Feasibility and Clinical Utility of Reporting Hereditary Cancer Predisposition Pathogenic Variants Identified in Research Germline Sequencing: A Prospective Interventional Study.

PURPOSE: Patients with cancer frequently undergo research-grade germline sequencing but clinically actionable results are not routinely disclosed. The objective of this study is to evaluate the feasibility of reporting clinically relevant secondary findings (SF) identified in germline research sequencing using the institutional molecular tumor board (MTB) and the treating oncology physician. METHODS: This prospective, interventional cohort study enrolled Total Cancer Care participants with any cancer diagnosis at a single institution. Patients underwent research-grade germline whole-exome sequencing, with bioinformatic analysis in a Clinical Laboratory Improvement Amendments-certified laboratory to verify pathogenic/likely pathogenic germline variants (PGVs) in any American College of Medical Genomics and Genetics SF v2.0 genes. After a protocol modification in consenting patients, the MTB reported PGVs to treating oncology physicians with recommendations for referral to a licensed genetic counselor and clinical confirmatory testing. RESULTS: Of the 781 enrolled participants, 32 (4.1%) harbored cancer predisposition PGVs, 24 (3.1%) were heterozygous carriers of an autosomal recessive cancer predisposition syndrome, and 14 (1.8%) had other hereditary disease PGVs. Guideline-directed testing would have missed 37.5% (12/32) of the inherited cancer predisposition PGVs, which included BRCA1, BRCA2, MSH6, SDHAF2, SDHB, and TP53 variants. Three hundred fifteen participants consented to reporting results; results for all living patients were reported to the clinical team with half referred to a licensed genetic counselor. There was concordance between all research variants identified in patients (n = 9) who underwent clinical confirmatory sequencing. CONCLUSION: MTB reporting of research-grade germline sequencing to the clinical oncology team is feasible. Over a third of PGVs identified using a universal testing strategy would have been missed by guideline-based approach, suggesting a role for expanding germline testing.

Familial 4p Interstitial Deletion Provides New Insights and Candidate Genes Underlying This Rare Condition.

Chromosome 4p deletions can lead to two distinct phenotypic outcomes: Wolf--Hirschhorn syndrome (a terminal deletion at 4p16.3) and less frequently reported proximal interstitial deletions (4p11-p16). Proximal 4p interstitial deletions can result in mild to moderate intellectual disability, facial dysmorphisms, and a tall thin body habitus. To date, only 35 cases of proximal 4p interstitial deletions have been reported, and only two of these cases have been familial. The critical region for this syndrome has been narrowed down to 4p15.33-15.2, but the underlying causative genes remain unclear. In this study, we report the case of a 3-year-old female with failure to thrive, developmental and motor delays, and morphological features. The mother also had a 4p15.2-p14 deletion, and the proband was found to have a 13.4-Mb 4p15.2-p14 deletion by chromosome microarray analysis. The deleted region encompasses 16 genes, five of which have a high likelihood of contributing to the phenotype: PPARGC1A, DHX15, RBPJ, STIM2, and PCDH7. These findings suggest that multiple genes are involved in this rare proximal 4p interstitial deletion syndrome. This case highlights the need for healthcare providers to be aware of proximal 4p interstitial deletions and the potential phenotypic manifestations.

Optimising clinical care through CDH1-specific germline variant curation: improvement of clinical assertions and updated curation guidelines.

BACKGROUND: Germline pathogenic variants in CDH1 are associated with increased risk of diffuse gastric cancer and lobular breast cancer. Risk reduction strategies include consideration of prophylactic surgery, thereby making accurate interpretation of germline CDH1 variants critical for physicians deciding on these procedures. The Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel (VCEP) developed specifications for CDH1 variant curation with a goal to resolve variants of uncertain significance (VUS) and with ClinVar conflicting interpretations and continues to update these specifications. METHODS: CDH1 variant classification specifications were modified based on updated genetic testing clinical criteria, new recommendations from ClinGen and expert knowledge from ongoing CDH1 variant curations. The CDH1 VCEP reviewed 273 variants using updated CDH1 specifications and incorporated published and unpublished data provided by diagnostic laboratories. RESULTS: Updated CDH1-specific interpretation guidelines include 11 major modifications since the initial specifications from 2018. Using the refined guidelines, 97% (36 of 37) of variants with ClinVar conflicting interpretations were resolved to benign, likely benign, likely pathogenic or pathogenic, and 35% (15 of 43) of VUS were resolved to benign or likely benign. Overall, 88% (239 of 273) of curated variants had non-VUS classifications. To date, variants classified as pathogenic are either nonsense, frameshift, splicing, or affecting the translation initiation codon, and the only missense variants classified as pathogenic or likely pathogenic have been shown to affect splicing. CONCLUSIONS: The development and evolution of CDH1-specific criteria by the expert panel resulted in decreased uncertain and conflicting interpretations of variants in this clinically actionable gene, which can ultimately lead to more effective clinical management recommendations.

Real-World Evaluation of a Population Germline Genetic Screening Initiative for Family Medicine Patients.

Hereditary factors contribute to disease development and drug pharmacokinetics. The risk of hereditary disease development can be attenuated or eliminated by early screening or risk reducing interventions. The purpose of this study was to assess the clinical utility of germline medical exome sequencing in patients recruited from a family medicine clinic and compare the mutation frequency of hereditary predisposition genes to established general population frequencies. At the University of Kentucky, 205 family medicine patients underwent sequencing in a Clinical Laboratory Improvement Amendments of 1988-compliant laboratory to identify clinically actionable genomic findings. The study identified pathogenic or likely pathogenic genetic variants-classified according to the American College of Medical Genetics and Genomics variant classification guidelines-and actionable pharmacogenomic variants, as defined by the Clinical Pharmacogenetics Implementation Consortium. Test results for patients with pharmacogenomic variants and pathogenic or likely pathogenic variants were returned to the participant and enrolling physician. Hereditary disease predisposition gene mutations in APOB, BRCA2, MUTYH, CACNA1S, DSC2, KCNQ1, LDLR, SCN5A, or SDHB were identified in 6.3% (13/205) of the patients. Nine of 13 (69.2%) underwent subsequent clinical interventions. Pharmacogenomic variants were identified in 76.1% (156/205) of patients and included 4.9% (10/205) who were prescribed a medication that had pharmacogenomic implications. Family physicians changed medications for 1.5% (3/205) of patients to prevent toxicity. In this pilot study, we found that with systemic support, germline genetic screening initiatives were feasible and clinically beneficial in a primary care setting.

Preparation of oral solid dosage forms based on homogenized spot melting technique.

Given the benefits of high printing precision and capability, the selective laser sintering technique has been used to manufacture medicines and implants with unique engineering and functional properties. Using homogenized beams with a reduced thermal gradient and a larger diameter as an alternative energy source, the thermal stability and production efficiency of powder bed fusion would be improved. Herein, a novel homogenized spot melting (HSM) technology for pharmaceutical preparation was developed in this study. The melting behavior of typical pharmaceutical polymers under a homogenized spot was determined. A crystalline polymer with a low melting point was used as a solid binder, and the HSM printability and formation of drug-loaded formulations were explored. Oral solid dosage forms with different morphological and dissolution designs were prepared and evaluated under optimal formulation and process conditions. It was observed that HSM reduced the surface temperature distribution of the powder bed and improved the printability of drugs and excipients. Crystalline PEG 8000 with suitable flowability and heat conduction efficiency in the molten state was preferable for HSM printing. Incorporating 40% PEG 8000 as a solid binder was an effective strategy for HSM processing of unfused or unstable powders. Solid preparations with different structures and dissolution behaviors were successfully printed, suggesting that HSM is a promisingtechnique for personalized medicine.

Sustained-release capsules coated via thermoforming techniques.

Capsule coatings have a wide range of applications as they afford protection to active pharmaceutical ingredients. However, few studies have focused on capsule coating owing to the sensitivity of hard gelatin shells to solvents and high temperature. In the present study, we aimed to coat capsules using two thermoforming coating techniques: vacuum forming coating (VFC) and centrifugal forming coating (CFC). Rheological and mechanical properties were investigated to comprehensively elucidate the processes and mechanisms underlying the two coating techniques. The corresponding coating integrity and drug release behavior were characterized and compared. Herein, we observed that a lower temperature was more suitable for the VFC process than the CFC process. The drug release rate decreased with the film thickness increased. Both optimal VFC and CFC capsules revealed a 24 h sustained-release property following Fick's diffusion law. The coating thickness distribution was more homogeneous for the VFC capsule than the CFC capsule. With the advantage solvent-free of functional capsule coatings, thermoforming coating techniques are convenient and efficient solutions for small-scale personalized coating of oral solid preparations.

Diagnosis of Classic Homocystinuria in Two Boys Presenting with Acute Cerebral Venous Thrombosis and Neurologic Dysfunction after Normal Newborn Screening.

Homocystinuria, caused by cystathionine ß-synthase deficiency, is a rare inherited disorder involving metabolism of methionine. Impaired synthesis of cystathionine leads to accumulation of homocysteine that affects several organ systems leading to abnormalities in the skeletal, cardiovascular, ophthalmic and central nervous systems. We report a 14-month-old and a 7-year-old boy who presented with neurologic dysfunction and were found to have cerebral venous sinus thromboses on brain magnetic resonance imaging (MRI)/magnetic resonance venogram (MRV) and metabolic and hypercoagulable work-up were consistent with classic homocystinuria. The 14-month-old boy had normal newborn screening. The 7-year-old boy initially had an abnormal newborn screen for homocystinuria but second tier test that consisted of total homocysteine was normal, so his newborn screen was reported as normal. With the advent of expanded newborn screening many treatable metabolic disorders are detected before affected infants and children become symptomatic. Methionine is the primary target in newborn screening for homocystinuria and total homocysteine is a secondary target. Screening is usually performed after 24-48 h of life in most states in the US and some states perform a second screen as a policy on all tested newborns or based on when the initial newborn screen was performed. This is done in hopes of detecting infants who may have been missed on their first screen. In the United Kingdom, NBS using dried blood spot is performed at 5 to 8 days after birth. It is universally known that methionine is a poor target and newborn screening laboratories have used different cutoffs for a positive screen. Reducing the methionine cutoff increases the sensitivity but not necessarily the specificity of the test and increasing the cutoff will miss babies who may have HCU whose levels may not be high enough to be detected at their age of ascertainment. It is not clear whether adjusting methionine level to decrease the false negative rates combined with total homocysteine as a second-tier test can be used effectively and feasibly to detect newborns with HCU. Between December 2005 and December 2020, 827,083 newborns were screened in Kentucky by MS/MS. Kentucky NBS program uses the postanalytical tools offered by the Collaborative Laboratory Integrated Reports (CLIR) project which considers gestational age and birthweight. One case of classical homocystinuria was detected and two were missed on first and second tier tests respectively. The newborn that had confirmed classical homocystinuria was one of twenty-three newborns that were referred for second tier test because of elevated methionine (cutoff is >60 µmol/L) and/or Met/Phe ratio (cutoff is >1.0); all 23 dried blood spots had elevated total homocysteine. One of the subjects of this case report had a normal methionine on initial screen and the other had a normal second-tier total homocysteine level. The performance of methionine and total homocysteine as screening analytes for homocystinuria suggest that it may be time for newborn screening programs to consider adopting next generation sequencing (NGS) platforms as alternate modality of metabolic newborn screening. Because of cost considerations, newborn screening programs may not want to adopt NGS, but the downstream healthcare cost incurred due to missed cases and the associated morbidity of affected persons far exceed costs to newborn screen programs. Since NGS is becoming more widely available and inexpensive, it may be feasible to change testing algorithms to use Newborn Metabolic NGS as the primary mode of testing on dry blood specimens with confirmation with biochemical testing. Some commercial laboratories have Newborn Screening Metabolic gene panel that includes all metabolic disorders on the most comprehensive newborn screening panel in addition to many other conditions that are not on the panel. A more targeted NGS panel can be designed that may not cost much and eventually help avoid the pitfalls associated with delayed diagnosis and cost of screening.

A Novel Approach to Improve Newborn Screening for Congenital Hypothyroidism by Integrating Covariate-Adjusted Results of Different Tests into CLIR Customized Interpretive Tools.

Newborn screening for congenital hypothyroidism remains challenging decades after broad implementation worldwide. Testing protocols are not uniform in terms of targets (TSH and/or T4) and protocols (parallel vs. sequential testing; one or two specimen collection times), and specificity (with or without collection of a second specimen) is overall poor. The purpose of this retrospective study is to investigate the potential impact of multivariate pattern recognition software (CLIR) to improve the post-analytical interpretation of screening results. Seven programs contributed reference data (N = 1,970,536) and two sets of true (TP, N = 1369 combined) and false (FP, N = 15,201) positive cases for validation and verification purposes, respectively. Data were adjusted for age at collection, birth weight, and location using polynomial regression models of the fifth degree to create three-dimensional regression surfaces. Customized Single Condition Tools and Dual Scatter Plots were created using CLIR to optimize the differential diagnosis between TP and FP cases in the validation set. Verification testing correctly identified 446/454 (98%) of the TP cases, and could have prevented 1931/5447 (35%) of the FP cases, with variable impact among locations (range 4% to 50%). CLIR tools either as made here or preferably standardized to the recommended uniform screening panel could improve performance of newborn screening for congenital hypothyroidism.

Oral preparations with tunable dissolution behavior based on selective laser sintering technique.

Selective laser sintering (SLS) sinters a powder layer by layer with a laser beam to prepare 3D printlets, which are widely used in the field of tissue engineering and personalized implants. To promote the SLS printing of oral solid preparations, the printability of commonly used drugs and excipients was evaluated using a 450 nm low energy laser. It was found that yellow drugs could absorb laser energy and sinter, while white drugs and pharmaceutical excipients had SLS printability when tartrazine lake was added as a photoabsorber. The printing mechanism of non-crystalline and crystalline polymers was powder sintering and powder melting, respectively. Increasing the laser energy density was beneficial to the printing efficiency but reduced printing accuracy. To ensure the integrity of multilayer printlets, the sintered thickness of each layer should be greater than the layer thickness. Furthermore, ibuprofen immediate-release tablets and metoprolol tartrate sustained-release tablets were prepared. Using 10% carboxy methyl starch sodium as an additive, the shell tablets with a side thickness of 1.5 mm had a good appearance and fast dissolution. Based on the optimized formulation, IBU preparations with various design shown good appearance and fast release property. There was no difference in the drug's crystalline state after SLS printing. Besides, the printed reservoir tablets with a sustained-released coating thickness of 3.5 mm had a good appearance and allowed 12 h sustained release. The results revealed that the SLS technique has great prospects in producing personalized oral preparations with immediate-release and sustained-release properties.

Acute undifferentiated leukemia: data on incidence and outcomes from a large population-based database.

Acute undifferentiated leukemia (AUL) is rare and defined by the absence of bona fide myeloid and lymphoid markers. Little is known about its incidence, survival and optimal management in the recent time period. Based on a case observed in our clinic, we queried the Surveillance, Epidemiology, and End Results database between 2000 and 2016. A total of 1,888 cases of AUL were diagnosed (1.34 per million person-years). The incidence of AUL has significantly decreased over time. Compared to other acute leukemias, patients with AUL have the highest median age (74 years); in contrast to acute myeloid leukemia (AML, 65) and acute lymphoblastic leukemia (ALL, 12). Excluding patients with preexisting malignancies, 1,444 patients with AUL were analyzed for survival. Only 35% of AUL patients had received chemotherapy. Comparatively, 94% of ALL and 71% of AML cases received chemotherapy. Among AUL patients who received chemotherapy, the median survival was 12 months as opposed to 1 month in the group who did not receive chemotherapy (or unknown status). Among adults, AUL patients had the worst prognosis, with a median overall survival (OS) of 9 months, compared to 27 months in ALL and 13 months in AML. Among children, the median OS was superior for all three groups of leukemias, the OS of AUL patients being better than in AML and very similar to ALL. On multivariate analysis, older age and time period were associated with worse outcome. We describe here the largest series of cases with AUL published to date.

Corrigendum to "First contiguous gene deletion causing biotinidase deficiency: The enzyme deficiency in three Sri Lankan children" [Mol. Genet. Metab. Rep. 2 (2016) 81-84].

[This corrects the article DOI: 10.1016/j.ymgmr.2015.01.005.].

A patient with constitutional ring 1 chromosome characterized by SNP array CGH.

We present a male patient with constitutional ring 1 chromosome and subsequent 6 Mb deletion at 1q43q44. The patient displays overlapping clinical features with reported patients with ring 1 chromosome and 1q43q44 microdeletion syndrome. To our knowledge, this is the first patient with ring 1 chromosome characterized by comparative genomic hybridization.

Isodicentric Y mosaicism involving a 46, XX cell line: Implications for management.

Carriers of isodicentric Y (idicY) mosaicism exhibit a wide range of clinical features, including short stature, gonadal abnormalities, and external genital anomalies. However, the phenotypic spectrum for individuals carrying an idicY and a 46, XX cell line is less clearly defined. A more complete description of the phenotype related to idicY is thus essential to guide management related to pubertal development, fertility, and gonadoblastoma risk in mosaic carriers. Findings from the evaluation of twin females with an abnormal karyotype, 48, XX, +idic(Yq) x2/47, XX, +idic(Yq)/46, XX, are presented to highlight the importance of interdisciplinary care in the management of multifaceted disorders of sex development.

Frontofacionasal Dysplasia in a Newborn with a De Novo Duplication of 7p15.2-p15.1.

We report a new case of frontofacionasal dysplasia or dysostosis (FFND) with a 1.5 Mb duplication in the region of 7p15.2-p15.1, and provide a review of the literature to understand the underlying pathogenesis better.

First contiguous gene deletion causing biotinidase deficiency: The enzyme deficiency in three Sri Lankan children.

We report three symptomatic children with profound biotinidase deficiency from Sri Lanka. All three children presented with typical clinical features of the disorder. The first is homozygous for a missense mutation in the BTD gene (c.98_104 del7insTCC; p.Cys33PhefsX36) that is commonly seen in the western countries, the second is homozygous for a novel missense mutation (p.Ala439Asp), and the third is the first reported instance of a contiguous gene deletion causing the enzyme deficiency. In addition, this latter finding exemplifies the importance of considering a deletion within the BTD gene for reconciling enzymatic activity with genotype, which can occur in asymptomatic children who are identified by newborn screening.

HER2 status in elderly women with breast cancer.

OBJECTIVES: HER2 (human epidermal growth factor receptor 2) is an important biomarker in breast cancer, but its prevalence in elderly women is not well established. Previous studies reported HER2 status based on either immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH) interchangeably. However, the tests may give discordant results. We report the prevalence of HER2 amplification in elderly women using only FISH. MATERIALS AND METHODS: We retrospectively identified women 65 years and older undergoing core biopsy, lumpectomy or mastectomy for primary breast malignancy at a single institution between 2009 and 2011. Data collected included age, histopathological type, hormone receptor status, and HER2 status. Descriptive statistics were performed using SAS Software, Version 9.2. RESULTS: One hundred fifty-eight women were included in the study. Most had invasive ductal carcinoma (74.7%), and were positive for either estrogen (ER) or progesterone (PgR) receptors (82.3% and 70.0%, respectively). Only 17% were negative for both ER and PgR; 11.4% were triple negative. Nineteen samples (12.0%) were positive for HER2. In univariate analyses, hormone receptor and HER2 status did not vary with age. When stratified by age, 60% of women with hormone receptor/HER2 positive tumors were younger than 70 years, compared with 22.2-33.3% of women in other subgroups. The difference was not statistically significant (p=0.20). CONCLUSION: This study adds to the knowledge of the biology of breast cancer in elderly women. Triple negative tumor incidence was similar to that previously reported for women under 70 years old, but HER2 positive tumors were less common. Additional prospective studies are needed to confirm our findings.

Nearly identical near-haploid karyotype in a peritoneal mesothelioma and a retroperitoneal malignant peripheral nerve sheath tumor.

The presence of a near-haploid karyotype is a rare finding in human malignancies, most frequently occurring in acute leukemia. In solid tumors, a near-haploid karyotype has been reported in fewer than 40 cases. We report two nearly identical near-haploid karyotypes from two distinctly different tumor types. The first case is a biphasic malignant mesothelioma from a 53-year-old white woman forming a large retroperitoneal mass. Cytogenetic evaluation revealed a primary hyperdiploid cell population as well as near-haploid and hypertetraploid populations with an overall karyotype of 27,XX,i(5)(p10),+7,add(15)(p11.2),+dic(1;20)(p13;p13)[2]/54,idemx2[90]/101-108,idemx4[19]. The second case is a large pelvic mass from a 48-year-old man. Histologic examination identified a malignant peripheral nerve sheath tumor displaying a karyotype of 26,X,+i(5)(p10),+7,der(15)t(1;15)(q12;p12),+20[5]/52,idemx2[20]. Herein we discuss the potential relationship between these two disparate neoplasms with nearly identical near-haploid karyotypes and present a literature review.