Biopsy Size Research Articles

Factors associated with residual tumor at time of Mohs micrographic surgery for basal cell and squamous cell carcinomas

BackgroundResidual tumor is not always clinically apparent following biopsy of cutaneous carcinomas, which may prompt patients to question the need for definitive treatment. ObjectiveWe investigated the percentage of cases in which residual tumor was histologically present at the time of Mohs micrographic surgery (MMS) for basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) and investigated factors associated with residual tumor. MethodsWe examined 483 MMS cases performed for biopsy-proven BCC (n=287) and SCC (n=196) between October 2022 and April 2023. Single-stage MMS specimens were step-sectioned en face to exhaust the block. Univariate and multivariable logistic regression models were created. ResultsResidual tumor was identified in 83.3% of BCC and 66.8% of SCC at time of MMS (p=0.01). In patients clinically appearing tumor-free following biopsy, residual histologic tumor was identified in 68.2% of BCC and 41.5% of SCC. Residual tumor was significantly more likely in men (p=0.04), high-risk sites (p=0.002), smaller biopsy sizes (p=0.0003), and larger pre-operative sizes (p<0.0001). LimitationsSingle center, retrospective cohort ConclusionThe majority of patients with BCC and SCC have residual histologic tumor at the time of MMS, oftentimes even when tumor is not clinically apparent. Multiple factors impact the presence/absence of residual tumor.

Advancements of non‐invasive imaging technologies for the diagnosis and staging of liver fibrosis: Present and future

AbstractLiver fibrosis is a reparative response triggered by liver injury. Non‐invasive assessment and staging of liver fibrosis in patients with chronic liver disease are of paramount importance, as treatment strategies and prognoses depend significantly on the degree of fibrosis. Although liver fibrosis has traditionally been staged through invasive liver biopsy, this method is prone to sampling errors, particularly when biopsy sizes are inadequate. Consequently, there is an urgent clinical need for an alternative to biopsy, one that ensures precise, sensitive, and non‐invasive diagnosis and staging of liver fibrosis. Non‐invasive imaging assessments have assumed a pivotal role in clinical practice, enjoying growing popularity and acceptance due to their potential for diagnosing, staging, and monitoring liver fibrosis. In this comprehensive review, we first delved into the current landscape of non‐invasive imaging technologies, assessing their accuracy and the transformative impact they have had on the diagnosis and management of liver fibrosis in both clinical practice and animal models. Additionally, we provided an in‐depth exploration of recent advancements in ultrasound imaging, computed tomography imaging, magnetic resonance imaging, nuclear medicine imaging, radiomics, and artificial intelligence within the field of liver fibrosis research. We summarized the key concepts, advantages, limitations, and diagnostic performance of each technique. Finally, we discussed the challenges associated with clinical implementation and offer our perspective on advancing the field, hoping to provide alternative directions for the future research.

Dispase/collagenase cocktail allows for coisolation of satellite cells and fibroadipogenic progenitors from human skeletal muscle.

Satellite cells (SCs) and fibroadipogenic progenitors (FAPs) are progenitor populations found in muscle that form new myofibers postinjury. Muscle development, regeneration, and tissue-engineering experiments require robust progenitor populations, yet their isolation and expansion are difficult given their scarcity in muscle, limited muscle biopsy sizes in humans, and lack of methodological detail in the literature. Here, we investigated whether a dispase and collagenase type 1 and 2 cocktail could allow dual isolation of SCs and FAPs, enabling significantly increased yield from human skeletal muscle. Postdissociation, we found that single cells could be sorted into CD56 + CD31-CD45- (SC) and CD56-CD31-CD45- (FAP) cell populations, expanded in culture, and characterized for lineage-specific marker expression and differentiation capacity; we obtained ∼10% SCs and ∼40% FAPs, with yields twofold better than what is reported in current literature. SCs were PAX7+ and retained CD56 expression and myogenic fusion potential after multiple passages, expanding up to 1012 cells. Conversely, FAPs expressed CD140a and differentiated into either fibroblasts or adipocytes upon induction. This study demonstrates robust isolation of both SCs and FAPs from the same muscle sample with SC recovery more than two times higher than previously reported, which could enable translational studies for muscle injuries.NEW & NOTEWORTHY We demonstrated that a dispase/collagenase cocktail allows for simultaneous isolation of SCs and FAPs with 2× higher SC yield compared with other studies. We provide a thorough characterization of SC and FAP in vitro expansion that other studies have not reported. Following our dissociation, SCs and FAPs were able to expand by up to 1012 cells before reaching senescence and maintained differentiation capacity in vitro demonstrating their efficacy for clinical translation for muscle injury.

Abstract 323: Optimization of a liquid biopsy assay to accommodate low cfDNA samples and increase throughput

Abstract Molecular testing that interrogates cell free DNA (cfDNA) from plasma has significant potential value across the cancer care continuum, including early cancer detection, driver mutation identification, and disease recurrence monitoring. To achieve this potential, liquid biopsy assays must be optimized to address multiple challenges. For one, cfDNA quantity in the blood is highly variable, with different amounts present depending on malignancy and stage of progression. This requires optimization to detect low concentrations of circulating tumor DNA (ctDNA) and to accommodate variable tumor fractions. In addition, minimizing assay costs will improve test accessibility and adoption. To address these issues, Genece Health is optimizing their lung cancer liquid biopsy assay to accommodate minimal cfDNA input into library preparation. Furthermore, Genece is testing algorithms that tolerate reduced read depths from low-pass whole genome sequencing (LP-WGS) while maintaining high performance. The Genece lung cancer liquid biopsy assay profiles fragment end-motif and size (FEMS) and fragment coverage signals using LP-WGS (Illumina) and proprietary machine learning algorithms. This study first tested the frequency of end motifs in pooled ctDNA after modifying library preparation (IDT) to reduce reagent volumes from 1X down to 0.5X, 0.25X, and 0.1X of manufacturer specification. A range of cfDNA input from 0.25 ng to 6 ng was tested with each library preparation volume. In addition, LP-WGS read coverage from 5X to 1X was evaluated to assess impact on end motif frequency and assay sensitivity. Performance characterization was further evaluated with a cohort of 50 clinical lung cancer and 100 healthy donor plasma samples tested at 1X and 0.25X library reagent volume. We observed an expected increase in library complexity at higher cfDNA input and library reagent volume. Assay performance was minimally impacted by variable cfDNA input and library reagent volume. However, reducing sequencing read depth is tolerated down to 1.25X depth before increasing the variability of end motif frequencies. These observations are corroborated in the 150 clinical sample cohort. This data shows that by altering standard library preparation conditions and reducing unique read depth, liquid biopsy assays can accommodate a wider range of cfDNA, while concurrently decreasing overall costs. Citation Format: Michael Salmans, Dasom Kim, Andrew Carson, Mengchi Wang, Byung In Lee, Bryan Leatham. Optimization of a liquid biopsy assay to accommodate low cfDNA samples and increase throughput [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 323.

Abstract 5660: Gene expression signatures from FFPE to predict recurrent metastatic nasopharyngeal cancer

Abstract Nasopharyngeal cancer (NPC) is an EBV-driven epithelial cancer endemic to Southeast Asia, Southern China and the Middle East. Accurate profiling of the gene expression of NPC has been challenging because of its intense inflammatory infiltrate, as well as the 3 - 4mm size of biopsies limiting the amount of available material. Here we present a large cohort of 339 micro-dissected gene expression libraries from formalin-fixed paraffin-embedded (FFPE) biopsies from NPC patients treated at the National University Health System, Singapore, as well as healthy controls. NPC tumors from patients who subsequently developed recurrent or metastatic disease (Group A), and patients who did not develop recurrent or metastatic disease (Group B) were profiled. Tumor epithelial and microenvironment compartments were separately obtained using laser-capture microdissection, followed by library preparation performed for RNA-Seq using an in-house specialised technique. Gene signatures for NPC tumor epithelial and microenvironment content were used to validate the purity of gene expression libraries. Our preliminary analysis revealed that primary, pre-treatment tumors from patients who subsequently developed recurrence (Group A) showed downregulation of processes related to interferon gamma and interferon alpha response (p-adj &amp;lt;0.001 for both), while processes related to epithelial-mesenchymal transition (p-adj &amp;lt; 0.05) were enriched compared to patients who remained healthy (Group B). In contrast to primary tumors, recurrent tumors in Group A were enriched for cellular respiration including oxidative phosphorylation (p-adj &amp;lt; 0.0001). Our approach highlights the utility of whole transcriptome profiling from small quantities of archival FFPE material. The gene signatures identified from the primary tumors of high-risk patients are biologically relevant and have the potential to be used in precision medicine to guide additional targeted intervention. Citation Format: Joshua K. Tay, Wei Keat Teo, Joseph W. Foley, Luvita Suryani, Bing Cheng Wu, Chor Hiang Siow, Kwok Seng Loh. Gene expression signatures from FFPE to predict recurrent metastatic nasopharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5660.

Assessing the effectiveness and safety of transbronchial lung cryobiopsy utilizing a flexible bronchoscope with an endobronchial blocker in diffuse parenchymal lung lesions.

Transbronchial lung cryobiopsy (TBLC) with flexible bronchoscope represents an encouraging modality to obtain a larger size specimen without crush artifact, and a higher diagnostic yield in patients with diffuse parenchymal lung lesions/diseases as compared to conventional transbronchial lung biopsy, and fewer complications as opposed to surgical lung biopsy. Artificial airway is preferred as it provides better airway protection in cases of severe bleeding. Although various researchers have published data on different modalities, the data is not sufficient to standardize a single technique. This study describes the procedural technique, safety, and yield of TBLC using a flexible bronchoscope with an endobronchial blocker. We performed a retrospective analysis of 100 consecutive patients who underwent TBLC using flexible bronchoscopy from May 2018 to June 2022. TBLC samples were obtained under moderate sedation without the use of artificial airway or fluoroscopy. Among the 100 patients, the majority were male (63%). The mean age of the enrolled patients was 44.43±15.92 years. The predominant diagnoses in our study were hypersensitivity pneumonitis (27%), followed by sarcoidosis (12%) and tuberculosis (10%). We obtained alveolated lung tissue in 90 out of 100 cases with a median biopsy size of 5 mm (in greatest dimension, interquartile range 5-4 mm), resulting in a specific histopathological diagnosis in 82 cases. The most frequent complications were bleeding and pneumothorax (13%). Mild bleeding occurred in 58% of the patients, and moderate bleeding occurred in 20% of the patients. There was no episode of severe/life-threatening bleeding. None of the patients required intensive care unit admission or endotracheal intubation. In conclusion, the use of TBLC through flexible bronchoscopy with an endobronchial blocker emerges as a minimally invasive, secure, time-efficient, and readily reproducible technique. Significantly, this procedure can be seamlessly executed in the bronchoscopy suite, eliminating the requirement for an artificial airway or general anesthesia.

A Subset of Classical Hodgkin Lymphoma Cases Demonstrate CD19 Expression on Hodgkin Reed-Sternberg Cells By Immunohistochemistry

Background: The neoplastic Hodgkin Reed-Sternberg (HRS) cells in Classical Hodgkin Lymphoma (CHL) are thought to lose their B-cell phenotype and lack expression of most B-cell markers. Improvements in antibodies and detection methods have revealed that a subset of CHL cases may express some B-cell markers, including CD19 and CD20, with currently unclear significance. Introduction of CD19-directed therapies has changed the landscape of salvage treatment in CD19 expressing Non-Hodgkin Lymphomas. As such, we sought to characterize CD19 expression in HRS cells and its clinical implications and potential utility as a therapeutic target. Methods: We conducted an Institutional Review Board-approved retrospective review of records for CHL cases from 01/2015 to 07/2020 at a tertiary care center. 42 cases met the criteria and had sufficient tissue for analysis: 39 CHL, 2 CHL mixed with other B-cell lymphomas, and 1 Epstein Barr Virus Post-Transplant Lymphoproliferative Disease (EBV-PTLD)-CHL type. Cases were assessed for CD19 immunohistochemical (IHC) staining using the EP169 Rabbit Monoclonal Antibody. HRS cells were identified via morphology and characteristic immunoprofile, with all HRS cells expressing strong CD30 and dim PAX-5. CD19 staining intensity (0-3+) and pattern of expression (cytoplasmic, Golgi body, membranous) in HRS cells was independently evaluated by three hematopathologists. Based on their consensus, CD19 staining was categorized as negative, weak (variable across HRS cells), or positive. CD19 staining was compared with histologic subtype, other B-cell marker staining, EBV association (via EBER-ISH), and clinical data and outcomes, including, age, stage, treatment, response, overall survival, and demise. Results: Histologically, cases were composed of the following subtypes: 26 nodular sclerosis, 8 mixed cellularity, 3 lymphocyte-rich, 1 lymphocyte-depleted, 2 other (EBV-PTLD and malignant B-cell lymphoma) and 2 unclassifiable (due to treatment effect and biopsy size). Nine of 42 cases (21%) were EBV-associated by EBER-ISH. CD19 staining of any kind in HRS cells was found in 13/42 cases (31%), with 10/42 (24%) demonstrating positive staining and 3/42 (7%) demonstrating weak staining. Staining intensity and pattern varied across positive cases (Table 1 and Figure 1). CD19 staining was seen in 4/26 nodular sclerosis (15%), 6/8 mixed cellularity (75%), 1/3 lymphocyte-rich (33%), and 1/1 lymphocyte-depleted (100%) cases. The strongest correlation with other markers was seen with EBV, with 7/9 (78%) EBV+ cases staining with CD19. Five cases with CD19 staining also demonstrated predominantly patchy/dim CD20 staining. The clinical characteristics of CHL cases with CD19 expression varied, with an age range of 10-78 years old and variable clinical stage at time of tissue sampling. Following initial treatment, 4/13 patients failed to achieve remissions after frontline chemotherapy (mostly ABVD), with 3 of these patients now deceased due to disease. Conclusions: A significant subset of CHL cases demonstrate CD19 expression on HRS cells, and EBV-associated CHL has a high rate of CD19 expression, which may reflect its different underlying disease mechanism. Larger studies are necessary to characterize the staining pattern and intensity that may be most amenable to specific therapies and to better elucidate the prognostic implications of CD19 expression. Given the accessibility of CD19 IHC and the growing list of effective CD19-directed therapies, it is worthwhile assessing CD19 expression, particularly in EBV associated CHL, and further research into the utility of CD19 expression on HRS as a potential therapeutic target is warranted.

Incidental prostate cancer after holmium laser enucleation of the prostate: Critical analysis of independent risk factors and impact on surgical outcomes.

The objectives of this study are to evaluate the impact of incidental prostate cancer (iPCa) and its different grade group (GG) on the surgical outcomes of holmium laser enucleation of the prostate (HoLEP) and, furthermore, to assess the independent risk factors associated with the detection of iPCa. A retrospective chart review was conducted at a single institution for HoLEP cases that were performed between 2017 and 2022. Patients with a preoperative diagnosis of prostate cancer and those without baseline prostate-specific antigen (PSA) levels within 1 year were excluded. Four hundred seventeen patients were divided into three groups: benign prostatic hyperplasia-377; clinically insignificant (GG 1)-29; and clinically significant prostate cancer (GG 2-5)-11. The preoperative parameters analysed included age, body mass index, race/ethnicity, use of 5-alpha-reductase inhibitors, PSA, prostate size, PSA density, and history of negative prostate biopsy. To evaluate the association between clinical and demographic variables, a multivariable-adjusted logistic regression analysis was performed. We also assessed intraoperative and post-operative outcomes among these three groups. A total of 417 patients were analysed; 40 (9.6%) patients had iPCa, of which 29 (72.5%) and 11 (27.5%) were clinically nonsignificant and significant prostate cancer, respectively. Of all the demographic and preoperative variables analysed, hypertension was significantly associated with overall prostate cancer diagnosis (p< 0.05), and no other variable including patient age, preoperative PSA, PSA density, prostate size, or prior prostate biopsy were associated with increased risk of overall prostate cancer or clinically significant prostate cancer diagnosis. Most cases of iPCa were GG1, and 34 (85%) were managed with active surveillance. The rate of iPCa after HoLEP was 9.6%, with most cases being GG 1 (72.5%) and managed through active surveillance. Age, prostate size, baseline PSA, and prior negative prostate biopsies were not associated with increased risk of iPCa.

Radial Endobronchial Ultrasound-guided Transbronchial Cryobiopsy versus Forceps Biopsy for the Diagnosis of Solitary Pulmonary Nodules: A Prospective Randomised Trial.

Improvements in pulmonary diagnostic imaging and the development of lung cancer screening are increasing the prevalence of Solitary pulmonary nodules (SPNs). Fluoroscopically guided radial endobronchial ultrasound (EBUS) with transbronchial forceps biopsy (TB-FB) has been the conventional diagnostic method. Transbronchial cryobiopsy (TB-CB) is an alternative biopsy method. We sought to compare transbronchial cryobiopsy to transbronchial forceps biopsy for the diagnosis of SPNs. A prospective, single-centre, randomised controlled trial was conducted at the Royal Adelaide Hospital (RAH). Patients with SPNs were randomised to either 5 transbronchial forceps biopsies or one transbronchial cryobiopsy. Complete blinding of investigators and participants was not possible, as transbronchial cryobiopsy required general anaesthesia. The primary outcome was diagnostic yield with secondary outcomes of specimen size, diagnostic yield for subsets challenging to access with forceps and safety. The overall diagnostic yield for the 28 enrolled subjects was 76.8%(22/28). The diagnostic yield was 91.7% (11/12 patients) for transbronchial cryobiopsy and 68.8% (11/16 patients) for forceps biopsy (p=0.14). Median biopsy sizes were consistently larger for the cryobiopsy arm at 7.0mm compared to 2.5mm(p<0.0001). An eccentric EBUS image signalling the probe was adjacent to the nodule occurred in 4/28 cases, and TB-CB confirmed a diagnosis in 3/3 randomised to this arm. There were no major complications with either technique. Transbronchial cryobiopsy under the guidance of fluoroscopy and radial EBUS facilitates larger biopsy specimens without a significant increase in major complications. Further research is required to confirm the effect on diagnostic yield; however, our study supports a role for TB-CB in the diagnosis of SPNs and small, nodule-adjacent biopsies. Reference number of R20160213(HREC/16/RAH/37).

Comparison between thoracoscopic pleural cryobiopsy and conventional forceps biopsy in diagnosis of exudative pleural effusion: Assiut University experience

Abstract Context Exudative pleural effusion is a diagnostic dilemma that includes many steps; one of them is obtaining a definite diagnosis through pleural biopsy. Cryoprobes are being increasingly used for obtaining larger specimens with fewer crush artifacts. However, the safety and feasibility of cryoprobe biopsy compared with standard forceps for pleural biopsy have not been fully assessed. Aims To evaluate the diagnostic value, size, and quality of the specimens obtained by flexible cryoprobe in comparison with those obtained by flexible forceps probe during medical thoracoscopy in patients with exudative pleural effusion and to assess the possible complications from the procedure. Settings and design This interventional prospective study was carried out at the endoscopy unit at Chest Department and Tuberculosis Assiut University Hospital. Patients and methods This study included 60 patients with undiagnosed exudative pleural effusion. Medical thoracoscopy was carried out for all the patients, and pleural biopsies were taken from the parietal pleura using a conventional rigid forceps probe and flexible cryoprobe in the same settings. Results Cryoprobe biopsy established a definite diagnosis in 55/60, with 91.6% diagnostic yield, whereas forceps biopsy had a definite diagnosis in 53/60, with 88.3% diagnostic yield. The size of cryoprobe biopsy was significantly larger in comparison with the forceps biopsy (26.56 ± 22.16 vs. 17.38 ± 12.08 mm2; P&lt;0.001). The depth of pleural biopsy was evaluated by the presence of extrapleural fat cells, which were significantly higher in cryoprobe biopsy in comparison with forceps biopsy [21 (35%) vs. 11 (18.3%); P=0.03]. There were no significant complications or procedure-related deaths. Conclusions Cryobiopsy is a possible safe and effective alternative to conventional forceps probe biopsy in the diagnosis of exudative pleural effusion with a larger, deeper, and less number of biopsies. It was also found that cryoprobe biopsy had a better diagnostic yield, sensitivity, and accuracy.

Virtual biopsies: Proof of concept for a novel quantitative approach to liver biopsy adequacy and pathology education.

To quantitatively measure liver biopsy adequacy requirements and the effect of a teaching intervention that uses a virtual biopsy platform. A library of virtual liver biopsies was created using digital whole-slide, trichrome-stained tissue sections from liver resection material and QuPath image analysis software. Blinded participants staged fibrosis on the virtual biopsies before and after a teaching intervention. This platform both modeled adequacy requirements for cirrhosis diagnosis on biopsy material and measured the effect of a teaching intervention on participant performance. Using this platform, diagnostic accuracy for cirrhosis could be modeled according to the function y = λ(1 ‒ e‒x/γ). The platform demonstrated that the relationship between biopsy size and diagnostic accuracy was statistically significant and that biopsies smaller than 6 mm long and 0.8 mm wide were insufficient to diagnosis cirrhosis. The platform also measured improvement in fibrosis staging accuracy among participants following a teaching intervention. These results provide proof of concept for a virtual biopsy method by which outstanding questions in anatomic pathology can be addressed quantitatively using open source software. Future work is needed to validate these findings in clinical practice.

Histomorphological characteristics of prostate specimens analyzed at a referral hospital in Kenya.

Background: Global shift to high energy foods is important risk factor for new variants of prostate cancer. Some prostate tumors have an indolent course while others have an aggressive course; therefore, knowledge of tumor subtypes can help in clinical decision-making based on the patient profile. Methods: The main aim of the study was to determine the histomorphological characteristics of prostate specimens analyzed at regional referral hospital. The study was a crosssectional retrospective study. The target specimens in this study consisted of prostate specimens that had prostate specific antigen level and were analyzed and reported between 2017 and 2022 at Jaramogi Odinga Oginga teaching and referral hospital, Kisumu Kenya. Results: Prostate color was not reported in eight reports. Of the 72 specimens observed, 50 (69.4%) were reported to be white, 12 (16.7%) were reported to be tan/white, 9 (12.5%) were tan brown, and 1 (1.4%) was tan grey. There was significant variation (p=0.001) in prostate specimen color. Of the 80 specimens, 47 (58.75%) had coarse surface, 16(20.00%) nodulated surfaces and 17 (21.25%) shrunken surfaces. There was variation in prostate surfaces (p&lt;0.00001, X2=23.275). Majority of specimens 55 (68.8%) measured between 0-29 mm, 12 (15%) measured 30-59 mm, 7 (8.8%) measured 60-89, 3 (3.8%) measured 90-119, 2 (2.5%) measured &gt;150 mm and 1 (1.3%) measured 120-149 mm. There was no significant difference in the prostate biopsy sizes in comparison to the mean (p=0.984, t=0.020, 95% CI). The majority 20 (25%), of specimens had prostate cancer 15 (18.75%) had atypical findings, and 2 (2.5%) had high-grade proliferative intraepithelial neoplasia. The microscopic features of the groups did not differ significantly (F2, 34 = 1.469, p = 0.244). There is positive correlation between Gleason scores and Prostate specific antigen levels (p = 0.004, r = 0.474). Conclusion: There is variation of specimen color in prostate specimen with prostate cancer implying that advanced prostate disease causes changes in prostate color. Atypical prostate findings are common in age 50-59 which may suggest that targeted prevention and intervention should focus on this age group. Higher Gleason scores are likely to be observed in patients with higher PSA levels among patients being evaluated for prostate tumors.

Immunohistochemical Patterns of Pituitary Adenomas in Southeastern Nigeria, a 10-year Histopathologic Review

ABSTRACT Introduction: Pituitary adenomas [PAs] constitute the third most common primary intracranial tumours, with a wide prevalence rate of 1% to 40%. Histologic (H & E) classification into acidophilic, basophilic and chromophobic adenomas have little clinical relevance but WHO recommended immunohistochemical subclassification has both therapeutic and prognostic significance. This immunohistochemical subclassification has not been done in our environment, making it imperative for us to evaluate the patterns in our environment. Aim: To determine the immunohistochemical patterns of PAs in Southeastern Nigeria. Materials and Methods: This was a 10-year retrospective review of all PA biopsies received at University of Nigeria Teaching Hospital Enugu, Memphys Hospital for Neurosurgery Enugu and Grace Pathology Consults Enugu, Nigeria. The age, sex, histologic, immunohistochemical subtypes and biopsy size of all diagnosed PAs were analyzed using Statistical Package for Social Sciences (SPSS) version 20 (New York: IBM Inc.) and the results were expressed in descriptive statistics. Results: One hundred cases of PAs were identified in this study constituting 19.6% of all primary intracranial tumors received at our study centers during the period under review. There were 45 (45.0%) females and 55 (55.0%) males giving a female to male ratio of 1:1.2, and a mean age of 45.3 years. The commonest histologic type was acidophilic adenoma (49.0%), followed by basophilic (40.0%) and chromophobic (11.0%) adenomas. Null cell adenomas were the most common immunohistochemical subtype (44.0%), followed by PRL-secreting adenomas (27.0%). Others were Luteinizing hormone (LH) − 13 (13.0%), follicle stimulating hormone (FSH) − 7 (7.0%), growth hormone (GH) − 3(3.0%), TSH − 2(2.0%) and ACTH − 1(1.0%) adenomas. Conclusion: PAs predominate amongst males, occurring mostly in the middle age groups in Southeastern Nigeria. Null cell adenoma is the commonest immunohistochemical subtype followed by PRL-secreting adenomas. Routine immunohistochemical characterization is required for accurate diagnosis and optimal patient care.

394 Gene expression based molecular test proves clinical validity as diagnostic aid for the differential diagnosis of psoriasis and eczema in formalin fixed and paraffin embedded tissue

Abstract Highly specific and efficient drugs have been developed to treat noncommunicable chronic inflammatory skin diseases (ncISD). Due to their specificity, these drugs require precise diagnostics to attribute the most efficient treatment to each patient. Diagnosis is complicated by the complex pathogenesis of ncISD and their clinical and histological overlap. Especially, precise diagnosis of psoriasis and atopic eczema is difficult in special cases, and molecular tools need to be developed to support gold standard diagnosis. Psoriasis and eczema are among the most prevalent skin diseases worldwide with a combined incidence rate of at least 2%. Although more than a dozen of immune-modulating therapies, particularly monoclonal antibodies targeting specific cytokines or their receptors, have become available over the last decade allowing highly effective treatment, the ultimate breakthrough would include accurate diagnostic tools. Diagnosis of both diseases largely depends on a subjective visual exam and histopathological examination. Due to phenotypical overlaps of both diseases, up to 50% of cases with a palmoplantar localization are misdiagnosed. To close this diagnostic gap, a gene expression-based classifier using NOS2 and CCL27 has been proposed and its clinical validity examined and proven in various patient cohorts. This study aims to develop a real-time based molecular classifier (MC) to distinguish psoriasis from atopic eczema in formalin-fixed, paraffin-embedded (FFPE)-fixed skin samples for diagnostic measures and to evaluate the potential of minimally invasive microbiopsies and noninvasive tape strips or sampling disks for molecular diagnosis. The FFPE, micro-(Ø1mm) and macrobiopsies (Ø4–6 mm), and tape strips were collected from psoriasis and eczema lesions and analysed by real-time polymerase chain reaction (PCR) for the expression of NOS2 and CCL27. A molecular classifier (MC) was established using a linear regression model. We transferred the RNAprotect-based molecular classifier to FFPE samples and were able to produce comparable results. Whereas the RNAprotect based MC distinguished psoriasis from eczema with a sensitivity and specificity for psoriasis of 97.7% and 100%, respectively, and an AUC of 0.99, the FFPE-based classifier determined probabilities for psoriasis with a sensitivity and specificity and of 92% and 100%, respectively, and an AUC of 0.97. To test if microbiopsies are also adequate tissue samples for the MC, we analysed gene expression in 83 pairs of macro- and microbiopsies by real-time quantitative-PCR. Delta-CT values showed no significant difference between macro- and microbiopsies. The MC led to reliable results in the vast majority of samples (91.57%) independent of biopsy size, with a small increase of misclassification (4.98%). The FFPE-based molecular classifier (MC) precisely separates eczema from psoriasis and efficiently identifies subtypes of both diseases. The reliability of the two markers NOS2 and CCL27 has been shown in FFPE tissue facilitating a possible implementation of this molecular diagnostic aid in routine clinical pathological practice. We further show that the gene expression profile of RNA later fixed microbiopsies is comparable to standard 4–6 mm biopsies and that microbiopsies are equally suited for the MC. We demonstrate the potential of tape strips of inflamed epidermis for molecular diagnostics of ncISD. Due to the minimally invasive sampling procedure, tape stripping may be particularly useful for the testing of visible and sensitive skin areas and in children as well as for repetitive sampling procedures necessary to monitor treatment responses over time. In summary, the MC discriminates psoriasis from eczema producing meaningful results in a broad range of skin tissue derived from invasive to minimally invasive sampling techniques. Further efforts are required to simplify and shorten the labor- and cost-intensive procedures of RNA isolation and real-time PCR. One possible solution is a fully automated and closed system, which would allow the integration of molecular diagnostics into the routine patient management at the point of care.

Predictors of Prostate Cancer at Fusion Biopsy: The Role of Positive Family History, Hypertension, Diabetes, and Body Mass Index

PSA density and an elevated PI-RADS score are among the strongest predictors of prostate cancer (PCa) in a fusion biopsy. Positive family history, hypertension, diabetes, and obesity have also been associated with the risk of developing PCa. We aim to identify predictors of the prostate cancer detection rate (CDR) in a series of patients undergoing a fusion biopsy. We retrospectively evaluated 736 consecutive patients who underwent an elastic fusion biopsy from 2020 to 2022. Targeted biopsies (2-4 cores per MRI target) were followed by systematic mapping (10-12 cores). Clinically significant PCa (csPCa) was defined as ISUP score ≥ 2. Uni- and multi-variable logistic regression analyses were performed to identify predictors of CDR among age, body mass index (BMI), hypertension, diabetes, positive family history, PSA, a positive digital rectal examination (DRE), PSA density ≥ 0.15, previous negative biopsy status, PI-RADS score, and size of MRI lesion. The median patients' age was 71 years, and median PSA was 6.6 ng/mL. A total of 20% of patients had a positive digital rectal examination. Suspicious lesions in mpMRI were scored as 3, 4, and 5 in 14.9%, 55.0%, and 17.5% of cases, respectively. The CDR was 63.2% for all cancers and 58.7% for csPCa. Only age (OR 1.04, p < 0.001), a positive DRE (OR 1.75, p = 0.04), PSA density (OR 2.68, p < 0.001), and elevated PI-RADS score (OR 4.02, p = 0.003) were significant predictors of the CDR in the multivariable analysis for overall PCa. The same associations were found for csPCa. The size of an MRI lesion was associated with the CDR only in uni-variable analysis (OR 1.07, p < 0.001). BMI, hypertension, diabetes, and a positive family history were not predictors of PCa. In a series of patients selected for a fusion biopsy, positive family history, hypertension, diabetes, or BMI are not predictors of PCa detection. PSA-density and PI-RADS score are confirmed to be strong predictors of the CDR.

Adjuvant treatment decisions among adults aged 65 years and older with early-stage hormone receptor positive breast cancer seen in a simple multidisciplinary clinic versus standard consultation

IntroductionRandomized studies support de-escalation of adjuvant therapy for a target population of older adults ≥65 years with stage I, estrogen-receptor (ER) positive breast cancer after breast conserving surgery. We sought to evaluate the impact of a simplified multidisciplinary clinic (s-MDC) in this population by comparing treatment patterns and patient perceptions of adjuvant radiation therapy (RT) and hormone therapy (HT) between patients seen in s-MDC vs. standard consultations. Materials and MethodsMedical records were retrospectively reviewed for patients in the above target population who underwent surgery between August 2020 and May 2022 at our institution. Two cohorts were included: (1) patients seen in s-MDC, and (2) patients seen in standard clinic separately by medical and radiation oncology (non-s-MDC cohort). The non-s-MDC patients declined, could not attend, and/or were not referred to the s-MDC. Patients in the s-MDC cohort were prospectively administered validated questionnaires to evaluate patient reported data including the Decision Autonomy Preference Scale (DAPS), e-Prognosis, and Medical Maximizing-Minimizing Scale (MMS). Chi square, t-tests, and non-parametric equivalents compared demographics, and logistic regression evaluated RT and HT use and survey score outcomes between cohorts. ResultsA total of 127 patients met inclusion criteria, with 33 s-MDC and 94 non-s-MDC patients. There was no difference between the cohorts in age, margin status, histology, grade, or focality. In the s-MDC cohort there were significantly more patients without sentinel lymph node biopsy (71.3% vs 42.4%, p = 0.003) and mean tumor size was smaller (0.69 vs. 0.96 cm, p < 0.003), and Charlson comborbidity index (CCI) was higher (5.21 vs 4.96, p = 0.038). There was no significant difference in receipt of RT (65% s-MDC vs 77% standard; odds ratio [OR] = 0.55, p = 0.189), HT (78% ss-MDC vs 72% standard; OR = 1.36, p = 0.513), or both (50% s-MDC vs 59% standard; OR = 0.7, p = 0.429). The s-MDC cohort was significantly more likely to undergo accelerated (vs. standard hypofractionated) RT (70% vs 39%; OR = 3.59, p = 0.020). In s-MDC patients with completed questionnaires (n = 33), all whose selected “mostly patient (n=6)” based decision making by DAPS chose RT while all “mostly doctor (n=1)” chose no RT. Based on e-Prognosis, there were lower odds of RT for increasing Schonberg score/ higher 10 yr mortality risk (OR 0.600, p = 0.048). MMS score ≥ 40 (“maximizer”) was strongly linked with the use of RT (OR 18.57, p = 0.011). DiscussionFor adults ≥65 years with early stage, ER positive breast cancer, s-MDC participation was not significantly associated with lower use of adjuvant RT or HT versus standard consultation but was significantly associated with shorter RT courses. DAPS and MMS results indicate that patient treatment preference may be predictable, highlighting an opportunity to tailor consultation discussions and recommendations based on intrinsic patient preferences and individual goals.

Abstract 2991: Complementarity of class I and II neoantigen mapping in MSI-high colorectal cancer in needle biopsy size tissue samples

Abstract Colorectal cancer (CRC) is currently the second most common cause of cancer related mortality in the world. One of the hallmarks of CRC is its complex mutational landscape that contributes to the derivation of neoantigens which aid the immunosurveillance of the cancer. Immunopeptides play an essential role in adaptive immunity by activating and ensuring T-cell specificity. Mass spectrometry (MS) is currently the only technology that can measure and identify the immunopeptide profiles of biological samples at a large scale, however, MS-based studies are frequently limited by sample input and poor scalability. Here, we introduce a semi-automated workflow requiring low sample input to robustly identify immunopeptides and apply it to a cohort of fresh frozen, high quality CRC samples (Indivumed) for immunopeptide profiling and neoantigen identification. We initially optimized a workflow that allowed the native lysis and sequential immunoprecipitation of class-I and class-II immunopeptides while ensuring scalability and reproducibility. The immunopeptides were thereafter subjected to our True Discovery FAIMS-DIA LC-MS/MS platform which we supported through a specific DDA library and searched the resulting data with Spectronaut (Biognosys) by applying a 1% FDR threshold. We utilized data from WGS on both tumor and adjacent normal tissue for the calling of high-confidence somatic variations (Indivumed) and the definition of the resulting neoantigens. We characterized 131,578 unique immunopeptides from 15 mg of fresh-frozen tumor tissue across 20 patients with CRC lesions that mapped to 12,488 genes. On average we identified 9,767 class-I and 16,445 class-II immunopeptides from all patients indicating significant inter-patient heterogeneity. Overall, we found the identification numbers of class-II immunopeptides to be more variable than that of class-I which might be linked to immune infiltration levels. From our pool of identified immunopeptides, we were able to detect 16 class-I and 29 class-II neoantigens, covering 87% of the microsatellite instability-high (MSI-H) samples in the cohort. Coverage of both classes was essential to increase neoantigen discovery as 53% MSI-H samples had neoantigens mapping to a single class. Interestingly we also detected a significant correlation between the mutational burden and the number of detected neoantigens in our patient cohort suggesting that the genetic set up of the cancer might influence the repertoire of presented neoantigens thereby controlling immunosurveillance. In summary, we have established a scalable and efficient pipeline for cell line and tissue immunopeptidomics for both class-I and II immunopeptides. Our pipeline generates high-quality identifications and can be deployed to help shed light on immunopeptidomics heterogeneity through large-scale profiling of patients as exemplified in the case of MSI-H CRC. Citation Format: Ilja E. Shapiro, Luca Räss, Christopher R. Below, Marco Tognetti, Tikira Temu, Oliver M. Bernhardt, Roland Bruderer, Jonathan Woodsmith, Lukas Reiter. Complementarity of class I and II neoantigen mapping in MSI-high colorectal cancer in needle biopsy size tissue samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2991.

260 Breast Cancer Biopsy Triage with Ex-Vivo Microscopy for Downstream Analysis

OBJECTIVES/GOALS: In this study, the ability of a pathologist to detect malignancy on digital pseudo-H&amp;E slides obtained via structured illumination microscopy (SIM) imaging of fresh diagnostic breast biopsies was assessed. The speed of imaging and processing was also assessed for potential clinical implementation. METHODS/STUDY POPULATION: This study was conducted in accordance with an Ochsner Medical Center of New Orleans IRB. 200 patients undergoing either stereotactic or ultrasound-guided diagnostic breast biopsies were consented and an additional core from the suspicious lesion was collected for research use. Research biopsies were transported to the lab and stained with DRAQ5 and Eosin-Y and imaged with SIM before being submitted for histology processing. Imaging and digital processing times were recorded. The resulting SIM images and histology slides were given to a pathologist for blind review to assess accuracy. RESULTS/ANTICIPATED RESULTS: The ex-vivo structured illumination microscopy images and subsequent histology slides from 79 research cores have been assessed to date. Some samples were excluded from the total data set and not included in the final assessment due to technical failures of the imaging protocol. Of the current set, the pathologist has a specificity of 88% and a sensitivity of 65%, as well as an NPV of 88% and a PPV of 65%. Staining time for each biopsy was completed within 3 and a half minutes and imaging at 20x magnification took between 4 and 12 minutes, depending on size and implementation of autofocus to the imaging system. Image processing took approximately 5 minutes per biopsy and is a direct function of biopsy size. DISCUSSION/SIGNIFICANCE: Decreased time between cancer suspicion and treatment will improve the prognosis of breast cancer patients. SIM imaging of fresh breast biopsies could ultimately allow primary and secondary histology to be performed simultaneously and minimize histopathology time, thus allowing clinicians and patients to implement treatment course more quickly.

Role of magnetic resonance imaging for preoperative prediction of early biochemical failure in localized prostate cancer.

The purpose of our study was to assess preoperative clinical biological and Magnetic Resonance Imaging (MRI) predictive factors of early biochemical failure (BF), defined as persistence of significant post-operative plasmatic prostate specific antigen (PSA) level after radical prostatectomy (RP) in patients with localized prostate cancer (PCa). In a retrospective cohort study we included 142 patients from our university hospital with newly diagnosed PCa, who underwent 3T multiparametric MRI prior to RP. Only the MRI target lesions [Prostate Imaging Reporting and Data System (PIRADS) ≥3] with histological correspondence were considered significant. Clinical, biological, MRI and pathological preoperative data were studied. We performed univariate and multivariate logistic regression analysis to identify significant parameters associated with early BF. Early BF occurred in 14% of patients (20/142). Patients with BF had higher PSA level at diagnosis, Gleason score, number of positive biopsies, size of the largest positive biopsy and higher National Comprehensive Cancer Network (NCCN) risk score (P<0.001 for all). According to MRI, they also had higher T stage and a higher size of capsular contact (P<0.001 for all). In contrast, there was no difference concerning neither ADC value, perfusion profile and zonal location of the index lesion. In multivariate analysis, the best combination of predictive factors of early BF was the association of preoperative Gleason score ≥4+3 [odds ratio (OR) =6.8 (1.4-32.5); P=0.002] and T stage ≥3 on preoperative MRI [OR =17.4 (3.2-94.9); P<0.001] with an area under the curve (AUC) of 0.89 [99% confidence interval (CI): 0.77-1], a negative predictive value of 94% and a positive predictive value of 75%. Combination of simple preoperative biomarkers as Gleason score and T stage according to MRI accurately stratify the risk of early BF following RP. These results emphasize the pivotal role of preoperative MRI for the management of localized PCa.

Quality parameters for RNA preparations from biopsies of ulcerated human skin.

Background: Obtaining high quality RNA from skin biopsies is complex due the physical composition and high content of nucleases of this tissue. This becomes particularly challenging when using compromised skin samples with necrotic, inflammed or damaged areas, such as those from patients suffering skin conditions, which affect more than 900 million people annually. We evaluated the impact of the biopsy size and tissue preservation method on the quality and quantity of RNA extracts. Methods: Skin lesion biopsies were obtained from patients with cutaneous leishmaniasis (CL). Biopsy specimens of 2 mm (n = 10) and 3 mm (n = 59) were preserved in Allprotect® reagent, and 4 mm biopsies in OCT (n = 54). Quality parameters were evaluated using Nanodrop and Bioanalyzer. The informativeness of the extracted samples for downstream analyses was evaluated using RT-qPCR and RNA-Seq. Results: The success rate, based on quality parameters of RNA extraction from tissue biopsies stored in OCT and 2 mm biopsies stored in Allprotect®, was 56% (30/54) and 30% (3/10), respectively. For 3 mm skin biopsies stored in Allprotect® was 93% (55/59). RNA preparations from 3 mm-Allprotect® biopsies had an average RIN of 7.2 ± 0.7, and their integrity was not impacted by sample storage time (up to 200 days at -20°C). RNA products were appropriate for qRT-PCR and RNA-seq. Based on these results, we propose a standardized method for RNA extraction from disrupted skin samples. This protocol was validated with lesion biopsies from CL patients (n = 30), having a success rate of 100%. Conclusions: Our results indicate that a biopsy size of 3 mm in diameter and preservation in Allprotect® for up to 200 days at -20°C, are best to obtain high quality RNA preparations from ulcerated skin lesion biopsy samples.