Phenylalanine hydroxylase deficiency treatment and management: A systematic evidence review of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: Elevated serum phenylalanine (Phe) levels due to biallelic pathogenic variants in phenylalanine hydroxylase (PAH) may cause neurodevelopmental disorders or birth defects from maternal phenylketonuria. New Phe reduction treatments have been approved in the last decade, but uncertainty on the optimal lifespan goal Phe levels for patients with PAH deficiency remains. METHODS: We searched Medline and Embase for evidence of treatment concerning PAH deficiency up to September 28, 2021. Risk of bias was evaluated based on study design. Random-effects meta-analyses were performed to compare IQ, gestational outcomes, and offspring outcomes based on Phe ≤ 360 µmol/L vs > 360 µmol/L and reported as odds ratio and 95% CI. Remaining results were narratively synthesized. RESULTS: A total of 350 studies were included. Risk of bias was moderate. Lower Phe was consistently associated with better outcomes. Achieving Phe ≤ 360 µmol/L before conception substantially lowered the risk of negative effect to offspring in pregnant individuals (odds ratio = 0.07, 95% CI = 0.04-0.14; P < .0001). Adverse events due to pharmacologic treatment were common, but medication reduced Phe levels, enabling dietary liberalization. CONCLUSIONS: Reduction of Phe levels to ≤360 µmol/L through diet or medication represents effective interventions to treat PAH deficiency.

Clonal cytopenia of undetermined significance (CCUS)-associated reversion of donor-derived, transient αß T-cell large granular clonal lymphocytosis, emerging post-transplant in a patient with a history of γδ T-cell large granular lymphocytic leukemia.

Autologous and allogeneic hematopoietic stem cell transplantation (HSCT) has revolutionized the therapy of hematolymphoid malignancies. Yet, how to best detect or predict the emergence of HSCT-related complications remain unresolved. Here, we describe a case of donor-derived, transient Alpha Beta (αß) T-cell large granular clonal lymphocytosis and cytopenia that emerged post-HSCT in a patient with a history of gamma delta (γδ) T-cell large granular lymphocytic leukemia (T-LGLL). Clonal unrelatedness of post-transplant T-LGL lymphocytosis to the patient's pretransplant T-LGLL was first identified by T-cell receptor (TCR) PCR showing different sized fragments of rearranged gamma chains, in addition to shift from γδ to αß TCR expression by flow cytometry analyses. Donor-derivation of the patient's post-transplant clonal lymphocytosis was confirmed by serial chimerism analyses of recipient's blood specimens demonstrating 100% donor DNA. Moreover, oncogenic DNMT3A and RUNX1 mutations were detected by next-generation sequencing (NGS) only in post-transplant specimens. Intriguingly, despite continued increase in DNMT3A and RUNX1 mutation load, the patient's clonal lymphocytosis and anemia eventually largely resolved; yet, the observed mutation profile with persistent thrombocytopenia indicated secondary clonal cytopenia of undetermined significance (CCUS) in the absence of overt morphologic evidence of myeloid neoplasm in the marrow. This case illustrates the utility of longitudinal chimerism analysis and NGS testing combined with flow cytometric immunophenotyping to evaluate emerging donor-derived hematolymphoid processes and to properly interpret partial functional engraftment. It may also support the notion that driver mutation-induced microenvironmental changes may paradoxically contribute to reestablishing tissue homeostasis.

Common clonal origin of three distinct hematopoietic neoplasms in a single patient: B-cell lymphoma, T-cell lymphoma, and polycythemia vera.

The potential for more than one distinct hematolymphoid neoplasm to arise from a common mutated stem or precursor cell has been proposed based on findings in primary human malignancies. Particularly, angioimmunoblastic T-cell lymphoma (AITL), which shares a somatic mutation profile in common with other hematopoietic malignancies, has been reported to occur alongside myeloid neoplasms or clonal B-cell proliferations, with identical mutations occurring in more than one cell lineage. Here we report such a case of an elderly woman who was diagnosed over a period of 8 years with diffuse large B-cell lymphoma, polycythemia vera, and AITL, each harboring identical somatic mutations in multiple genes. Overall, at least five identical nucleotide mutations were shared across multiple specimens, with two identical mutations co-occurring at variable variant allele frequencies in all three specimen types. These findings lend credence to the theory that a common mutated stem cell could give rise to multiple neoplasms through parallel hematopoietic differentiation pathways.

Proficiency Testing of Standardized Samples Shows High Interlaboratory Agreement for Clinical Next Generation Sequencing-Based Hematologic Malignancy Assays With Survey Material-Specific Differences in Variant Frequencies.

CONTEXT.­: As laboratories increasingly turn from single-analyte testing in hematologic malignancies to next-generation sequencing-based panel testing, there is a corresponding need for proficiency testing to ensure adequate performance of these next-generation sequencing assays for optimal patient care. OBJECTIVE.­: To report the performance of laboratories on proficiency testing from the first 4 College of American Pathologists Next-Generation Sequencing Hematologic Malignancy surveys. DESIGN.­: College of American Pathologists proficiency testing results for 36 different engineered variants and/or allele fractions as well as a sample with no pathogenic variants were analyzed for accuracy and associated assay performance characteristics. RESULTS.­: The overall sensitivity observed for all variants was 93.5% (2190 of 2341) with 99.8% specificity (22 800 of 22 840). The false-negative rate was 6.5% (151 of 2341), and the largest single cause of these errors was difficulty in identifying variants in the sequence of CEBPA that is rich in cytosines and guanines. False-positive results (0.18%; 40 of 22 840) were most likely the result of preanalytic or postanalytic errors. Interestingly, the variant allele fractions were almost uniformly lower than the engineered fraction (as measured by digital polymerase chain reaction). Extensive troubleshooting identified a multifactorial cause for the low variant allele fractions, a result of an interaction between the linearized nature of the plasmid and the Illumina TruSeq chemistry. CONCLUSIONS.­: Laboratories demonstrated an overall accuracy of 99.2% (24 990 of 25 181) with 99.8% specificity and 93.5% sensitivity when examining 36 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 10% or greater. The data also highlight an issue with artificial linearized plasmids as survey material for next-generation sequencing.

Incidental splenic findings in pancreatosplenectomy specimens resected for primary pancreatic lesions.

AIMS: Little has been written on the frequency and nature of incidental splenic lesions diagnosed on histopathological examination of pancreatosplenectomy specimens. METHODS AND RESULTS: For 191 such specimens, incidental histological findings after haematopathologist re-review were tabulated. Cases suspicious for lymphoid malignancy underwent molecular analysis for immunoglobulin heavy and kappa light chain rearrangement. Follow-up was obtained on selected cases. In five cases (3%), the spleen was sampled but not mentioned in the original microscopic report; all were normal on re-review. Otherwise, most (171 of 186, 92%) were initially diagnosed as normal, with 160 (94%) remaining so on re-review. Findings on re-review not initially described (n = 11, 6%) included four cases with splenic morphology suspicious for possible leukaemia/lymphoma involvement. Additional findings included abscess formation, foamy macrophages, necrotising granulomas and simple cysts. Fifteen spleens were initially diagnosed as abnormal; the histopathological process was confirmed in all, including non-necrotising granulomas, cysts, Gamna-Gandy bodies, foamy macrophages, involvement by pancreatic neoplasm and involvement by known chronic lymphocytic leukaemia (CLL). Molecular analysis was performed on the five cases of known/suspected lymphoma and two were positive for monoclonal gene rearrangement, including the known CLL and a previously undiagnosed case with similar immunophenotype. CONCLUSIONS: Incidental splenic findings are not uncommon in pancreatosplenectomy specimens. While most are of limited clinical significance, low-grade lymphoproliferative disorders may go undetected if the spleen is overlooked. We recommend careful observation of splenic findings in these specimens, with a low threshold for haematopathological consultation when in doubt.

Performance Comparison of Different Analytic Methods in Proficiency Testing for Mutations in the BRAF, EGFR, and KRAS Genes: A Study of the College of American Pathologists Molecular Oncology Committee.

CONTEXT.­: The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories ("home brews"), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee. OBJECTIVE.­: To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes: BRAF, EGFR, and KRAS. DESIGN.­: There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences for BRAF, EGFR, and KRAS. Performance was analyzed by test method, kit manufacturer, variants tested, and preanalytic and postanalytic practices. RESULTS.­: Of 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses for BRAF; 848 [97.5%] acceptable of 870 total responses for EGFR; 942 [97.0%] acceptable of 971 total responses for KRAS). Next-generation sequencing platforms (summed across all analytes and 2 platforms) demonstrated 99.4% accuracy for these analytes (165 [99.4%] acceptable of 166 total next-generation sequencing responses). Slight differences in performance were noted among select commercial assays, dependent upon the particular design and specificity of the assay. Wide differences were noted in the lower limits of neoplastic cellularity laboratories accepted for testing. CONCLUSIONS.­: These data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.

Comparison of Laboratory-Developed Tests and FDA-Approved Assays for BRAF, EGFR, and KRAS Testing.

Importance: The debate about the role of the Food and Drug Administration (FDA) in the regulation of laboratory-developed tests (LDTs) has focused attention on the analytical performance of all clinical laboratory testing. This study provides data comparing the performance of LDTs and FDA-approved companion diagnostics (FDA-CDs) in proficiency testing (PT) provided by the College of American Pathologists Molecular Oncology Committee. Objective: To compare the analytical performance of LDTs and FDA-CDs on well-characterized PT samples and to compare the practice characteristics of laboratories using these assays. Design, Setting, and Participants: This comparison of PT responses examines the performance of laboratories participating in the College of American Pathologists PT for 3 oncology analytes for which both FDA-CDs and LDTs are used: BRAF, EGFR, and KRAS. A total of 6897 PT responses were included: BRAF (n = 2524; 14 PT samples), EGFR (n = 2216; 11 PT samples), and KRAS (n = 2157, 10 PT samples). US Food and Drug Administration companion diagnostics and LDTs are compared for both accuracy and preanalytic practices of the laboratories. Main Outcomes and Measures: As per the College of American Pathologists PT standards, results were scored and the percentages of acceptable responses for each analyte were compared. These were also broken down by the specific variants tested, by kit manufacturer for laboratories using commercial reagents, and by preanalytic practices. Results: From analysis of 6897 PT responses, this study demonstrates that both LDTs and FDA-CDs have excellent performance overall, with both test types exceeding 97% accuracy for all 3 genes (BRAF, EGFR, and KRAS) combined. Rare variant-specific differences did not consistently favor LDTs or FDA-CDs. Additionally, more than 60% of participants using an FDA-CD reported adapting their assay from the approved procedure to allow for a greater breadth of sample types, minimum tumor content, and instrumentation, changing the classification of their assay from FDA-CD to LDT. Conclusions: This study demonstrates the high degree of accuracy and comparable performance of both LDTs and FDA-CDs for 3 oncology analytes. More significantly, the majority of laboratories using FDA-CDs have modified the scope of their assay to allow for more clinical practice variety, rendering them LDTs. These findings support both the excellent and equivalent performance of both LDTs and FDA-CDs in clinical diagnostic testing.

Is Tubulocystic Renal Cell Carcinoma Real?: Genomic Analysis Confirms the World Health Organization Classification.

This commentary highlights the article by Lawrie et al that validates that tubulocystic renal cell carcinoma is a distinct type of renal neoplasm.

p53 alteration in morphologically normal/benign breast luminal cells in BRCA carriers with or without history of breast cancer.

Germline mutations in BRCA genes have been shown to predispose patients to breast cancer. Studies have suggested that p53 alteration is a necessary step in tumorigenesis in BRCA carriers. Our previous study showed p53 alteration in morphologically normal/benign breast luminal cells in sporadic breast cancer patients, the so-called breast p53 signature. Here, we studied p53 status in 66 BRCA1/2 carriers' breasts: 29 patients with breast carcinoma (2 patients with bilateral breast carcinomas) and 37 without. Seven of the 12 (58%) triple-negative breast carcinomas in BRCA carriers were positive for p53 alteration (immunohistochemical stain and/or sequencing), the same frequency as in sporadic triple-negative breast carcinomas. Focal p53 positivity in adjacent normal/benign luminal cells was identified in 4 of the 7 cases with p53-positive carcinomas but not in breasts with p53-negative carcinomas, indicating that p53 positivity in normal/benign breast luminal cells is not a random event. Furthermore, in BRCA carriers' prophylactic mastectomies, 12 of the 94 (12.77%) breasts had focal p53 positivity in normal/benign luminal cells, with 2 cases in bilateral breasts, significantly higher than in previously studied mammoplasty specimens (0%). Our study suggests that germline BRCA gene mutations could result in genomic instability and an elevated gene mutation rate (such as the p53 gene) in breast luminal cells compared with the general population, predisposing BRCA carriers to develop p53-positive/triple-negative breast carcinomas.

Pigmented epithelioid melanocytoma (animal-type melanoma): An institutional experience.

BACKGROUND: Pigmented epithelioid melanocytoma (PEM) is an uncommon, recently described entity with unknown biologic behavior. There is a high rate of regional metastases, but limited evidence of distant metastases or disease-related death. OBJECTIVE: We sought to report our series of patients given a diagnosis of PEM at our institution and provide mutational analysis of genes commonly implicated in melanoma in 2 cases. METHODS: The pathology database was queried for cases of PEM diagnosed at the University of Rochester. Charts were reviewed for follow-up information. Mutational analysis of melanoma-associated genes was performed on 2 cases. RESULTS: Nine cases of PEM were retrieved in a 10-year retrospective review. Five patients underwent sentinel lymph node biopsy with 3 of 5 having a positive sentinel lymph node. All 9 patients are alive and disease-free with average follow-up of 38.75 months. Two tumors were tested for common melanoma-associated mutations, and were negative, except for a telomerase reverse transcriptase promoter deletion detected in 1 sample. The deletion has not been associated with melanoma, and therefore its biologic significance is unclear. LIMITATIONS: Small sample size, retrospective nature, and single institution experience are limitations. CONCLUSIONS: PEM appears to have an indolent behavior. However, currently the evidence is too limited to provide insight into its true biologic potential.

Next-Generation Sequencing for Minimal Residual Disease Surveillance in Acute Lymphoblastic Leukemia: An Update.

Monitoring minimal residual disease (MRD) is an important predictor of outcome in acute lymphoblastic leukemia (ALL) and is used in risk stratification, prognosis determination, and therapy guidance. Several laboratory techniques have proven utility for characterizing leukemic cells and following MRD through diagnosis, remission and possible recurrence. Methods for determining MRD are based on the detection of leukemia-specific aberrant immunophenotypes by mulitparameter flow cytometry or the evaluation of leukemia-specific rearranged immunoglobulin or T-cell receptor sequences by quantitative real-time PCR. Next-generation sequencing (NGS) is emerging as a new flexible and sensitive tool to detect MRD, which allows identification of clonal composition and scalable sensitivity depending on sequence coverage. As NGS becomes more accessible and affordable, guidelines should be established for its application to MRD surveillance.

Index case of acute myeloid leukemia in a family harboring a novel CEBPA germ line mutation.

The persistence of a CEBPA mutation at the time of complete remission warrants germ line analysis.Not all patients harboring germ line CEBPA mutations have a family history of AML.

The Spectrum of Clinical Utilities in Molecular Pathology Testing Procedures for Inherited Conditions and Cancer: A Report of the Association for Molecular Pathology.

Clinical utility describes the benefits of each laboratory test for that patient. Many stakeholders have adopted narrow definitions for the clinical utility of molecular testing as applied to targeted pharmacotherapy in oncology, regardless of the population tested or the purpose of the testing. This definition does not address all of the important applications of molecular diagnostic testing. Definitions consistent with a patient-centered approach emphasize and recognize that a clinical test result's utility depends on the context in which it is used and are particularly relevant to molecular diagnostic testing because of the nature of the information they provide. Debates surrounding levels and types of evidence needed to properly evaluate the clinical value of molecular diagnostics are increasingly important because the growing body of knowledge, stemming from the increase of genomic medicine, provides many new opportunities for molecular testing to improve health care. We address the challenges in defining the clinical utility of molecular diagnostics for inherited diseases or cancer and provide assessment recommendations. Starting with a modified analytic validity, clinical validity, clinical utility, and ethical, legal, and social implications model for addressing clinical utility of molecular diagnostics with a variety of testing purposes, we recommend promotion of patient-centered definitions of clinical utility that appropriately recognize the valuable contribution of molecular diagnostic testing to improve patient care.

p53 alteration in morphologically normal/benign breast tissue in patients with triple-negative high-grade breast carcinomas: breast p53 signature?

p53 alterations have been identified in approximately 23% of breast carcinomas, particularly in hormone receptor-negative high-grade carcinomas. It is considered to be an early event in breast carcinogenesis. Nevertheless, the putative precursor lesion of high-grade breast carcinoma remains elusive. Breast excision specimens from 93 triple-negative high-grade invasive ductal carcinomas, 48 estrogen receptor (ER)-positive/progesterone receptor-positive/Her2-negative non-high-grade invasive ductal carcinomas, and 50 mammoplasty breasts were selected. At least 2 tissue blocks with tumor and adjacent benign tissue were sectioned and subjected to immunohistochemistry staining for p53. TP53 gene sequencing was performed on select tumors. Further immunohistochemistry staining for ER and Ki-67 was performed on consecutive sections of tissue with p53-positive normal/benign cells. Of the 93 high-grade carcinomas, 51 (55%) were positive for p53 alteration, whereas only 3 (6.25%) of the 48 non-high-grade carcinomas were p53 altered. Focal p53 positivity in adjacent normal/benign breast tissue was identified in 19 cases, and 18 of them also had p53 alteration in their carcinomas. Only 1 case had focal p53 staining in normal/benign tissue, but the tumor was negative for p53 alteration. No p53 staining positivity was identified in the mammoplasty specimens. The p53-stained normal/benign cells were ER negative and did not show an increase in the Ki-67 labeling index. These findings indicate that the p53 staining positivity in normal/benign breast tissue is not a random event. It could be considered as the "p53 signature" in breast and serve as an indicator for future potential risk of p53-positive high-grade breast carcinoma.

Evolution to plasmablastic lymphoma evades CD19-directed chimeric antigen receptor T cells.

A patient with relapsed and refractory chronic lymphocytic leukaemia with Richter transformation was treated with chimeric antigen receptor (CAR)-modified T cells targeted for CD19 but later relapsed with a clonally related plasmablastic lymphoma. The loss of most routine markers of pre-plasma cell or B lymphoid differentiation (including CD19) highlights the ability of such mature lymphomas to evade lineage-specific targeted immunotherapy by differentiating along pathways comparable to their normal cellular counterparts. Molecular genetic evaluation demonstrated multiple independent lines of CD19-negative disease that eventually evolved in this single patient. Such plasticity represents potential challenges for antigen-directed CAR-T cell therapy, while serving as a testament to the selective pressure exerted by these engineered T cells over time.

Reporting incidental findings in genomic scale clinical sequencing--a clinical laboratory perspective: a report of the Association for Molecular Pathology.

Advances in sequencing technologies have facilitated concurrent testing for many disorders, and the results generated may provide information about a patient's health that is unrelated to the clinical indication, commonly referred to as incidental findings. This is a paradigm shift from traditional genetic testing in which testing and reporting are tailored to a patient's specific clinical condition. Clinical laboratories and physicians are wrestling with this increased complexity in genomic testing and reporting of the incidental findings to patients. An enormous amount of discussion has taken place since the release of a set of recommendations from the American College of Medical Genetics and Genomics. This discussion has largely focused on the content of the incidental findings, but the laboratory perspective and patient autonomy have been overlooked. This report by the Association of Molecular Pathology workgroup discusses the pros and cons of next-generation sequencing technology, potential benefits, and harms for reporting of incidental findings, including the effect on both the laboratory and the patient, and compares those with other areas of medicine. The importance of genetic counseling to preserve patient autonomy is also reviewed. The discussion and recommendations presented by the workgroup underline the need for continued research and discussion among all stakeholders to improve our understanding of the effect of different policies on patients, providers, and laboratories.

Standardized assessment of seizures in patients with juvenile neuronal ceroid lipofuscinosis.

AIM: To evaluate seizure phenomenology, treatment, and course in individuals with juvenile neuronal ceroid lipofuscinosis (JNCL). METHOD: Data from an ongoing natural history study of JNCL were analyzed using cross-sectional and longitudinal methods. Seizures were evaluated with the Unified Batten Disease Rating Scale, a disease-specific quantitative assessment tool. RESULTS: Eighty-six children (44 males, 42 females) with JNCL were assessed at an average of three annual visits (range 1-11). Eighty-six percent (n=74) experienced at least one seizure, most commonly generalized tonic-clonic, with mean age at onset of 9 years 7 months (SD 2y 10mo). Seizures were infrequent, typically occurring less often than once every 3 months, and were managed with one to two medications for most participants. Valproate (49%, n=36) and levetiracetam (41%, n=30) were the most commonly used seizure medications. Myoclonic seizures occurred infrequently (16%, n=14). Seizure severity did not vary by sex or genotype. Seizures showed mild worsening with increasing age. INTERPRETATION: The neuronal ceroid lipofuscinoses (NCLs) represent a group of disorders unified by neurodegeneration and symptoms of blindness, seizures, motor impairment, and dementia. While NCLs are considered in the differential diagnosis of progressive myoclonus epilepsy, we show that myoclonic seizures are infrequent in JNCL. This highlights the NCLs as consisting of genetically distinct disorders with differing natural history.

Mutations of the TERT promoter are common in basal cell carcinoma and squamous cell carcinoma.

Telomerase is frequently expressed in cancer and contributes to carcinogenesis. Two recent publications report the identification of a set of recurrent mutations in melanoma in the promoter of the telomerase reverse transcriptase gene (TERT) that appears to be the result of mutagenesis from ultraviolet (UV) radiation. Both groups reported that the mutations increase the transcription of TERT. This prompted our search for similar mutations in two other UV-related skin cancers, basal cell carcinoma, and squamous cell carcinoma. We found that the activating TERT promoter mutations reported in melanoma are also frequent in squamous cell carcinoma (50%) and basal cell carcinoma, the latter including both sporadic tumors (78%) and tumors from patients with nevoid basal cell carcinoma syndrome (68%). These mutations were found in only 1 of 11 Bowen's disease (squamous cell carcinoma in situ) specimens, and in none of 15 non-malignant skin specimens and 57 blood specimens. The mutations were frequently homozygous or hemizygous, with little or no normal signal at the mutated positions. These data suggest that TERT promoter mutations are the most frequent putative oncogenic mutations in cutaneous cancer.