Molecular characterization and survival analysis of a cohort of glioblastoma, IDH-wildtype.

Glioblastoma, IDH-wild type, the most common malignant primary central nervous system tumor, represents a formidable challenge in clinical management due to its poor prognosis and limited therapeutic responses. With an evolving understanding of its underlying biology, there is an urgent need to identify prognostic molecular groups that can be subject to targeted therapy. This study established a cohort of 124 sequential glioblastomas from a tertiary hospital and aimed to find correlations between molecular features and survival outcomes. Comprehensive molecular characterization of the cohort revealed prevalent alterations as previously described, such as TERT promoter mutations and involvement of the PI3K-Akt-mTOR, CK4/6-CDKN2A/B-RB1, and p14ARF-MDM2-MDM4-p53 pathways. MGMT promoter methylation is a significant predictor of improved overall survival, aligned with previous data. Conversely, age showed a marginal association with higher mortality. Multivariate analysis to account for the effect of MGMT promoter methylation and age showed that, in contrast to other published series, this cohort demonstrated improved survival for tumors harboring PTEN mutations, and that there was no observed difference for most other molecular alterations, including EGFR amplification, RB1 loss, or the coexistence of EGFR amplification and deletion/exon skipping (EGFRvIII). Despite limitations in sample size, this study contributes data to the molecular landscape of glioblastomas, prompting further investigations to examine these findings more closely in larger cohorts.

Molecular Pathology Education: A Suggested Framework for Primary Care Resident Training in Genomic Medicine: A Report of the Association for Molecular Pathology Training and Education Committee.

Developments in genomics are profoundly influencing medical practice. With increasing use of genetic and genomic testing across every aspect of the health care continuum, patients and their families are increasingly turning to primary care physicians (PCPs) for discussion and advice regarding tests, implications, and results. Yet, with the rapid growth of information, technology, and applications, PCPs are finding it challenging to fill the gaps in knowledge and support the growing needs of their patients. A critical component in expanding PCP genomic literacy lies in the education of physicians in training and in practice. Although a framework for developing physician competencies in genomics has already been developed, the Association for Molecular Pathology is uniquely situated to actively utilize the skills of its members to engage and support PCPs in this effort. This report provides an overview and a suggested basic teaching framework, which can be used by molecular professionals in their individual institutions as a starting point for educational outreach.

Cellular Fibroma of Tendon Sheath With Novel TNC-USP6 Gene Fusion Clinically Mimicking Arthritis in a 7-Year-Old Boy.

Cellular fibroma of tendon sheath (CFTS) is a rare, benign myofibroblastic neoplasm of tenosynovial soft tissues closely resembling nodular fasciitis (NF), but is histomorphologically distinct from classic fibroma of tendon sheath (FTS). We report a case of a pediatric patient with thumb swelling clinically concerning for arthritis with a biopsy demonstrating myofibroblastic proliferation with features consistent with NF/CFTS, and molecular studies confirming the presence of a USP6 gene fusion (TNC-USP6). This case highlights a unique clinical presentation of CFTS in a pediatric patient mimicking an inflammatory or reactive/non-neoplastic musculoskeletal disorder and the increasingly crucial role of molecular testing to differentiate a reactive myofibroblastic process from a neoplasm. Moreover, this report identifies TNC as a new fusion partner to USP6 fusion partner adding to our growing understanding of the USP6-rearranged family of tumors.

A Recursive Partitioning Analysis Demonstrating Risk Subsets for 8-Year Biochemical Relapse After Margin-Positive Radical Prostatectomy Without Adjuvant Hormone or Radiation Therapy.

PURPOSE: The cohort of patients with locally advanced prostate cancer (PC) and positive surgical margin(s) at radical prostatectomy (RP) who would benefit from salvage or adjuvant treatment is unclear. This study examines the risk of prostate-specific antigen (PSA) relapse in a large population of men with PC after margin-positive RP. METHODS AND MATERIALS: Using a multi-institutional database, patients with clinically localized PC who underwent RP between 2002 and 2010 with recorded follow-up PSA were retrospectively selected. Patients were excluded for pathologic seminal vesicle or lymph node involvement, metastatic disease, pre-RP PSA ≥ 30, or adjuvant (nonsalvage) radiation therapy or hormone therapy. The primary endpoint was biochemical relapse free survival (bRFS), where PSA failure was defined as PSA > 0.10 ng/mL and rising, or at salvage intervention. The Kaplan-Meier method was employed for bRFS estimates; recursive partitioning analysis using cumulative or single maximal margin extent (ME) and Gleason grade (GG) at RP was applied to identify variables associated with bRFS. RESULTS: At median follow-up of 105 months, 210 patients with positive margins at RP were eligible for analysis, and 89 had experienced PSA relapse. Median age was 61 years (range, 43-76), and median pre-RP PSA 5.8 ng/mL (1.6-26.0). Recursive partitioning analysis yielded 5 discrete risk groups, with the lowest risk group (GG1, ≤ 2 mm ME) demonstrating a bRFS of 92% at 8 years compared with the highest risk group (GG3-5, ≥ 3 mm ME) of 11%. CONCLUSIONS: This retrospective study suggests that it may be possible to risk-stratify patients undergoing margin-positive RP using commonly acquired clinical and pathologic variables. Patients with low-grade tumors and minimally involved margins have a very low recurrence risk and may be able to forego postprostatectomy radiation. Meanwhile, those with higher grade and greater involvement could benefit from adjuvant or early salvage radiation therapy.

Validation of Optical Genome Mapping for the Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy.

The molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD) relies on detecting contractions of the unique D4Z4 repeat array at the chromosome 4q35 locus in the presence of a permissive 4q35A haplotype. Long, intact DNA molecules are required for accurate sizing of D4Z4 repeats. We validated the use of optical genome mapping to determine size and haplotype of D4Z4 alleles for FSHD analysis. The cohort included 36 unique DNA specimens from fresh blood samples or archived agarose plugs. High-molecular- weight DNA underwent sequence-specific labeling followed by separation and image analysis with data collection on the Saphyr system. D4Z4 allele sizes were calculated and haplotypes determined from the labeling patterns. Each specimen had previous diagnostic testing using restriction enzyme digests with EcoRI, EcoRI/BlnI, XapI, or HindIII, followed by pulsed field gel electrophoresis and Southern blot analysis with appropriate probes. Optical genome mapping detected 4q35 and 10q26 alleles ranging from 1 to 79 D4Z4 repeats and showed strong correlation with Southern blot allele sizing (R2 = 0.95) and haplotyping (133 of 134; 99.4% haplotype match). Analysis of inter-assay and intra-assay runs showed high reproducibility (0.03 to 0.94 %CV). Subsequent optical genome mapping for routine clinical testing from 315 clinical FSHD cases compared favorably with historical result trends. Optical genome mapping is an accurate and highly reproducible method for chromosomal abnormalities associated with FSHD.

A Curriculum for Genomic Education of Molecular Genetic Pathology Fellows: A Report of the Association for Molecular Pathology Training and Education Committee.

Molecular genetic pathology (MGP) is a subspecialty of pathology and medical genetics and genomics. Genomic testing, which is defined as that which generates large data sets and interrogates large segments of the genome in a single assay, is increasingly recognized as essential for optimal patient care through precision medicine. The most common genomic testing technologies in clinical laboratories are next-generation sequencing and microarray. It is essential to train in these methods and to consider the data generated in the context of the diagnosis, medical history, and other clinical findings of individual patients. Accordingly, updating the MGP fellowship curriculum to include genomics is timely, important, and challenging. At the completion of training, an MGP fellow should be capable of independently interpreting and signing out results of a wide range of genomic assays and, given the appropriate context and institutional support, of developing and validating new assays in compliance with applicable regulations. The Genomics Task Force of the MGP Program Directors, a working group of the Association for Molecular Pathology Training and Education Committee, has developed a genomics curriculum framework and recommendations specific to the MGP fellowship. These recommendations are presented for consideration and implementation by MGP fellowship programs with the understanding that MGP programs exist in a diversity of clinical practice environments with a spectrum of available resources.

Impact of routine mismatch repair screening on genetic counseling and surgical management in colorectal cancer patients.

BACKGROUND: Mismatch repair (MMR) deficiency in colorectal cancer (CRC) should prompt consideration of genetic counseling (GC) as a Lynch syndrome (LS) diagnosis may have several implications for the patient and family. The study aims were to examine how routine MMR testing influences the rate of GC and surgical resection extent. METHODS: A single-institution retrospective review was performed on CRC specimens (including colonoscopic biopsies) routinely screened for MMR deficiency from 2012 to 2018. MLH1-deficient cancers with mutated BRAF or MLH1-promoter hypermethylation were excluded. RESULTS: MMR deficiency was identified in 295 of 1139 CRC specimens. After exclusions, 57 patients remained. Forty-two patients (74%) were identified preoperatively, and 35 (83%) were referred to GC: 16 were seen preoperatively, 9 postoperatively. Eight patients were diagnosed with Lynch syndrome (LS) preoperatively: 2 had no resection, 2 underwent segmental resection and 4 underwent extended resection. CONCLUSIONS: Most MMR-deficient patients were identified and referred to GC preoperatively, though not all were seen. Of the preoperatively diagnosed LS patients, half underwent extended resection. Barriers to GC and decision-making around resection extent bears further study.

The clinical characteristics of melanoma with BRAF V600R mutation: a case series study.

Currently, several targeted therapy regimens are approved as first-line treatment in V600E/K-mutant advanced and metastatic melanoma. Patients with the third most common pathologic variant in the BRAF gene, V600R, were not included in BRAF/MEK inhibitors clinical trials, so there is lack of information about the clinical characteristics and predictive value of this mutation in systemic therapy of unresectable disease. We retrospectively reviewed clinical BRAF mutation testing results and the records of melanoma patients at the University of Iowa Hospitals and Clinics from 2011 to 2017. DNA from formalin-fixed, paraffin-embedded tumor specimens were sequenced using a next-generation sequencing panel or dye terminator sequencing covering exon 15 of the BRAF gene. The study protocol was approved by the University of Iowa Institutional Review Board. Nine patients (5.3% of 168 cases with BRAF mutation) were found to have the V600R mutation. We report our experience in treatment of seven patients with V600R-mutant melanoma, whose clinical records were available for review. Four patients in our cohort received BRAF inhibitors. Three patients demonstrated partial objective response to BRAF/MEK targeted therapy. V600R-mutant melanoma accounts for a significant number of cases even in single-institution practices. We believe that testing for BRAF-mutation status should include rare variants of this mutation. From our experience, the high rate of ulceration, male predominance and advanced age at diagnosis are features of melanoma with V600R mutation, which are similar to those reported for V600K mutation. We observed objective response to BRAF/MEK inhibitors in three cases with V600R variant.

BCR-NTRK2 fusion in a low-grade glioma with distinctive morphology and unexpected aggressive behavior.

A 52-yr-old man was found to have a 6.6-cm left frontotemporal mass. Biopsy revealed a low-grade astrocytic neoplasm with significant infiltration and an unusual morphologic appearance. Only rare mitotic figures were seen and the Ki-67 proliferative index was very low. Unexpectedly, the low-grade astrocytoma showed rapid progression within a short time, but subsequent resection showed similar histologic findings to the original biopsy with only slightly more mitoses and a marginally increased Ki-67 proliferative index. Molecular testing performed on the tumor showed no alterations in the IDH1, IDH2, EGFR, or BRAF genes by sequencing, intact 1p/19q by FISH, and a novel BCR-NTRK2 fusion transcript by reverse transcription and anchored multiplex PCR. The patient underwent standard-of-care therapy, both first and second line, for a high-grade glioma because of the aggressive behavior, but the glioma continued to progress despite treatment, and the patient died within 13.5 mo of the original diagnosis. At the time of diagnosis, the BCR-NTRK2 fusion transcript had not been described in solid tumors; however, a recent publication described this fusion transcript in two glioblastomas. Although no approved therapy was available for this patient, FDA approval has now been given for solid tumors with any NTRK gene family fusions. This unexpected molecular finding in a deceptively low-grade-appearing glioma supports the use of expanded molecular testing in gliomas and solid tumors, particularly in instances where targeted therapies are available.

Is More Always Better? An Assessment of the Impact of Lymph Node Yield on Outcome for Clinically Localized Prostate Cancer with Low/Intermediate Risk Pathology (pT2-3a/pN0) Managed with Prostatectomy Alone.

The clinical impact of lymph node dissection extent remains undetermined in the contemporary setting, as reflected in care pattern variations. Despite some series demonstrating a direct relationship between number of lymph nodes identified and detection of nodal involvement, the correlation between lymph node yield and disease control or survival outcomes remains unclear. Patients with clinically localized prostate cancer, pre-RP PSA <30, and pT2-3a/N0 disease at RP were retrospectively identified from two databases for inclusion. Those who received pre- or post-RP radiotherapy or hormone therapy were excluded. Kaplan-Meier method was employed for survival probability estimation. Cox regression models were used to assess bRFS differences between subsets. From 2002 to 2010, 667 eligible patients were identified. The median age was 61 yrs. (range, 43-76), with median PSA 5.6 ng/dL (0.9-28.0). At RP, most patients had pT2c (64%) disease with Gleason Score (GS) ≤6 (43%) or 7 (48%); 218 (33%) patients had positive margins (M+). At median clinical and PSA follow-up of 96 and 87 months, respectively, 146 patients (22%) experienced PSA failure with an estimated bRFS of 81%/76% at 5/8 years. For patients who underwent LND, univariable analysis identified PSA (at diagnosis), higher GS (≥7, at biopsy or RP), intermediate/high risk stratification, M+ as adversely associated with bRFS (all p < 0.01). A higher number of LNs excised was not associated with improved bRFS for the entire cohort (HR = 0.97, p = 0.27), nor for any clinical risk stratum, biopsy GS, or RP GS subgroup. This study did not demonstrate an association between LN yield and bRFS in patients with clinically localized pT2-3a/pN0 prostate cancer managed with RP alone, either in the entire population or with substratification by clinical risk stratum or GS.

A case of primary cutaneous Ewing sarcoma in a neutropenic patient.

Primary cutaneous Ewing sarcoma is a rare clinical presentation of Ewing sarcoma, usually occurring as a small, localized tumor on the extremities of young adults and associated with favorable prognosis. We report a case of primary cutaneous Ewing sarcoma, which presented on the sole of the foot of a 27-year-old patient with relapsed acute myeloid leukemia and neutropenia. Diagnosis was determined through histological features and staining, as well as fluorescence in situ hybridization and molecular testing. The patient underwent wide-local excision with plan to begin targeted chemotherapy, but unfortunately died from adenovirus pneumonia while neutropenic before targeted chemotherapy was initiated.

Incidental prostate cancer diagnosed at radical cystoprostatectomy for bladder cancer: disease-specific outcomes and survival.

BACKGROUND: The current standard of care for men with muscle-invasive bladder cancer is radical cystoprostatectomy (RCP). One-third of RCP specimens demonstrate incidental prostate cancer, primarily reported in small series with limited follow-up. The aim of this study is to report mature outcomes, including patterns of failure and disease-specific recurrence rates, and survival, for a large cohort of men with incidental prostate cancer at RCP performed at a tertiary referral center. METHODS: This retrospective study describes cancer control and survival rates for men who underwent RCP for bladder cancer and were found incidentally to have prostate cancer. Analysis of patient-, tumor-, and treatment-specific factors were analyzed for association with disease control and survival endpoints. RESULTS: Between 2002 and 2010, 94 patients with incidental discovery of prostate cancer postRCP were identified for inclusion in this study. Forty-five patients (45%) underwent RCP for recurrent (rather than initial presentation of) bladder carcinoma. At a median follow-up of 40.3 months (71.2 months for survivors; range, 8.9-155.5 months), 42 patients were alive without recurrence and 52 patients had died (25 associated with disease). The estimated 5-year bladder cancer disease-free, urinary tract malignancy disease-free, and prostate specific antigen (PSA) relapse-free survivals were 76% [95% confidence interval (CI), 65-84%], 64% (52-74%), and 97% (79-100%), respectively. The estimated 5-year urinary tract malignancy-specific and overall survivals were 61% (49-71%) and 52% (41-62%), respectively. Univariate analysis demonstrated associations between pathologic T/N-stage and nodal ratio with bladder cancer disease-free, urinary tract malignancy disease-specific, and overall survivals, with patient age at diagnosis as an additional adverse factor associated with overall survival. Multivariate analysis confirmed pN-stage and age as independently associated with worse survival. CONCLUSION: For men undergoing RCP for bladder cancer, the present study suggests that incidentally discovered prostate cancers, irrespective of pathologic stage, Gleason score, or clinical significance, do not impact 5-year disease control or survival outcomes.

Individualization of Adjuvant Therapy After Radical Prostatectomy for Clinically Localized Prostate Cancer: Current Status and Future Directions.

Radiation therapy indications in the postprostatectomy setting are evolving. Several retrospective series have identified a number of "high-risk" pathologic features associated with an elevated risk of disease recurrence after radical prostatectomy. More recently, several randomized phase III trials demonstrated superior biochemical relapse-free survival for adjuvant radiation therapy after prostatectomy for patients with these high-risk pathologic features, including positive margin status, extraprostatic extension, and/or seminal vesicle invasion. These series further suggested improvement in distant metastasis control and overall survival after 15 years. However, not all patients with high-risk features experience disease recurrence after surgery alone, and some subsets of patients experience suboptimal disease control and survival despite immediate postoperative radiotherapy. Furthermore, some patients without high-risk features will develop recurrence. The present review discusses the current data and potential future directions to improve individualization of therapy after prostatectomy.

Esophageal eosinophilia in pediatric patients with celiac disease: is it a causal or an incidental association?

OBJECTIVES: Celiac disease (CD) and eosinophilic esophagitis (EoE) are 2 distinct disease entities affecting the gastrointestinal tract of pediatric patients. Recently it has been suggested that EoE is more prevalent in patients with celiac disease than in the general population. We studied the association between these 2 disease entities in our pediatric patients. METHODS: We reviewed our hospital files for suspected or confirmed cases of CD. Only cases with both duodenal and esophageal biopsies in pediatric patients were included. A total of 120 patients who met these criteria were included as the disease group. We also selected 100 patients with no clinical suspicion of CD and included them as a control group. Slides were reviewed using established criteria for diagnosis of both conditions. Duodenal biopsies were categorized as positive, negative, and suspicious for CD, whereas esophageal biopsies were classified as either positive or negative for esophageal eosinophilia (EE). Serologic and clinical data were additionally collected. RESULTS: Sixty-two (62) cases were considered positive for CD in the disease group; among those 4 (6.5%) showed EE. In the control group, 91 cases were negative for CD, histologically, and 7 of those had EE (7.7%). Although 6 patients in the control group were histologically suspicious for CD, none of them had evidence of EE. CONCLUSIONS: Our findings show that, in our patient population, patients with CD are not more likely to have EE than patients undergoing upper endoscopy for other reasons. Therefore, we suggest that the association between CD and EE is likely incidental and not causal.

Expression of cadherin 17 in well-differentiated neuroendocrine tumours.

AIMS: Cadherin 17 (CDH17) is expressed primarily in normal intestinal epithelium and digestive tract tumours, and has limited expression in other neoplasms. The aims of this study were to examine CDH17 expression in well-differentiated neuroendocrine tumours (WDNETs) from various primary sites, representing the foregut, midgut, and hindgut, and tumours metastasizing to the liver, and to correlate the differences between the expression of CDH17, CDX2, and thyroid transcription factor 1 (TTF1). METHODS AND RESULTS: We investigated CDH17 immunohistochemical expression in 150 primary WDNETs from eight anatomical sites, including 68 from the foregut, 70 from the midgut, and 12 from the hindgut, and 15 metastases. CDH17 immunoreactivity increased significantly from foregut to hindgut WDNETs (P < 0.0001). Pancreatic WDNETs expressed CDH17 at a significantly higher frequency than other foregut tumours. Within the midgut, appendiceal and small-intestinal WDNETs were more frequently positive for CDH17 than for CDX2. All hindgut WDNETs expressed CDH17, in contrast to CDX2 (positive in one rectal case). CDH17 expression in liver metastases was similar to that of the primary tumours. CONCLUSIONS: This study is the first to comprehensively examine CDH17 expression in WDNETs from different sites. CDH17 is a sensitive marker for midgut WDNETs, and the CDH17+/CDX2-/TTF1- phenotype was found to be sensitive (92%) and specific (91%) for hindgut WDNETs.

A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing.

BACKGROUND: Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. METHODS: THREE DNA EXTRACTION METHODS WERE COMPARED: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student's t-test. RESULTS: The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. CONCLUSIONS: Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

MDM2 copy numbers in well-differentiated and dedifferentiated liposarcoma: characterizing progression to high-grade tumors.

OBJECTIVES: MDM2 gene amplification is associated with well-differentiated (WDL) and dedifferentiated liposarcomas (DDL). Using fluorescent in situ hybridization (FISH), we sought to characterize various patterns of MDM2 amplification among the morphologic spectrum of liposarcoma. METHODS: Forty-six cases of liposarcoma in various sites were examined and included 22 WDLs, 14 DLLs, and 10 negative control subjects. RESULTS: The MDM2 amplification ratio (MDM2/CEP12) was lower in WDL (10.2) compared with DDL (18.3) cases (P = .0000002). An amplification ratio of 16 showed optimal sensitivity (0.86) and specificity (0.96) as a cutoff point for progression to DDL. Borderline areas, defined as tumors with increased cellularity and atypia but with preserved lipomatous differentiation, showed a significantly higher MDM2 ratio (17.5; P = .0007) compared with WDL. Central (retroperitoneal and intra-abdominal) tumors also showed a significantly higher MDM2 ratio than peripheral ones (P = .02). CONCLUSIONS: Differences in MDM2 amplification profiles among liposarcomas could help further define and predict progression to high-grade neoplasia.