Therapy-Related Acute Promyelocytic Leukemia: A Case Report and a Review of Literature.

Acute promyelocytic leukemia (APL) is a subgroup of acute myeloid leukemia (AML), and while not a common form of cancer, it does make up a modest portion of acute leukemia. The genetic hallmark of APL is the t(15;17)(q24.1;q21.2) promyelocytic leukemia/retinoic acid receptor alpha (PML/RARA) protein. We present the case of a patient who had undergone prior therapy for stage IIIC squamous cell carcinoma of the anorectal region with 5-fluorouracil, mitomycin C, and radiation and developed therapy-related acute promyelocytic leukemia about 18 months later. We also review the clinical features and management of APL while also highlighting that therapy-related APL, although uncommon, can develop from chemoradiation. The specific diagnosis of therapy-related APL is its own distinct diagnosis, but its treatment remains the same as primary APL.

The Intestinal Tract Brush Border in Young Children Uniformly Expresses Guanylate Cyclase C.

The present study examined staining of guanylate cyclase C (GCC/GUCY2C) in the small and large intestines of children younger than age 7 years. Normal intestinal tissue from children aged 0 to 7 years was stained using GCC, uroguanylin, and villin antibodies and scored for staining intensity. A subset underwent quantitative real-time polymerase chain reaction. Data were analyzed using t test of independent means, descriptive statistics, and logistic regression. Four hundred sixty-four specimens underwent immunohistochemistry; 291 specimens underwent real-time polymerase chain reaction. GCC, villin, and uroguanylin were detected across age groups and anatomic sites. No significant differences were identifiable across age groups. GUCY2C and uroguanylin mRNA was detected in all samples, with no variability of statistical significance of either target-to-villin normalization between any age cohorts. A gradient of expression of GCC across age groups does not seem to exist.

Mild Idiopathic Infantile Hypercalcemia-Part 1: Biochemical and Genetic Findings.

CONTEXT: Idiopathic infantile hypercalcemia (IIH), an uncommon disorder characterized by elevated serum concentrations of 1,25 dihydroxyvitamin D (1,25(OH)2D) and low parathyroid hormone (PTH) levels, may present with mild to severe hypercalcemia during the first months of life. Biallelic variants in the CYP24A1 or SLC34A1 genes are associated with severe IIH. Little is known about milder forms. OBJECTIVE: This work aims to characterize the genetic associations and biochemical profile of mild IIH. METHODS: This is a cross-sectional study including children between age 6 months and 17 years with IIH who were followed in the Calcium Clinic at the Hospital for Sick Children (SickKids), Toronto, Canada. Twenty children with mild IIH on calcium-restricted diets were evaluated. We performed a dietary assessment and analyzed biochemical measures including vitamin D metabolites and performed a stepwise molecular genetic analysis. Complementary biochemical assessments and renal ultrasounds were offered to first-degree family members of positive probands. RESULTS: The median age was 16 months. Median serum levels of calcium (2.69 mmol/L), urinary calcium:creatinine ratio (0.72 mmol/mmol), and 1,25(OH)2D (209 pmol/L) were elevated, whereas intact PTH was low normal (22.5 ng/L). Mean 1,25(OH)2D/PTH and 1,25(OH)2D/25(OH)D ratios were increased by comparison to healthy controls. Eleven individuals (55%) had renal calcification. Genetic variants were common (65%), with the majority being heterozygous variants in SLC34A1 and SLC34A3, while a minority showed variants of CYP24A1 and other genes related to hypercalciuria. CONCLUSION: The milder form of IIH has a distinctive vitamin D metabolite profile and is primarily associated with heterozygous SLC34A1 and SLC34A3 variants.

Oncostatin-M Does Not Predict Treatment Response in Inflammatory Bowel Disease in a Pediatric Cohort.

OBJECTIVES: This study aimed to determine whether mRNA expression of oncostatin-M (OSM) and its receptor (OSMR) in initial, pre-treatment intestinal biopsies is predictive of response to tumor necrosis factor antagonists (anti-TNF) in a pediatric inflammatory bowel disease (IBD) cohort. Secondary outcomes correlated OSM and OSMR expression with demographic variables; IBD type, extent, phenotype, and severity; laboratory values; and endoscopic findings. METHODS: A retrospective chart review was conducted on 98 pediatric patients. Patients' clinical courses were stratified as follows: failed anti-TNF (n = 14), quiescent on anti-TNF (n = 36), anti-TNF naïve (n = 19), and age-matched non-IBD controls (n = 29). The mRNA from each patient's pre-treatment ileal or colonic biopsy was isolated, and expression of OSM and OSMR was analyzed. RESULTS: There was no difference in OSM or OSMR expression among the three IBD groups; however, expression was significantly higher in patients with IBD than non-IBD controls (P < 0.001). OSM and OSMR were more highly expressed in patients with ulcerative colitis (UC) with a Mayo score of 3 (P = 0.0092 and P = 0.0313, respectively). High OSM expression correlated with severe disease activity indices at diagnosis (P = 0.002), anemia at diagnosis (P = 0.0236), and need for immunomodulators (P = 0.0193) and steroids (P = 0.0273) during patients' clinical courses. CONCLUSIONS: OSM and OSMR expression were not predictive of response to anti-TNF in our pediatric cohort. OSM expression did correlate with IBD compared with healthy controls as well as with several clinical indicators of severe IBD.

Detection of SMN1 to SMN2 gene conversion events and partial SMN1 gene deletions using array digital PCR.

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutations of survival motor neuron 1 (SMN1) but retention of one or more copies of the paralog SMN2. Within the SMA population, there is substantial variation in SMN2 copy number (CN); in general, those individuals with SMA who have a high SMN2 CN have a milder disease. Because SMN2 functions as a disease modifier, its accurate CN determination may have clinical relevance. In this study, we describe the development of array digital PCR (dPCR) to quantify SMN1 and SMN2 CNs in DNA samples using probes that can distinguish the single nucleotide difference between SMN1 and SMN2 in exon 8. This set of dPCR assays can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 CN and disease severity. We can detect SMN1-SMN2 gene conversion events in DNA samples by comparing CNs at exon 7 and exon 8. Partial deletions of SMN1 can also be detected with dPCR by comparing CNs at exon 7 or exon 8 with those at intron 1. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 CNs from SMA samples as well as identify gene conversion events and partial deletions of SMN1.

Molecular Genetic Testing for Kidney Disorders During the COVID-19 Pandemic.

Chronic kidney disease (CKD) has major morbidity and mortality for children and adults. While in adults CKD often is associated with diabetic complications, genetic variants can be the underlying cause in both populations. Beginning in 2016 with the emergence of more affordable next-generation sequencing (NGS) technologies, the Molecular Diagnostics Lab at Nemours Children's Hospital-Delaware developed the first clinically actionable pediatric NGS kidney panel comprised of 46 genes including APOL1. Apolipoprotein L1 (APOL1) associated nephropathy is reported along a spectrum of non-diabetic kidney disease. It is significantly associated with two "risk alleles" defined as G1 and G2 and typically found in individuals of African descent. In early 2020, as COVID-19 spread across the globe, reports of patients with kidney failure began to emerge. A collapsing glomerulopathy in Black patients with COVID-19 was found to be associated with the APOL1 predisposition of the known G1 and/or G2 risk variants. We identified genetic variants in 11 genes (NPHS1; NPHS2; LAMB2; WT1; COL4A4; COL4A5; COQ8B; CUBN; MEFV; PMM2; SMARCAL1) known to be associated with pediatric onset nephrotic syndrome, or detection of the high-risk haplotype of APOL1, in the majority (78%) of patients tested. These clinically actionable results guided medical care and improved patient outcomes.

The novel duplication HRAS c.186_206dup p.(Glu62_Arg68dup): clinical and functional aspects.

Specific activating missense HRAS variants cause Costello syndrome (CS), a RASopathy with recognizable facial features. The majority of these dominant disease causing variants affect the glycine residues in position 12 or 13. A clinically suspected CS diagnosis can be confirmed through identification of a dominant pathogenic HRAS variant. A novel HRAS variant predicting p.(Glu62_Arg68dup) was identified in an individual with hypertrophic cardiomyopathy, Chiari 1 malformation and ectodermal findings consistent with a RASopathy. Functional studies showed that the p.Glu62_Arg68dup alteration affects HRAS interaction with effector protein PIK3CA (catalytic subunit of phosphoinositide 3-kinase) and the regulator neurofibromin 1 (NF1) GTPase-activating protein (GAP). HRASGlu62_Arg68dup binding with effectors rapidly accelerated fibrosarcoma (RAF1), RAL guanine nucleotide dissociation stimulator (RALGDS) and phospholipase C1 (PLCE1) was enhanced. Accordingly, p.Glu62_Arg68dup increased steady-state phosphorylation of MEK1/2 and ERK1/2 downstream of RAF1, whereas AKT phosphorylation downstream of PI3K was not significantly affected. Growth factor stimulation revealed that expression of HRASGlu62_Arg68dup abolished the HRAS' capacity to modulate downstream signaling. Our data underscore that different qualities of dysregulated HRAS-dependent signaling dynamics determine the clinical severity in CS.

Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer.

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.

Medically actionable comorbidities in adults with Costello syndrome.

Costello syndrome (CS) is an autosomal-dominant condition caused by activating missense mutations in HRAS. There is little literature describing health concerns specific to adults with CS. Parents of individuals with CS need to know what to anticipate as their children age. We surveyed a group of 20 adults and older adolescents with CS regarding their medical concerns and lifestyle characteristics. We identified several previously undescribed actionable medical concerns in adults with CS. First, the high prevalence of anxiety in this cohort indicates that screening for anxiety is warranted since this is a treatable condition that can have a significant impact on quality of life. Second, adults with CS should be monitored for progressive contractures or other problems that could decrease mobility. This is especially important in a population that seems to have increased risk for osteopenia. Finally, the lack of cancer diagnoses in adulthood is of interest, although the cohort is too small to draw definitive conclusions about cancer risk in adults with CS. Ongoing follow-up of the current cohort of adults with CS is necessary to delineate progressive medical and physical problems, which is essential for providing targeted management recommendations and anticipatory guidance to families.

GGC Repeat Expansion and Exon 1 Methylation of XYLT1 Is a Common Pathogenic Variant in Baratela-Scott Syndrome.

Baratela-Scott syndrome (BSS) is a rare, autosomal-recessive disorder characterized by short stature, facial dysmorphisms, developmental delay, and skeletal dysplasia caused by pathogenic variants in XYLT1. We report clinical and molecular investigation of 10 families (12 individuals) with BSS. Standard sequencing methods identified biallelic pathogenic variants in XYLT1 in only two families. Of the remaining cohort, two probands had no variants and six probands had only a single variant, including four with a heterozygous 3.1 Mb 16p13 deletion encompassing XYLT1 and two with a heterozygous truncating variant. Bisulfite sequencing revealed aberrant hypermethylation in exon 1 of XYLT1, always in trans with the sequence variant or deletion when present; both alleles were methylated in those with no identified variant. Expression of the methylated XYLT1 allele was severely reduced in fibroblasts from two probands. Southern blot studies combined with repeat expansion analysis of genome sequence data showed that the hypermethylation is associated with expansion of a GGC repeat in the XYLT1 promoter region that is not present in the reference genome, confirming that BSS is a trinucleotide repeat expansion disorder. The hypermethylated allele accounts for 50% of disease alleles in our cohort and is not present in 130 control subjects. Our study highlights the importance of investigating non-sequence-based alterations, including epigenetic changes, to identify the missing heritability in genetic disorders.

Cytotoxicity of Zardaverine in Embryonal Rhabdomyosarcoma from a Costello Syndrome Patient.

Costello syndrome (CS) patients suffer from a very high 10% incidence of embryonal rhabdomyosarcoma (ERMS). As tools to discover targeted therapeutic leads, we used a CS patient-derived ERMS cell line (CS242 ERMS) harboring a homozygous p.G12A mutation in HRAS, and a control cell line derived from the same patient comprising non-malignant CS242 fibroblasts with a heterozygous p.G12A HRAS mutation. A library of 2,000 compounds with known pharmacological activities was screened for their effect on CS242 ERMS cell viability. Follow-up testing in a panel of cell lines revealed that various compounds originally developed for other indications were remarkably selective; notably, the phosphodiesterase (PDE) inhibitor zardaverine was at least 1,000-fold more potent in CS242 ERMS than in the patient-matched non-malignant CS242 fibroblasts, other ERMS, or normal fibroblasts. Chronic treatment with zardaverine led to the emergence of resistant cells, consistent with CS242 ERMS comprising a mixed population of cells. Many PDE inhibitors in addition to zardaverine were tested on CS242 ERMS, but almost all had no effect. Interestingly, zardaverine and analogs showed a similar cytotoxicity profile in CS242 ERMS and cervical carcinoma-derived HeLa cells, suggesting a mechanism of action common to both cell types that does not require the presence of an HRAS mutation (HeLa contains wild type HRAS). Two recent studies presented possible mechanistic explanations for the cytotoxicity of zardaverine in HeLa cells. One revealed that zardaverine inhibited a HeLa cell-based screen measuring glucocorticoid receptor (GR) activation; however, using engineered HeLa cells, we ruled out a specific effect of zardaverine on signaling through the GR. The second attributed zardaverine toxicity in HeLa cells to promotion of the interaction of phosphodiesterase 3A and the growth regulatory protein Schlafen 12. We speculate that this work may provide a possible mechanism for zardaverine action in CS242 ERMS, although we have not yet tested this hypothesis. In conclusion, we have identified zardaverine as a potent cytotoxic agent in a CS-derived ERMS cell line and in HeLa. Although we have ruled out some possibilities, the mechanism of action of zardaverine in CS242 ERMS remains to be determined.

Tumor lysis syndrome and metastatic melanoma.

Tumor lysis syndrome (TLS) is a potential emergent complication of oncologic treatment. TLS is commonly reported in hematological malignancies with rapid cell turnover rates, but is relatively rare in solid tumors. TLS is most frequently a result of cancer treatment in combination with a large tumor burden, but has occasionally been reported to occur spontaneously, especially in cases of advanced or metastatic disease. In this article, we describe the case of a patient with newly diagnosed metastatic melanoma that developed TLS two days after initiation of corticosteroids. In addition, we present a brief literature review of melanoma-associated TLS and review the etiology, diagnosis, and management of TLS.

Paternal uniparental disomy with segmental loss of heterozygosity of chromosome 11 are hallmark characteristics of syndromic and sporadic embryonal rhabdomyosarcoma.

Costello syndrome (CS) arises from a typically paternally derived germline mutation in the proto-oncogene HRAS, and is considered a rasopathy. CS results in failure-to-thrive, intellectual disabilities, short stature, coarse facial features, skeletal abnormalities, congenital heart disease, and a predisposition for cancer, most commonly embryonal rhabdomyosarcoma (ERMS). The goal of this study was to characterize CS ERMS at the molecular level and to determine how divergent it is from sporadic ERMS. We characterized eleven ERMS tumors from eight unrelated CS patients, carrying paternally derived HRAS c.34G>A (p.Gly12Ser; 6) or c.35G>C (p.Gly12Ala; 2) mutations. Loss of heterozygosity (LOH) was evaluated in all CS ERMS by microarray and/or short tandem repeat (STR) markers spanning the entire chromosome 11. Eight CS ERMS tumors displayed complete paternal uniparental disomy of chromosome 11 (pUPD11), whereas two displayed UPD only at 11p and a second primary ERMS tumor showed UPD limited to 11p15.5, the classical hallmark for ERMS. Three sporadic ERMS cell lines (RD, Rh36, Rh18) and eight formalin fixed paraffin embedded (FFPE) ERMS tumors were also analyzed for RAS mutations and LOH status. We found a higher than anticipated frequency of RAS mutations (HRAS or NRAS; 50%) in sporadic ERMS cell lines/tumors. Unexpectedly, complete uniparental disomy (UPD11) was observed in five specimens, while the other six showed LOH extending across the p and q arms of chromosome 11. In this study, we are able to clearly demonstrate complete UPD11 in both syndromic and sporadic ERMS. © 2016 Wiley Periodicals, Inc.