A multi-center analysis of individuals with a 47,XXY/46,XX karyotype.

INTRODUCTION: Klinefelter syndrome (KS), a sex chromosome aneuploidy, is associated with a 47,XXY chromosomal complement and is diagnosed in ∼1:600 live male births. Individuals with a 46,XX cell line in addition to 47,XXY are less common with a limited number of published case reports. METHODOLOGY: To better understand the implications of a 47,XXY/46,XX karyotype, we conducted a retrospective, multi-center analysis of the cytogenetic findings and associated clinical records of 34 patients diagnosed with this SCA across 14 institutions. RESULTS: Presence of the XX cell line ranged from 5-98% in patient specimens. Phenotypes also exhibited significant heterogeneity with some reporting a single reason for referral and others presenting with a constellation of symptoms, including ambiguous genitalia and ovotestes. Ovotestes were present in 12% of individuals in this cohort, who had a significantly higher percentage of XX cells. Notably, two patients were assigned female sex at birth DISCUSSION: These findings highlight the variability of the clinical phenotypes associated with this SCA as well as the challenges of clinical management for this population. Karyotype or FISH analysis, which offer single-cell resolution, rather than chromosomal microarray or molecular testing, is the ideal test strategy in these instances as mosaicism can occur at low levels.

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma.

INTRODUCTION: NK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. "suicide mechanisms" regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. OBJECTIVES: We sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. METHODS: Primary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. RESULTS: AZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. CONCLUSIONS: mTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Detection of chromosomal alteration after infusion of gene-edited allogeneic CAR T cells.

A chromosome 14 inversion was found in a patient who developed bone marrow aplasia following treatment with allogeneic chimeric antigen receptor (CAR) Tcells containing gene edits made with transcription activator-like effector nucleases (TALEN). TALEN editing sites were not involved at either breakpoint. Recombination signal sequences (RSSs) were found suggesting recombination-activating gene (RAG)-mediated activity. The inversion represented a dominant clone detected in the context of decreasing absolute CAR Tcell and overall lymphocyte counts. The inversion was not associated with clinical consequences and wasnot detected in the drug product administered to this patient or in any drug product used in this or other trials using the same manufacturing processes. Neither was the inversion detected in this patient at earlier time points or in any other patient enrolled in this or other trials treated with this or other product lots. This case illustrates that spontaneous, possibly RAG-mediated, recombination events unrelated to gene editing can occur in adoptive cell therapy studies, emphasizes the need for ruling out off-target gene editing sites, and illustrates that other processes, such as spontaneous V(D)J recombination, can lead to chromosomal alterations in infused cells independent of gene editing.

Pediatric Donor Cell Acute Lymphoblastic Leukemia Following Bone Marrow Transplant for GATA2 Mutation.

Donor cell leukemia is a rare complication following hematopoietic stem cell transplant (HSCT). There are currently few reports in children and only rare, reported cases of donor-derived myelodysplastic syndrome/acute myeloid leukemia in patients with an underlying germline GATA2 mutation. Most reported cases are myeloid in origin and occur following related HSCT. We present a 3-year-old female who developed a donor-derived B-cell acute lymphoblastic leukemia 2 years post unrelated HSCT for GATA2 germline mutation.

STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

Mosaic trisomy 1q: a recurring chromosome anomaly that is a diagnostic challenge and is associated with a Fryns-like phenotype.

OBJECTIVE: Trisomy of the long arm of chromosome 1 is a very rare cytogenetic anomaly that is difficult to diagnose because of tissue-limited mosaicism. This study aimed to further characterize the prenatal and post-natal findings associated with this anomaly, including the first reported chromosomal microarray finding. METHOD: This is a retrospective study of six cases of mos 46,X,der(Y)t(Y;1)(q12;q21)/46,XY, diagnosed both prenatally and post-natally. Detailed clinical features and pregnancy outcome were documented. RESULTS: Recurrent prenatal and post-natal features of our case series, as well as the previously reported cases, were described, suggesting a Fryns-like phenotype. A diagnosis of mosaic trisomy 1q is difficult to confirm post-natally in some cases because of the tissue provided for analysis, emphasizing the need to study multiple tissue types in cases of fetal loss with a suspected underlying chromosomal imbalance. CONCLUSION: The overlap of clinical features between mosaic trisomy 1q and Fryns syndrome emphasizes the need to obtain appropriate samples for genetic analysis. The present cases and a review of the literature suggest that partial trisomy of the long arm of chromosome 1 is a distinct de novo clinical entity with low recurrence risk. © 2017 John Wiley & Sons, Ltd.

Fusion tyrosine kinase NPM-ALK Deregulates MSH2 and suppresses DNA mismatch repair function novel insights into a potent oncoprotein.

The fusion tyrosine kinase NPM-ALK is central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL). We recently identified that MSH2, a key DNA mismatch repair (MMR) protein integral to the suppression of tumorigenesis, is an NPM-ALK-interacting protein. In this study, we found in vitro evidence that enforced expression of NPM-ALK in HEK293 cells suppressed MMR function. Correlating with these findings, six of nine ALK(+)ALCL tumors displayed evidence of microsatellite instability, as opposed to none of the eight normal DNA control samples (P = 0.007, Student's t-test). Using co-immunoprecipitation, we found that increasing levels of NPM-ALK expression in HEK293 cells resulted in decreased levels of MSH6 bound to MSH2, whereas MSH2·NPM-ALK binding was increased. The NPM-ALK·MSH2 interaction was dependent on the activation/autophosphorylation of NPM-ALK, and the Y191 residue of NPM-ALK was a crucial site for this interaction and NPM-ALK-mediated MMR suppression. MSH2 was found to be tyrosine phosphorylated in the presence of NPM-ALK. Finally, NPM-ALK impeded the expected DNA damage-induced translocation of MSH2 out of the cytoplasm. To conclude, our data support a model in which the suppression of MMR by NPM-ALK is attributed to its ability to interfere with normal MSH2 biochemistry and function.

Fine-needle aspiration diagnosis of clonal extramedullary hematopoiesis in a case of myeloproliferative neoplasm.

Extramedullary hematopoiesis (EMH)-the proliferation of hematopoietic progenitors outside of the bone marrow (BM) is a well-known phenomenon in myeloproliferative neoplasms (MPN). Abundant literature describes EMH at various body sites in cases of MPN, and some studies showed the presence of cytogenetic changes associated with MPN in the EMH tissues. We present a case of an 80-year-old female, with a history of MPN, presenting with mediastinal adenopathy. The transbronchial fine-needle aspiration (FNA) of the mediastinal lymph node showed EMH with atypical megakaryocytes and del(13q) demonstrated by fluorescence in situ hybridization. The subsequent BM biopsy demonstrated myelofibrosis with atypical megakaryocytes harboring the same cytogenetic abnormality. Our case highlights the capability of FNA cytology for providing accurate morphologic, immunohistochemical, and cytogenetic diagnosis of clonal EMH.

A complex KMT2A::AFF3 fusion resulting from a three-way chromosomal rearrangement in pediatric B lymphoblastic leukemia.

The KMT2A::AFF3 fusion, t(2;11)(q11.2;q23.2), is a very rare fusion occurring in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Our patient is a 2-year-old male who presented with three weeks of intermittent fever. Bone marrow biopsy showed 82% blasts and cytogenetic analysis demonstrated a complex 3-way chromosomal rearrangement involving KMT2A and an unknown fusion partner. Molecular testing identified the fusion partner as AFF3, a FLT3-TKD non-D835 mutation, and an NF1 mutation. This case demonstrates a highly complex three-way variant translocation resulting in the rare KMT2A::AFF3 fusion with only a few cases previously described in the literature.

It Takes a Village: Providing International Pediatric Pathology Services in a Resource-Limited Setting

OBJECTIVES: Partnerships between low- to middle-income countries (LMICs) and high-income countries (HICs) is one strategy to mitigate observed health disparities. Cambodia's Angkor Hospital for Children (AHC), an LMIC institution, faces shortages in health care resources, including pathology services. A partnership was created with Children's Wisconsin (CW), an HIC hospital, including provision of pathology services. We describe our established pathology workflow, examine cases seen in AHC patients, and evaluate the impact of CW's interpretations. METHODS: AHC provides clinical history and impression and ships samples to CW, which processes the samples, and pathologists provide interpretations, sending reports electronically to AHC. For analysis, final diagnoses were considered "concordant," "refined," or "discordant" based on agreement with the clinical impression. Cases were also classified as "did not change management" or "changed management" based on how CW interpretation affected clinical management. RESULTS: We included 347 specimens (177 malignant, 146 benign, 24 insufficient for diagnosis). Of these cases, 31% were discordant and 44% of cases with clinical follow-up had a change in management with CW interpretation. CONCLUSIONS: Inclusion of pathology services in LMIC-HIC partnerships is crucial for resolving health disparities between the institutions involved. The described partnership and established pathology workflow can be adapted to the needs and resources of many institutions.

Patient-Derived Ovarian Cancer Spheroids Rely on PI3K-AKT Signaling Addiction for Cancer Stemness and Chemoresistance.

Ovarian cancer is the most lethal gynecological malignancy among women worldwide and is characterized by aggressiveness, cancer stemness, and frequent relapse due to resistance to platinum-based therapy. Ovarian cancer cells metastasize through ascites fluid as 3D spheroids which are more resistant to apoptosis and chemotherapeutic agents. However, the precise mechanism as an oncogenic addiction that makes 3D spheroids resistant to apoptosis and chemotherapeutic agents is not understood. To study the signaling addiction mechanism that occurs during cancer progression in patients, we developed an endometrioid subtype ovarian cancer cell line named 'MCW-OV-SL-3' from the ovary of a 70-year-old patient with stage 1A endometrioid adenocarcinoma of the ovary. We found that the cell line MCW-OV-SL-3 exhibits interstitial duplication of 1q (q21-q42), where this duplication resulted in high expression of the PIK3C2B gene and aberrant activation of PI3K-AKT-ERK signaling. Using short tandem repeat (STR) analysis, we demonstrated that the cell line exhibits a unique genetic identity compared to existing ovarian cancer cell lines. Notably, the MCW-OV-SL-3 cell line was able to form 3D spheroids spontaneously, which is an inherent property of tumor cells when plated on cell culture dishes. Importantly, the tumor spheroids derived from the MCW-OV-SL-3 cell line expressed high levels of c-Kit, PROM1, ZEB1, SNAI, VIM, and Twist1 compared to 2D monolayer cells. We also observed that the hyperactivation of ERK and PI3K/AKT signaling in these cancer cells resulted in resistance to cisplatin. In summary, the MCW-OV-SL3 endometrioid cell line is an excellent model to study the mechanism of cancer stemness and chemoresistance in endometrioid ovarian cancer.

Correction: Parashar et al. Patient-Derived Ovarian Cancer Spheroids Rely on PI3K-AKT Signaling Addiction for Cancer Stemness and Chemoresistance. Cancers 2022, 14, 958.

The authors would like to correct the author byline to include Dr [...].

Failure of NIPT to detect constitutional chromoanasynthesis involving chromosome 21 in a case of fetal hydrops-A case report.

We report a case of a de novo ring 21 complex chromosomal rearrangement in a fetus presenting with hydrops. Noninvasive prenatal testing (NIPT) failed to detect the imbalance. This case highlights the need to understand the various limitations and strengths of NIPT technology when counseling patients.

Identification of two novel phenotypically distinct breast cancer cell subsets based on Sox2 transcription activity.

Sox2 (sex-determining region Y-box protein 2) is a transcription factor regulating pluripotency in embryonic stem cells. Sox2 is aberrantly expressed in breast and other cancers, though its biological significance remains widely unexplored. To understand the significance of this aberrancy, we assessed the transcription activity of Sox2 in two Sox2-expressing breast cancer cell lines, MCF7 and ZR751, using a lentiviral Sox2 GFP reporter vector. Surprisingly, Sox2 transcription activity, as measured by GFP expression encoded in a Sox2 reporter construct, was detectable only in a small subset of cells in both cell lines. Purification of GFP+ cells (cells with Sox2 activity) and GFP- cells (cells without Sox2 activity) was enriched for two phenotypically distinct cell populations in both MCF7 and ZR751 cell lines. Specifically, GFP+ cells formed significantly more colonies in methylcellulose and more mammospheres in vitro compared to GFP- cells. These phenotypic differences are directly linked to Sox2 as siRNA knockdown of Sox2 in GFP+ cells abolished these abilities. To provide a mechanistic explanation to our observations, we performed gel shift and chromatin immunoprecipitation studies; Sox2 was found to bind to its DNA binding consensus sequence and the promoters of Cyclin D1 and Nanog (two known Sox2 downstream targets) only in GFP+ cells. GFP+ cells also up-regulated CD49f, phospho-GSK3ß, and ß-catenin. In summary, we have identified two novel phenotypically distinct cell subsets in two breast cancer cell lines based on their differential Sox2 transcription activity. We demonstrate that Sox2 transcription activity, and not its protein expression alone, underlies the tumorigenicity and cancer stem cell-like phenotypes in breast cancers.