High-grade neuroendocrine carcinoma of the salivary glands is a rare malignancy that can be difficult to distinguish from metastatic neuroendocrine (Merkel cell) carcinoma of the skin, which often ...occurs on the head and neck and may metastasize to lymph nodes in or adjacent to salivary glands, particularly the parotid gland. As the 2 tumors have morphologic and immunophenotypic overlap, additional diagnostic tools may be clinically useful. Merkel cell carcinoma is known to harbor Merkel cell polyomavirus in up to 80% of cases. However, the presence or absence of this virus in salivary gland neuroendocrine carcinomas has not been investigated. We evaluated 7 primary salivary gland high-grade neuroendocrine carcinomas (all from the parotid) for the virus by both immunohistochemistry (CM2B4 clone) and real-time polymerase chain reaction directed against the conserved small T antigen. Five of the tumors had small cell morphology, and 2 had large cell morphology. All were either chromogranin and/or synaptophysin positive. Four of the 5 small cell (80%) and 1 of the 2 large cell (50%) carcinomas were cytokeratin 20 positive. All but 1 case had cervical lymph node metastases at presentation. Merkel cell polyomavirus T antigen was not detected in any of the 7 tumors, either by immunohistochemistry or by polymerase chain reaction with adequate controls. These observations suggest that primary parotid high-grade neuroendocrine carcinoma arises from a biological pathway that is different from that of cutaneous Merkel cell carcinomas. Furthermore, viral testing may aid in distinguishing the 2 tumor types, as a positive result would favor a metastasis.
Progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML) is associated with the acquisition and expansion of subclones. Our understanding of subclone evolution ...during progression, including the frequency and preferred order of gene mutation acquisition, remains incomplete. Sequencing of 43 paired MDS and secondary AML samples identified at least one signaling gene mutation in 44% of MDS and 60% of secondary AML samples, often below the level of standard sequencing detection. In addition, 19% of MDS and 47% of secondary AML patients harbored more than one signaling gene mutation, almost always in separate, coexisting subclones. Signaling gene mutations demonstrated diverse patterns of clonal evolution during disease progression, including acquisition, expansion, persistence, and loss of mutations, with multiple patterns often coexisting in the same patient. Multivariate analysis revealed that MDS patients who had a signaling gene mutation had a higher risk of AML progression, potentially providing a biomarker for progression.
Subclone expansion is a hallmark of progression from MDS to secondary AML. Subclonal signaling gene mutations are common at MDS (often at low levels), show complex and convergent patterns of clonal evolution, and are associated with future progression to secondary AML. See related article by Guess et al., p. 316 (33). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.
In silico approaches for next-generation sequencing (NGS) data modeling have utility in the clinical laboratory as a tool for clinical assay validation. In silico NGS data can take a variety of ...forms, including pure simulated data or manipulated data files in which variants are inserted into existing data files. In silico data enable simulation of a range of variants that may be difficult to obtain from a single physical sample. Such data allow laboratories to more accurately test the performance of clinical bioinformatics pipelines without sequencing additional cases. For example, clinical laboratories may use in silico data to simulate low variant allele fraction variants to test the analytical sensitivity of variant calling software or simulate a range of insertion/deletion sizes to determine the performance of insertion/deletion calling software. In this article, the Working Group reviews the different types of in silico data with their strengths and limitations, methods to generate in silico data, and how data can be used in the clinical molecular diagnostic laboratory. Survey data indicate how in silico NGS data are currently being used. Finally, potential applications for which in silico data may become useful in the future are presented.
Introduction: Clonal cytopenias of undetermined significance (CCUS) and myelodysplastic syndromes (MDS) represent a disease continuum that are distinguished by the presence of dysplasia in >10% of ...blood cells. However, morphologic dysplasia is subject to high inter-observer variability, suggesting that dysplasia may not be ideal to differentiate CCUS from MDS. Defining the clonal architecture of samples from cytopenic patients may provide a more accurate objective measure of disease status than dysplasia. We hypothesize that the number and size of subclones is reduced in patients with CCUS compared to MDS, suggesting that increased subclonal diversity is a hallmark of higher-risk disease. Methods: We performed whole genome sequencing with higher exome coverage (eWGS) on bone marrow (n=58) or peripheral blood (n=4) and paired normal DNA from baseline banked samples from patients with CCUS (n=13), MDS (n=29; IPSS-R very low/low 3, intermediate 5, high/very high 19, no score 2), and secondary AML (sAML) (n=20), including 32 previously reported patients, to define clonal architecture. Putative somatic variants, including 76 genes that are recurrently mutated in myeloid neoplasms, were validated in hematopoietic and paired normal samples, and available serial samples (n=23), using orthogonal target enrichment sequencing platforms achieving higher coverage (~600-1000x). Clonality of validated somatic mutations was defined using SciClone. Results: The absolute neutrophil count, hemoglobin, and platelet counts trended lower in MDS vs CCUS patients (median, 1.3 vs 2 K/ul ns; 9.3 vs 11.35 g/dL p<0.01; 63 vs 109.5 K/uL ns, respectively). The ages and median number of total validated somatic mutations identified by eWGS at baseline was similar for CCUS (n=428), MDS (n=300), and sAML patients (n=420.5). We first defined the founding clone (i.e., the dominant clone with the highest median variant allele frequency VAF) and subclones. While the number of total clones per sample was not different between CCUS, MDS, and sAML (median, n=3 for each), the median VAF of all the mutations present in the founding clone (i.e., measure of the maximal molecular disease burden) was significantly lower in CCUS (21.3%) compared to MDS (39.2%, p<0.05) and sAML samples (45.2%, p<0.001), but MDS and sAML were not different (p=0.2) (Figure 1A). In contrast, there was no significant difference in the median VAF of subclones in CCUS and MDS samples (13.4% vs 16.8%), which were both lower than sAML (25.3%, p<= 0.01). However, the proportion of patients with no subclones (i.e., only the founding clone detected) was higher for CCUS (5/13 38.5%) compared to MDS and sAML (4/29 13.8% and 0/20 0%, respectively, p 0.0006), suggesting that CCUS samples have reduced subclonal diversity compared to MDS. Consistent with this, genes commonly mutated in subclones occurred less frequently in CCUS vs MDS samples, including activated signaling (0/13 vs 6/29) and transcription factor genes (3/13 vs 9/29, respectively). Nine CCUS patients had serial samples sequenced (median 2 serial samples, range 1-5) at a median follow-up of 539 days (range, 84-4066 days). In CCUS patients with serial samples, there was minimal change in the founding clone (0.16%, range -3.7-23.9%) or the subclone median VAFs (0.2%, range -22.4-22.2%), with only 2 patients having a founding clone or subclone VAF increase of >5%. UPN 529198 had the acquisition of a new SRSF2(P95H)-mutant subclone 4066 days after initial banking but remained with stable cytopenias and no MDS (Figure 1B). One CCUS patient developed MDS 70 days after banking with a blast count that increased from 2% to 15% but had a negligible change in the founding clone VAF (2.8% increase). However, 3 subclones did increase in size (max VAF increase of 6.1%). In summary, while the total number of mutations is similar between CCUS, MDS and sAML samples, the clonal architecture varies across diseases. CCUS has a lower total molecular disease burden and a lower proportion of patients with a subclone compared to MDS patients in our cohort. The data suggest that defining clonal architecture and incorporating subclonal complexity in the evaluation of cytopenic patients could provide an objective measure to characterize and monitor disease burden rather than relying on the presence or absence of dysplasia, especially when considering the diagnosis of CCUS and lower-risk MDS.
Background Relapse in patients with acute myeloid leukemia (AML) has been, in part, attributed to the ability of leukemic blasts to escape immune surveillance through mechanisms such as MHC class II ...antigen modulation, alteration of the cytokine milieu, and upregulation of inhibitory and immune checkpoint ligands that promote T-cell suppression. Yet, immune checkpoint inhibitors (ICI) used in AML have yielded variable and unpredictable results. Moreover, evaluation of T-cell function is not part of the diagnostic assessment of patients with AML. Methods To better characterize the T-cell dysfunction in AML, we conducted a prospective study on freshly harvested, bone marrow aspirates from patients with AML at presentation, collected under an Institutional IRB-approved banking protocol over the past two years. Using a multiparameter flow cytometry-based assay, we analyzed T-cell activation potential in response to CD3/CD28 (bead-based), T-cell receptor (TCR) stimulation, both alone (n= 40 AMLs and 10 healthy donors) or in combination with a library of ICI (n=15 AML and 5 healthy donors). We selected OX40 (CD134/TNFRSF4) and ICOS (Inducible T-cell costimulator) as surrogate markers for T-cell activation based on their expression pattern post bead-based stimulation in healthy donors' bone marrows. The ICI library comprised 31 therapeutic antibody inhibitors of known immune checkpoints, signaling pathways, and chemokines reported to be implicated in T-cell suppression in AML and other solid tumors. Results Healthy T-cells displayed the highest surface expression of OX40 and ICOS at 5 days post-bead-stimulation, which was selected as the endpoint for our in vitro analyses. We found that bone marrow-derived T-cells from healthy donors upregulated the expression of the markers ICOS and/or OX40 in at least 20% of T-cells post-stimulation (n= 10, ICOS: median 36.9%, range 20-72%; OX40: median 59.3%, range 21.4 -83%). Overall, AML samples displayed a lower degree of ICOS and OX40 T-cell expression in response to TCR stimulation (n=40, ICOS: median 20.5%, range 4-65%; OX-40: median 30.3%, range 0.3-80%). Based on this result, T-cell activation was defined as >20% CD3+ cells ICOS+/OX-40+ with stimulation, with greater than 2-fold changes from baseline ICOS and OX40 expression. Using this criterion, patients with AML were then classified as activators or non-activators. T-cell activation status correlated with ELN 2022 risk (1) (Figure 1A). For the intermediate risk group, we observed that the activators group had a significantly longer relapse-free survival when compared to the non-activators (Figure 1B, n=10, Mantel-Cox log-rank = 0.02, follow up 400 days at the time of writing). While a similar trend was observed in the favorable and the poor risk groups, it did not reach significance in these ELN categories. To assess if the T-cell activation could be rescued or augmented upon immune checkpoint inhibition, we tested the effect of each of the 31 compounds of the ICI library on the degree of T-cell activation in diagnosis bone marrow aspirates of 15 patients (7 activators and 8 non activators) and in 5 healthy donors. Matched peripheral blood (PB) samples were available for 5 of the 15 patients with AML. All tested normal donors reached the threshold for T-cell activation, which was not significantly affected by ICI treatment. The 7 AML activators behaved similarly to the healthy donors. In 2 of the 8 AML non-activators, selected ICI rescued T-cell activation in absence of TCR stimulation, while in the remaining 6 cases, T-cell activation was not achieved under any condition. Similar, but overall dampened activation trends, were observed in PB samples. Conclusions Consistent with the findings of our previous retrospective study (2), the ongoing prospective analysis of T-cell activation of diagnosis bone marrows of patients with AML suggests that testing TCR-mediated T-cell activation potential at diagnosis may help refine risk stratification of the heterogenous ELN intermediate risk group. AML immune suppression seems to be reversible in a minority of cases with the use of selected ICI inhibitors. References 1. Blood(2022) 140 (12): 1345-1377 2. Proc Natl Acad Sci U S A. 2021 Dec 7;118(49)
Alterations in epigenetic regulators are increasingly recognized as early events in tumorigenesis; thus, patients with acquired or inherited variants in epigenetic regulators may be at increased risk ...for developing multiple types of cancer. DNMT3A overgrowth syndrome (DOS), caused by germline pathogenic variants in the DNA methyltransferase gene
, has been associated with a predisposition toward development of hematopoietic and neuronal malignancies. DNMT3A deficiency has been described to promote keratinocyte proliferation in mice. Although altered DNA methylation patterns are well-recognized in melanoma, the role of DNA methyltransferases in melanoma pathogenesis is not clear. We report the case of an adult DOS patient with a germline
loss-of-function mutation, who developed an early-onset melanoma with regional lymph node metastatic disease. Exome sequencing of the primary tumor identified an additional acquired, missense
mutation in the dominant tumor clone, suggesting that the loss of DNMT3A function was relevant for the development of this tumor.
Background: Previous studies indicate that mutations in signaling (e.g., receptor tyrosine kinases and RAS pathway members) and transcription factor genes are more common in secondary acute myeloid ...leukemia (sAML) than myelodysplastic syndrome (MDS), suggesting a role in disease progression. However, our understanding of the timing and order of mutation acquisition in these genes remains incomplete without analyzing paired MDS and sAML samples from the same patient. Defining the role of signaling gene mutations during progression should provide biologic insight into clonal evolution and help define prognostic markers for MDS progression.
Methods: We banked paired MDS and sAML (and matched skin) samples from 44 patients (median time to progression: 306 days, range 21-3568). We sequenced 44 sAML (+ skin) samples for 285 recurrently mutated genes (RMGs) and 12 samples were selected for enhanced whole genome sequencing (eWGS, genome with deep exome coverage) of MDS and sAML samples (+ skin) to determine clonal hierarchy. Somatic mutations in these 12 samples were validated with high coverage error-corrected sequencing, and clonality was defined in MDS and sAML samples using mutation variant allele frequencies (VAFs). Additionally, error-corrected sequencing for all sAML RMG mutations, plus 40 additional genes, was performed on 43 of the MDS samples. Single cell DNA sequencing (scDNAseq, Mission Bio) was performed on 6 samples.
Results: We identified 32 signaling gene mutations in 15 of the 44 sAML samples, with only 11 of 32 mutations (34%) detected in the initial, paired MDS sample (limit of detection; <0.1% VAF). This was significantly less than the percentage of sAML transcription factor gene mutations present at MDS (17 of 23, 74%, p=0.006). We used eWGS data to define clonal hierarchies for 12 patients, and found that both signaling and transcription factor gene mutations were in subclones (9 of 9, and 7 of 8 clones, respectively), with signaling gene mutations occurring as terminal events during clonal evolution. Finally, 8 of 9 subclones with signaling gene mutations expanded at progression. Together, the data confirm that both signaling and transcription factor mutations occur in subclones, but with a preferred order of mutation acquisition.
We next asked if low-level (<1% VAF) signaling gene mutations were present in MDS samples. Using error-corrected sequencing, we identified 22 signaling gene mutations that were present at MDS and absent at sAML (avg VAF: 0.8%; range 0.05%-11.7%). Combined with sAML-defined signaling genes, 33 total signaling gene mutations were detected at MDS in 19 patients, but only 11 (33%) were present after progression. We observed 5 distinct patterns of clonal evolution for signaling genes: 1) MDS mutations persist and expand at sAML (n=6), 2) ≥2 mutations are present at MDS, at least one mutation persists (and expands) and another contracts at sAML (n=4), 3) MDS mutations contract and a new mutation emerges at sAML (n=2), 4) MDS mutations collapse at sAML (n=7), and 5) no MDS mutations, but ≥1 mutation emerges at sAML (n=5). These diverse patterns of clonal evolution suggest that MDS cells undergo strong selective pressure to acquire a signaling gene mutation, but only mutations in the correct context contribute to progression.
Finally, we observed that several MDS (n=6) and sAML (n=10) samples had multiple signaling gene mutations, and it was not always clear whether they occurred in the same subclone. We performed scDNAseq of 6 sAML samples with multiple signaling gene mutations (2-4/case). In 5 of 6 cases the signaling gene mutations did not occur in the same subclone. One sample contained 2 subclones with a NRAS and a PTPN11 mutation, with a separate subclone harboring an additional NRAS mutation. In sum, the co-occurrence of two signaling gene mutations in the same subclone is rare, indicating that the presence of multiple signaling gene mutations may be functionally redundant or detrimental to leukemia cells.
Conclusions: Rare cells containing signaling gene mutations are present in nearly half of MDS patients who progress to sAML. The high frequency of signaling gene mutations and diverse patterns of clonal evolution (including the loss of one mutation and acquisition of another), suggest that signaling genes are a major driver of progression to sAML. The paucity of subclones with multiple signaling gene mutations suggests a therapeutic vulnerability for mutant cells.
DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Jacoby:AbbVie: Research Funding; Jazz Pharmaceuticals: Research Funding.
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