Expanding the understanding of telomere biology disorder with reports from two families harboring variants in ZCCHC8 and TERC.

Telomere biology disorder (TBD) can present within a wide spectrum of symptoms ranging from severe congenital malformations to isolated organ dysfunction in adulthood. Diagnosing TBD can be challenging given the substantial variation in symptoms and age of onset across generations. In this report, we present two families, one with a pathogenic variant in ZCCHC8 and another with a novel variant in TERC. In the literature, only one family has previously been reported with a ZCCHC8 variant and TBD symptoms. This family had multiple occurrences of pulmonary fibrosis and one case of bone marrow failure. In this paper, we present a second family with the same ZCCHC8 variant (p.Pro186Leu) and symptoms of TBD including pulmonary fibrosis, hematological disease, and elevated liver enzymes. The suspicion of TBD was confirmed with the measurement of short telomeres in the proband. In another family, we report a novel likely pathogenic variant in TERC. Our comprehensive description encompasses hematological manifestations, as well as pulmonary and hepatic fibrosis. Notably, there are no other reports which associate this variant to disease. The families expand our understanding of the clinical implications and genetic causes of TBD.

Risk of cancer in relatives of patients with myelodysplastic neoplasia and acute leukemias.

BACKGROUND: The risk of cancer among relatives of patients with either myelodysplastic neoplasia (MDS), acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) has not been thoroughly examined. METHODS: We linked the Danish Civil Registration System with the Danish Cancer Registry, the Danish National Acute Leukemia Registry, and the Danish Myelodysplastic Syndrome Database to estimate the relative risk of cancer among relatives of patients with MDS/AML/ALL. We used standardized incidence ratios (SIRs), i.e., the ratio of observed to expected number of cancers among the relatives as a measure of relative risk. RESULTS: We identified 13010 first-degree (FDR) and 22051 second-degree (SDR) relatives of 8386 patients with MDS/ALL/AML. Disregarding basal cell carcinoma (BCC), the relative risk for cancer overall was increased in both FDR (SIR=1.3; 95% confidence interval (CI) 1.1-1.4) and SDR (SIR=1.5; 95% CI 1.2-1.8). SIRs among FDRs were statistically significantly increased for malignant melanoma, BCC and for the combined groups of cancers of the male genital organs, urinary tract, and MDS/AML/ALL. Among SDRs, SIRs were statistically significantly increased for malignant melanoma, BCC, and cancers in the digestive organs and peritoneum. CONCLUSIONS: We observed an increased risk of cancer among FDR and SDR of patients with MDS/AML/ALL.

National clinical Genetic Networks - GENets - Establishment of expert collaborations in Denmark.

Genetic conditions are often familial, but not all relatives receive counseling from the same institution. It is therefore necessary to ensure consistency in variant interpretation, counseling practices, and clinical follow up across health care providers. Furthermore, as new possibilities for gene-specific treatments emerge and whole genome sequencing becomes more widely available, efficient data handling and knowledge sharing between clinical laboratory geneticists and medical specialists in clinical genetics are increasingly important. In Denmark, these needs have been addressed through the establishment of collaborative national networks called Genetic Expert Networks or "GENets". These networks have enhanced patient and family care significantly by bringing together groups of experts in national collaborations. This promotes coordinated clinical care, the dissemination of best clinical practices, and facilitates the exchange of new knowledge.

ProGraME: A novel flow cytometry algorithm for the diagnosis of low-risk myelodysplastic syndromes in patients with cytopenia.

OBJECTIVES: Flow cytometry (FC) is, together with morphology, genetics, and cytogenetics, used in the diagnostic assessment of cytopenia, as its value in evaluating bone marrow dysplasia been highlighted by several studies. However, despite the development of algorithms and guidelines, there is still a lack of standardization of the FC assessment of bone marrow dysplasia. METHODS: By combining FC, together with morphological analysis and cytogenetic/molecular assessment in a training cohort of 209 patients, we created a novel score, ProGraME, which includes four parameters, each from a different cell lineage (Progenitor cells, Granulocytes, Monocytes, Erythroid precursors), solely based on relevant population gating. Points for ProGraME were attained for: lymphoid precursors ≤5% of all CD34+ cells (1.5 point); a granulocyte-to-lymphocyte side-scatter ratio ≤6 (1 point); a monocyte CD33-CV% ≥ 63 (2 points), and an erythroid precursor CD36-CV% ≥ 65 (2 points). RESULTS: Using a cutoff of ≥2 as suggestive of dysplasia, ProGraME showed a sensitivity of 91% and a specificity of 81% in the training cohort and 95% and 75%, respectively, in an independent validation cohort of 159 patients. In addition, ProGraME had a very high negative predictive value of 97.1% and 97.8% in the training and validation cohorts, respectively, offering a useful tool for excluding bone marrow dysplasia. Finally, among the 23 CCUS patients that scored positive for dysplasia with ProGraME in the training cohort, 16 of them (69%) carried high-risk mutations, suggesting that FC might help identify early changes of dysplasia. CONCLUSIONS: ProGraME can potentially optimize the FC diagnosis of low-risk myelodysplasia without minimal requirements of flow analysis other than accurate population gating.

Size matters in telomere biology disorders ‒ expanding phenotypic spectrum in patients with long or short telomeres.

The end of each chromosome consists of a DNA region termed the telomeres. The telomeres serve as a protective shield against degradation of the coding DNA sequence, as the DNA strand inevitably ‒ with each cell division ‒ is shortened. Inherited genetic variants cause telomere biology disorders when located in genes (e.g. DKC1, RTEL1, TERC, TERT) playing a role in the function and maintenance of the telomeres. Subsequently patients with telomere biology disorders associated with both too short or too long telomeres have been recognized. Patients with telomere biology disorders associated with short telomeres are at increased risk of dyskeratosis congenita (nail dystrophy, oral leukoplakia, and hyper- or hypo-pigmentation of the skin), pulmonary fibrosis, hematologic disease (ranging from cytopenia to leukemia) and in rare cases very severe multiorgan manifestations and early death. Patients with telomere biology disorders associated with too long telomeres have in recent years been found to confer an increased risk of melanoma and chronic lymphocytic leukemia. Despite this, many patients have an apparently isolated manifestation rendering telomere biology disorders most likely underdiagnosed. The complexity of telomere biology disorders and many causative genes makes it difficult to design a surveillance program which will ensure identification of early onset disease manifestation without overtreatment.

A predictive model for bone marrow disease in cytopenia based on noninvasive procedures.

Bone marrow specimens are the core of the diagnostic workup of patients with cytopenia. To explore whether next-generation sequencing (NGS) could be used to rule out malignancy without bone marrow specimens, we incorporated NGS in a model to predict presence of disease in the bone marrow of patients with unexplained cytopenia. We analyzed the occurrence of mutations in 508 patients with cytopenia, referred for primary workup of a suspected hematologic malignancy from 2015 to 2020. We divided patients into a discovery (n = 340) and validation (n = 168) cohort. Targeted sequencing, bone marrow biopsy, and complete blood count were performed in all patients. Mutations were identified in 267 (53%) and abnormal bone marrow morphology in 188 (37%) patients. Patients with isolated neutropenia had the lowest frequency of both mutations (21%) and abnormal bone marrow morphology (5%). The median number of mutations per patient was 2 in patients with abnormal bone marrow morphology compared with 0 in patients with a nondiagnostic bone marrow morphology (P < .001). In a multivariable logistic regression, mutations in TET2, SF3B1, U2AF1, TP53, and RUNX1 were significantly associated with abnormal bone marrow morphology. In the validation cohort, a model combining mutational status and clinical data identified 34 patients (20%) without abnormal bone marrow morphology with a sensitivity of 100% (95% confidence interval: 93%-100%). Overall, we show that NGS combined with clinical data can predict the presence of abnormal bone marrow morphology in patients with unexplained cytopenia and thus can be used to assess the need of a bone marrow biopsy.

Improved survival after allogeneic transplantation for acute lymphoblastic leukemia in adults: a Danish population-based study.

We investigated trends of survival in a population-based cohort study of all 181 adults who received HCT for ALL in Denmark between 2000-2019. Patients had a median (min-max) age of 36 (18-74) years at HCT and were followed for a median of eight years. Overall survival (OS) improved over time with an estimated 2-year OS of 49% (CI 27-66%) in year 2000 versus 77% (CI 59-88%) in year 2019. More patients achieved cure over time (OR for cure per year 1.07, CI 1.00-1.15), while the rate of death in non-cured patients remained stable (HR of excess mortality per year 0.99, CI 0.93-1.06). Relapse decreased over time (HR 0.92 per year, CI 0.87-0.98), whereas non-relapse mortality did not change notably (HR 0.98 per year, CI 0.93-1.04). In conclusion, survival after HCT in adults with ALL has improved over the past two decades, primarily due to more patients achieving cure.

Epigenetic therapy in combination with a multi-epitope cancer vaccine targeting shared tumor antigens for high-risk myelodysplastic syndrome - a phase I clinical trial.

BACKGROUND: Standard care for patients with high-risk myelodysplastic syndrome (MDS) is hypomethylating agents such as azacitidine (AZA), which can induce expression of methylated tumor-associated antigens and therefore potentiate immunotherapeutic targeting. METHOD: In this phase 1 trial, we combined AZA with a therapeutic peptide vaccine targeting antigens encoded from NY-ESO-1, MAGE-A3, PRAME, and WT-1, which have previously been demonstrated to be upregulated by AZA treatment. RESULT: Five patients who had responded to AZA monotherapy were included in the study and treated with the vaccine. The combination therapy showed only few adverse events during the study period, whereof none classified as serious. However, no specific immune responses could be detected using intracellular cytokine staining or ELISpot assays. Minor changes in the phenotypic composition of immune cells and their expression of stimulatory and inhibitory markers were detected. All patients progressed to AML with a mean time to progression from inclusion (TTP) of 5.2 months (range 2.8 to 7.6). Mean survival was 18.1 months (range 10.9 to 30.6) from MDS diagnosis and 11.3 months (range 4.3 to 22.2) from inclusion. Sequencing of bone marrow showed clonal expansion of malignant cells, as well as appearance of novel mutations. CONCLUSION: The patients progressed to AML with an average time of only five months after initiating the combination therapy. This may be unrelated to the experimental treatment, but the trial was terminated early as there was no sign of clinical benefit or immunological response. Why the manuscript is especially interesting This study is the first to exploit the potential synergistic effects of combining a multi-peptide cancer vaccine with epigenetic therapy in MDS. Although our results are negative, they emphasize challenges to induce immune reactivity in patients with high-risk MDS.

The real-world outcomes of multiple myeloma patients treated with daratumumab.

Most patients cannot be included in randomized clinical trials. We report real-world outcomes of all Danish patients with multiple myeloma (MM) treated with daratumumab-based regimens until 1 January 2019. METHODS: Information of 635 patients treated with daratumumab was collected retrospectively and included lines of therapy (LOT), hematologic responses according to the International Myeloma Working Group recommendations, time to next treatment (TNT) and the cause of discontinuation of treatment. Baseline characteristics were acquired from the validated Danish Multiple Myeloma Registry (DMMR). RESULTS: Daratumumab was administrated as monotherapy (Da-mono) in 27.7%, in combination with immunomodulatory drugs (Da-IMiD) in 57.3%, in combination with proteasome inhibitors (Da-PI) in 11.2% and in other combinations (Da-other) in 3.8% of patients. The median number of lines of therapy given before daratumumab was 5 for Da-mono, 3 for Da-IMiD, 4 for Da-PI, and 2 for Da-other. In Da-mono, overall response rate (ORR) was 44.9% and median time to next treatment (mTNT) was 4.9 months. In Da-IMiD, ORR was 80.5%, and mTNT was 16.1 months. In Da-PI, OOR was 60.6% and mTNT was 5.3 months. In patients treated with Da-other, OOR was 54,2% and mTNT was 5.6 months. The use of daratumumab in early LOT was associated with longer TNT (p<0.0001). Patients with amplification 1q had outcome comparable to standard risk patients, while patients with t(4;14), t(14;16) or del17p had worse outcome (p = 0.0001). Multivariate analysis indicated that timing of treatment (timing of daratumumab in the sequence of all LOT that the patients received throughout the course of their disease) was the most important factor for outcome (p<0.0001). CONCLUSION: The real-world outcomes of multiple myeloma patients treated with daratumumab are worse than the results of clinical trials. Outcomes achieved with daratumumab were best when daratumumab was used in combination with IMIDs and in early LOT. Patients with high-risk CA had worse outcomes, but patients with amp1q had similar outcomes to standard-risk patients.

[Germ line predisposition for myeloid neoplasia in adults].

Myeloid neoplasms with germ line predisposition (hMN) are likely underdiagnosed and are estimated to constitute a substantial fraction of patients with myelodysplastic syndrome and acute myeloid leukaemia. Correct diagnosis of hMN is vital, as it can influence treatment decisions, facilitate genetic counselling and help identify family members at risk. In this review, we describe the symptoms associated with hMN and present an example of the underlying molecular mechanism. Furthermore, we summarise the current knowledge and recommendations for diagnosis, surveillance and treatment of hMN.

Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelomonocytic Leukemia: Clinical and Molecular Genetic Prognostic Factors in a Nordic Population.

Chronic myelomonocytic leukemia (CMML) is an aggressive disease in which survival after allogeneic hematopoietic stem cell transplantation (HCT) remains relatively poor. An assessment of prognostic factors is an important part of treatment decision making and has the potential to be greatly improved by the inclusion of molecular genetics. However, there is a significant knowledge gap in the interpretation of mutational patterns. This study aimed to describe outcomes of allogeneic HCT in patients with CMML in relation to clinical and molecular genetic risk factors. This retrospective study included 64 patients with CMML who underwent allogeneic HCT between 2008 and 2018, with a median follow-up of 5.4 years. Next-generation sequencing using targeted myeloid panels was carried out on saved material from 51 patients from the time of transplantation. Kaplan-Meier and Cox regression were used for analysis of overall survival (OS), and cumulative incidence with competing risks and Fine and Gray models were used for analysis of relapse and nonrelapse mortality (NRM). Mutations were detected in 48 patients (94%), indicating high levels of minimal residual disease (MRD) positivity at transplantation, even among those in complete remission (CR) (n = 14), 86% of whom had detectable mutations. The most frequently mutated genes were ASXL1 (37%), TET2 (37%), RUNX1 (33%), SRSF2 (26%), and NRAS (20%). Risk stratification using the CMML-specific Prognostic Scoring System molecular score (CPSS-Mol) resulted in 45% of patients moving to a higher risk-group compared with risk stratification using the CPSS. High leucocyte count (≥13 × 109/L), transfusion requirement, and previous intensive chemotherapy were associated with higher incidence of relapse. Being in CR was not linked to better outcomes. Neither ASXL1 nor RUNX1 mutation was associated with a difference in OS, relapse, or NRM, despite being high risk in the nontransplantation setting. TET2 mutations were associated with a significantly higher 3-year OS (73% versus 40%; P = .039). Achieving MRD-negative CR was rare in this CMML cohort, which may explain why we did not observe better outcomes for those in CR. This merits further investigation. Our analyses suggest that the negative impact of ASXL1 and RUNX1 mutations can be overcome by allogeneic HCT; however, risk stratification is complex in CMML and requires larger cohorts and multivariate models, presenting an ongoing challenge in this rare disease.

Outcome of treatment with carfilzomib before and after treatment with daratumumab in relapsed or refractory multiple myeloma patients.

Real world evidence is important since most patients cannot be included in randomized clinical trials (RCTs). In a nationwide, cohort of relapsed/refractory multiple myeloma patients treated with daratumumab (N = 635), we retrospective studied patients treated with carfilzomib (N = 251). Data were collected by audit of medical records. We compared characteristics of patients treated with carfilzomib before daratumumab (Car-Da; N = 150) and after daratumumab (Da-Car; N = 101) with those not treated with carfilzomib (N = 384). Furthermore, we examined effectiveness and safety of carfilzomib. The group of patients treated with carfilzomib differed from patients not treated with carfilzomib in the following parameters: They were younger, more were treated up-front with high dose melphalan and autologous stem cell transplantation (HDM-ASCT)and had relapse within 18 months thereafter, and more had high-risk cytogenetic abnormalities (CA) and amplification 1q (amp1q). In patients treated with Car-Da, 30.3% had high-risk CA and 30.1% had amp1q and in Da-Car it was 43.3% and 41%, respectively. In the Car-Da cohort, 34.4% experienced early relapse after HDM-ASCT versus 47.4% in the Da-Car cohort. The percentage of patients with very good partial remission was higher in patients treated with Car-Da compared to Da-Car (31.7% vs. 17.4%). The median duration of treatment and time to next treatment (TNT) of Car-Da/Da-Car were 4.6/4.3 months and 7.1/4.3 months and only a trend toward superior TNT for Car-Da was found (p = 0.06). Toxicity of carfilzomib was the same as reported in RCT. A similar poor TNT of daratumumab was found when used before (5.6 months) or after carfilzomib (4.9 months). In this cohort of patients with sequential treatment with carfilzomib and daratumumab or vice versa, a high percentage of patients were high-risk by CA, amp1q, and early relapse after HDM-ASCT. Outcome of Car-DA and outcome of Da-Car were equally poor. These patients should be considered for new promising treatment strategies.

The molecular profile of mucosal melanoma.

Herein, we wanted to explore the molecular landscape of mucosal melanoma from different sites and identify potential molecular targets for future therapy. Mucosal melanomas (N = 40) from different sites (conjunctiva, sinonasal cavity, rectum, and vagina) were investigated. Targeted next-generation sequencing along with Nanostring gene expression profiling was performed. Genetically, conjunctival melanoma was characterized by BRAF-V600E (30%) and NF1 mutations (17%). Mucosal melanomas at nonsun-exposed sites harbored alterations in NRAS, KIT, NF1, along with atypical BRAF mutations. When comparing the gene expression profile of conjunctival melanoma and nonsun-exposed mucosal melanoma, 41 genes were found to be significantly deregulated. Programmed death-ligand 1 (PD-L1) presented a significant sixfold upregulation in conjunctival melanoma compared to the other mucosal melanomas. While melanomas of the sinonasal cavity, vagina, and rectum are molecularly similar, conjunctival melanoma is characterized by a higher frequency of BRAF-V600E mutations and differential expression of several genes involved in the immune response.

The Number of Signaling Pathways Altered by Driver Mutations in Chronic Lymphocytic Leukemia Impacts Disease Outcome.

PURPOSE: Investigation of signaling pathways altered by recurrent gene mutations and their clinical impact in a consecutive cohort of patients with newly diagnosed chronic lymphocytic leukemia (CLL). The heterogeneous clinical course and genetic complexity of CLL warrant improved molecular prognostication. However, the prognostic value of recurrent mutations at the time of diagnosis remains unclear. EXPERIMENTAL DESIGN: We sequenced samples from 314 consecutive, newly diagnosed patients with CLL to investigate the clinical impact of 56 recurrently mutated genes assessed by next-generation sequencing. RESULTS: Mutations were identified in 70% of patients with enrichment among IGHV unmutated cases. With 6.5 years of follow-up, 15 mutated genes investigated at the time of diagnosis demonstrated significant impact on time to first treatment (TTFT). Carrying driver mutations was associated with shorter TTFT and poor overall survival. For outcome from CLL diagnosis, the number of signaling pathways altered by driver mutations stratified patients better than the number of driver mutations. Moreover, we demonstrated gradual impact on TTFT with increasing number of altered pathways independent of CLL-IPI risk. Thus, a 25-gene, pathway-based biomarker assessing recurrent mutations refines prognostication in CLL, in particular for CLL-IPI low- and intermediate-risk patients. External validation emphasized that a broad gene panel including low burden mutations was key for the biomarker based on altered pathways. CONCLUSIONS: We propose to include the number of pathways altered by driver mutations as a biomarker together with CLL-IPI in prospective studies of CLL from time of diagnosis for incorporation into clinical care and personalized follow-up and treatment.

Orbital precursor B-lymphoblastic lymphoma involving the extraocular muscles in a 56-year-old male and a review of the literature.

The aim of the present study was to describe a rare case of orbital precursor B-lymphoblastic lymphoma (B-LBL) in an adult. A 56-year-old male in complete remission of a gastric precursor B-LBL was referred to our orbital clinic due to rapid development of left-sided painless periorbital swelling, diplopia, and proptosis. Complete ophthalmoplegia was observed. Notably, magnetic resonance imaging showed swelling of the medial and inferior rectus muscles in the left orbit and biopsies were performed. Following histological diagnosis of precursor B-LBL, the patient was treated with radiotherapy (2Gy × 20) and chemotherapy according to the NOPHO ALL 2008 protocol. The disease progressed and the patient succumbed after 5 months. Histomorphologically, a lymphoblastic infiltrate was observed within the skeletal muscle tissue. The tumor cells were small and immature, and stained strongly for cluster of differentiating (CD)10, CD79a, paired box 5 and B cell lymphoma-2. The Ki-67 proliferative index was 90%. Multiplex ligation-dependent probe amplification and array comparative genomic hybridization detected whole chromosomal gain of X and 12, and both hemizygous and homozygous deletion on 9p comprising cyclin dependent kinase inhibitor 2A/B. Furthermore, array comparative genomic hybridization detected copy number imbalances consisting of focal or smaller deletions on chromosomes 1, 9, 10, 11 and 20. The final diagnosis was precursor B-LBL relapse in the extraocular muscles. Orbital precursor B-LBL is extremely rare in adults, and the diagnosis may be challenging to make. It is recommended to obtain material for cytogenetic and molecular analyses.

Bilateral diffuse uveal melanocytic proliferation: Case report and literature review.

Bilateral diffuse uveal melanocytic proliferation (BDUMP) is a rare paraneoplastic intraocular disease that causes progressive visual loss in patients driven by an IgG factor associated with an underlying malignancy. Characteristic ocular findings include exudative retinal detachment, rapid cataract formation and uveal melanocytic tumours. The awareness and documentation of BDUMP has increased during the past decade, and the increasing amount of data collected demonstrates the effect of treatment with plasmapheresis and the value of diagnostic tools in BDUMP such as genetic and immunologic investigations. The literature of BDUMP has not been reviewed since 2003, and there is a growing need for an updated review on diagnosis and management of BDUMP. We review the literature and report a case of BDUMP with a white ciliary body tumour, iris rubeosis, increased iris pigmentation and cataract.

Mutations in idiopathic cytopenia of undetermined significance assist diagnostics and correlate to dysplastic changes.

Cytopenia is common in the elderly population and etiology may be difficult to assess. Here, we investigated the occurrence of mutations in patients with idiopathic cytopenia of undetermined significance and the usefulness in improving diagnostics. We included 60 patients with persistent cytopenia > 6 months without definite diagnosis of hematological neoplasm after routine assessment. Bone marrow material underwent a blinded morphology review and DNA was sequenced with a targeted 20 gene panel representing the most commonly mutated genes in myelodysplastic syndrome. Thirty seven (62%) patients carried at least one mutation at inclusion, and of these 95% carried a mutation in TET2, ASXL1, SRSF2, or DNMT3A. The most commonly mutated gene was TET2 observed in 43% of all patients. During one to eight years follow-up seven patients progressed to a myeloid neoplasm and six of these had a detectable mutation at study entry. Median time to progression was 53 months (range 10-78), and at time of progression each patient had at least two mutations detected. Mutations in TP53 and NRAS were not present in patients at inclusion, but identified as secondary hits triggering progression. The morphology review was concordant in 68% of all cases, and 93% of the cases reclassified into the group "highly suspicious for MDS" had a mutation. All patients who had a concordant review "highly suspicious for MDS" had at least two mutations detected. Overall, we show that morphology examination is challenging in this heterogeneous group and targeted sequencing helps identify patients at risk of progression. Am. J. Hematol. 91:1234-1238, 2016. © 2016 Wiley Periodicals, Inc.