Our aim in this study was to explore certain clinical profiles involving severe immunosuppression that may help explain the prolonged persistence of viable viruses. We present three severely immunosuppressed cases (A, B, and C) with a history of lymphoma and prolonged SARS-CoV-2 shedding (2, 4, and 6 months), two of whom finally died. Whole-genome sequencing of 9 and 10 specimens from Cases A and B revealed extensive within-patient acquisition of diversity, 12 and 28 new single nucleotide polymorphisms, respectively, which suggests ongoing SARS-CoV-2 replication. This diversity was not observed for Case C after analysing 5 sequential nasopharyngeal specimens and one plasma specimen, and was only observed in one bronchoaspirate specimen, although viral viability was still considered based on constant low Ct values throughout the disease and recovery of the virus in cell cultures. The acquired viral diversity in Cases A and B followed different dynamics. For Case A, new single nucleotide polymorphisms were quickly fixed (13–15 days) after emerging as minority variants, while for Case B, higher diversity was observed at a slower emergence: fixation pace (1–2 months). Slower SARS-CoV-2 evolutionary pace was observed for Case A following the administration of hyperimmune plasma. This work adds knowledge on SARS-CoV-2 prolonged shedding in severely immunocompromised patients and demonstrates viral viability, noteworthy acquired intra-patient diversity, and different SARS-CoV-2 evolutionary dynamics in persistent cases.

Molecular diagnosis of myeloid neoplasms (MN) is based on the detection of multiple genetic alterations using various techniques. Next-generation sequencing (NGS) has been proved as a useful method for analyzing many genes simultaneously. In this context, we analyzed diagnostic samples from 121 patients affected by MN and ten relapse samples from a subset of acute myeloid leukemia patients using two enrichment-capture NGS gene panels. Pathogenicity classification of variants was enhanced by the development and application of a custom onco-hematology score. A total of 278 pathogenic variants were detected in 84% of patients. For structural alterations, 82% of those identified by cytogenetics were detected by NGS, 25 of 31 copy number variants and three out of three translocations. The detection of variants using NGS changed the diagnosis of seven patients and the prognosis of 15 patients and enabled us to identify 44 suitable candidates for clinical trials. Regarding AML, six of the ten relapsed patients lost or gained variants, comparing with diagnostic samples. In conclusion, the use of NGS panels in MN improves genetic characterization of the disease compared with conventional methods, thus demonstrating its potential clinical utility in routine clinical testing. This approach leads to better-adjusted treatments for each patient.

Development of de novo hematologic malignancies in donor cells after allogeneic stem cell transplantation (allo-SCT) provides a useful in vivo model to study the process of leukemogenesis. A systematic analysis of the cases reported in the literature was performed to identify risk factors and mechanisms involved in the pathogenesis of donor cell-derived hematologic neoplasms (DCHN) and leukemogenic transformation. Relevant data were extracted from 137 cases. Cases of DCHN show a wide heterogeneity with regard to recipient/donor age, sex mismatch, and conditioning regimen. Some characteristics, such as the type of primary disease, the type of hematologic malignancy of the DCHN, and the stem cell source used in the transplant procedure, differ from those expected. Mechanisms involved in the pathogenesis of DCHN are complex, and several hypotheses have been proposed, such as pre-existing hematologic neoplasms or premalignant clones in the donor, decreased immune surveillance, and damage to bone marrow microenvironment in the recipient. Most likely several if not all these mechanisms play a role in DCHN development. Novel approaches, such as next-generation sequencing to study consecutive samples after allo-SCT in these patients, appear to be promising to decipher the mechanisms of leukemogenesis.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for patients with hematologic malignances. Haploidentical HSCT (Haplo-HSCT) is an alternative option for patients who do not have an HLA-matched donor. The use of post-transplantation high dose cyclophosphamide (PT-Cy) is commonly employed for graft-versus-host disease (GVHD) prophylaxis in haplo-HSCT. Cyclophosphamide (Cy) is an alkylating agent with antineoplastic and immunosuppressive activity, whose bioactivation requires the activity of polymorphic enzymes in the liver to produce phosphoramide mustard, which is a DNA alkylating agent. To identify polymorphisms in the genes of Cy metabolism and correlate them with post-HSCT complications [GVHD, sinusoidal obstruction syndrome (SOS), hemorrhagic cystitis (HC) and transplant-related mortality (TRM)], we designed a custom next-generation sequencing panel with Cy metabolism enzymes. We analyzed 182 patients treated with haplo-HSCT with PT-Cy from 2007 to 2019, detecting 40 variants in 11 Cy metabolism genes. Polymorphisms in CYP2B6, a major enzyme involved in Cy activation, were associated with decreased activity of this enzyme and a higher risk of Graf-versus-host disease (GVHD). Variants in other activation enzymes (CYP2A6, CYP2C8, CYP2C9, CYP2C19) lead to decreased enzyme activity and were associated with GVHD. Polymorphisms in detoxification genes such as glutathione S-transferases decreased the ability to detoxify cyclophosphamide metabolites due to lower enzyme activity, which leads to increased amounts of toxic metabolites and the development of III-IV acute GVHD. GSMT1*0 a single nucleotide polymorphism previously recognized as a risk factor for SOS was associated with a higher risk of SOS. We conclude that polymorphisms of genes involved in the metabolism of cyclophosphamide in our series are associated with severe grades of GVHD and toxicities (SOS and TRM) after haplo-HSCT and could be used to improve the clinical management of transplanted patients.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only medical intervention that has led to an HIV cure. Whereas the HIV reservoir sharply decreases after allo-HSCT, the dynamics of the T cell reconstitution has not been comprehensively described. We analyzed the activation and differentiation of CD4⁺ and CD8⁺ T cells, and the breadth and quality of HIV- and CMV-specific CD8⁺ T cell responses in 16 patients with HIV who underwent allo-HSCT (including five individuals who received cells from CCR5Δ32/Δ32 donors) to treat their underlying hematological malignancy and who remained on antiretroviral therapy (ART). We found that reconstitution of the T cell compartment after allo-HSCT was slow and heterogeneous with an initial expansion of activated CD4⁺ T cells that preceded the expansion of CD8⁺ T cells. Although HIV-specific CD8⁺ T cells disappeared immediately after allo-HSCT, weak HIV-specific CD8⁺ T cell responses were detectable several weeks after transplant and could still be detected at the time of full T cell chimerism, indicating that de novo priming, and hence antigen exposure, occurred during the time of T cell expansion. These HIV-specific T cells had limited functionality compared with CMV-specific CD8⁺ T cells and persisted years after allo-HSCT. In conclusion, immune reconstitution was slow, heterogeneous, and incomplete and coincided with de novo detection of weak HIV-specific T cell responses. The initial short phase of high T cell activation, in which HIV antigens were present, may constitute a window of vulnerability for the reseeding of viral reservoirs, emphasizing the importance of maintaining ART directly after allo-HSCT.