Negative Immunohistochemistry Research Articles

Impact of Driver Mutations on Metastasis-Free Survival in Uveal Melanoma: A Meta-Analysis.

The prognosis of uveal melanoma is significantly influenced by the risk of metastasis, which varies according to clinical and genetic features. Driver mutations can predict the likelihood of disease progression and survival, although the data in the literature are inconsistent. This meta-analysis aimed to evaluate the prognostic significance of driver mutations, including GNAQ, GNA11, BAP1, and SF3B1, in the advancement of uveal melanoma. A comprehensive search of databases yielded relevant studies, and data from 13 studies (848 eyes) were synthesized to assess the impact of these mutations on metastasis-free survival. The BAP1 mutation and negative immunohistochemistry were associated with a higher risk of metastasis (logHR = 1.44, 95% CI 1.05-1.83). GNAQ, GNA11, and SF3B1 mutations did not show a significant increase in risk. In summary, BAP1 has proven to reliably predict the likelihood of disease progression in uveal melanoma, while further studies are needed to establish the significance of other driver mutations.

Primary renal synovial sarcoma with negative immunohistochemistry: a case report and literature review

Just less than 200 cases of primary renal synovial sarcoma (PRSS) are known, a very rare neoplasm of the kidney. The diagnosis can be very challenging because of its similarity with other neoplasms, and is based on anatomopathological analyses, especially immunohistochemistry and cytogenetics. Due to the rarity, there are no standard protocols for treatment, although radical nephrectomy is considered the primary treatment and can be associated or not with chemotherapy. We present a case of PRSS with negative immunohistochemistry: the importance of both methods for a correct diagnosis is underlined, and a literature review of this rare entity is presented.

Effect of breast cancer receptor subtypes and CSF cytology status on survival for patients with leptomeningeal disease.

2025 Background: Leptomeningeal disease (LMD) in metastatic breast cancer (MBC) carries a poor prognosis. Data on the effect of BC sub types and CSF cytology results on overall survival (OS) are lacking. This single institution retrospective study compares OS among MBC with LMD across BC sub types and CSF cytology results. Methods: This study included patients (pts) diagnosed with MBC LMD from Jan 2010 to Jan 2023. BC sub types were defined as (1) ER positive (+)/HER2 negative (-), HER2+, triple-negative BC (TNBC), (2) HER2 0 [immunohistochemistry (IHC) score 0], HER 2 low [ IHC 2+ with fluorescence in-situ hybridization negative OR IHC 1+], HER2+. ER and HER2 positivity were defined per ASCO/CAP criteria. CSF cytology sub types were CSF+ (for malignant cells), CSF-, and CSF not tested. Baseline characteristics were compared using Fisher’s Exact and Wilcoxon rank-sum tests. OS among sub types was evaluated using Kaplan-Meier analysis and compared using log rank test. For multivariate analysis, a step wise model selection was performed after inclusion of sub type of interest; and variables meeting entry (p<0.10) and staying criteria (p<0.05) were included in final Cox model. Results: Out of 219 patients screened, 69 pts were registered. Median OS and associated 95% confidence interval (CI) within BC sub types in analyses 1 and 2 and CSF sub type analysis are shown in the table. OS in analysis 1 was not significantly different by BC sub types. In multivariate analysis, TNBC sub type had significantly shorter OS than ER+/HER2- group [Hazard Ratio (HR) 2.64, 95% CI (1.23, 5.80) (p=0.04)] after adjusting for interval from MBC to LMD diagnosis and CSF cytology status. OS by BC sub types in analysis 2 was not significantly different in unadjusted and multivariate analyses. CSF- group had significantly better OS compared to CSF+ or CSF not tested group in unadjusted analysis (Table) and multivariate analysis [CSF+ vs CSF- HR 4.5 95% CI (1.75-12.11); CSF not tested vs CSF- HR 2.91 95% CI (1.45-6.26), overall p=0.002]. Conclusions: This single institution study on MBC pts with LMD showed that BC receptor sub types were significantly associated with OS when adjusted for the interval from MBC to LMD diagnosis and CSF cytology group. This study showed significantly improved OS among patients with negative CSF cytology compared to those with positive cytology or those not tested, indicating a novel prognostic value of CSF cytology (currently on diagnostic value) for MBC LMD. These findings will be further validated. [Table: see text]

Abstract PO2-13-03: Spatial HER2 heterogeneity on HER2-PET in HER2-low and negative disease: potential biomarker for antibody-drug conjugate effect

Abstract Background: Anti-HER2 antibody drug conjugates (ADCs) can assert an effect in part of patients with HER2-low breast cancer. In light of patient burden and financial sustainability, biomarkers to select responding patients are urgently needed, but so far, none have been identified. In the IMPACT trial (NCT01957332), patients with newly diagnosed and non-rapidly progressive metastatic breast cancer (MBC) of all subtypes (including HER2-negative, -low, and –positive disease) were included. We assessed the performance of Zirconium-89 (89Zr)-trastuzumab positron emission tomography (HER2-PET) in HER2-low and negative MBC. Methods: All patients received extensive workup, including a metastasis biopsy and HER2-PET at baseline. HER2 status was determined by immunohistochemistry (IHC) and in situ hybridization. ⁸⁹Zr-trastuzumab uptake was quantified as maximum and mean standardized uptake values (SUVmax, SUVmean) in metastases and healthy background tissue. Quantitative ⁸⁹Zr-trastuzumab uptake of all metastases and corresponding biopsied metastasis, was related to HER2 IHC status. Results: In 200 patients, ⁸⁹Zr-trastuzumab uptake was quantified in 5,163 metastases. With increasing HER2 IHC status, uptake was higher (geometric mean SUVmax 7.0, 7.6, 7.3, and 17.4 with HER2 IHC 0, 1, 2, or 3+, in respectively 71, 71, 20, and 24 biopsied metastases (P  0.001 between IHC 0 and 3+). SUVmax in lesions was heterogeneous within and between patients for all HER2 IHC groups. All HER2 IHC groups, also HER2-negative and -low, had uptake exceeding that of the healthy background (geometric mean tumor-to-background ratio SUVmax >1 in all groups, increasing with higher HER2 IHC). Of patients with a HER2 IHC 0 biopsy, 27.8% had at least one metastasis elsewhere in the body, with uptake exceeding the overall 90th SUVmax percentile of 14.6; in HER2 low IHC1+ and 2+, this was 31.9% and 30%. Conclusions: In this largest series with HER2-PET in patients with MBC, we show that ±30% of patients with HER2-low or -negative IHC in the biopsy, in fact, have metastases with high HER2 uptake elsewhere in the body. This spatial heterogeneity provides novel insights into HER2-negative and -low disease compared to standard HER2 IHC of a single biopsy. As HER2-PET uptake is related to ADC response in HER2 positive MBC1, HER2-PET is a potential biomarker for anti-HER2 ADC effect in HER2-low and -negative MBC as well. Reference: 1. Gebhart G, et al. Ann Oncol 2016;27(4): 619-624. Supported by the Dutch Cancer Society grant 2012-5565 Citation Format: Carolien Schroder, Jasper van Geel, Bertha Eisses, Adrienne Brouwers, Sjoerd Elias, Frederike Bensch, Evelien Kuip, Agnes Jager, Andor Glaudemans, Bert van der Vegt, Willemien Menke-van der Houven van Oordt, Elisabeth de Vries. Spatial HER2 heterogeneity on HER2-PET in HER2-low and negative disease: potential biomarker for antibody-drug conjugate effect [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-03.

Abstract PO4-20-11: Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay

Abstract A 72-year-old female presented to the emergency department with sudden onset of imbalance and cognitive decline. Two years ago, she was diagnosed with stage III estrogen receptor (ER) positive, progesterone receptor (PR) negative and human epidermal growth factor receptor 2 (HER2) negative (immunohistochemistry score: 0) invasive breast cancer. She subsequently underwent lumpectomy, adjuvant radiation and chemotherapy. She had been taking adjuvant letrozole at the time of presentation. Magnetic resonance imaging (MRI) of the brain showed obstructive hydrocephalus without any parenchymal lesion or leptomeningeal enhancement. MRI of spine, computerized tomography of chest/abdomen/pelvis and bone scan were unrevealing except a left adrenal mass which was equivocal for metastasis. Cerebrospinal fluid (CSF) analysis showed rare malignant cells. Based on the morphology of malignant cells and the patient’s prior history of breast cancer, the diagnosis of leptomeningeal disease with breast primary was made. There were insufficient cells for additional studies. CNSide, a microfluidic platform that uses antibody capture method to detect CSF tumor cells, showed 4678 tumor cells per milliliter (ml) of CSF. Immunocytochemistry of these cells showed that the cells were ER negative (criterion: < 2.6% ER positive cells) and programmed cell death ligand 1 (PD-L1) negative (criterion: < 3.4% PD-L1 positive cells). Fluorescence in situ hybridization (FISH) testing showed 22% of tumor cells with HER2 amplification (either HER2 to CEP17 ratio >/= 2.0 or HER2 copy number >/= 6). She underwent ventriculoperitoneal (VP) shunt placement with complete recovery of her neurological symptoms. Five weeks later, she was started on trastuzumab deruxtecan (T-DXd) mainly because of the HER2 FISH results from CNSide. She did not receive central nervous system(CNS) irradiation due to complete resolution of neurological symptoms with VP shunt placement. After two cycles of T-DXd, MRI brain showed mild leptomeningeal enhancement along bilateral superior cerebellar sulci. Positron emission tomography/computed tomography(PET/CT) scan showed a new sclerotic rib focus, thought to represent treatment response. It also showed decrease in size of the adrenal mass. The adrenal mass was hypermetabolic on PET, consistent with malignancy. CSF cytology was negative, while CSF tumor cell count significantly reduced to 6.72 cells/ml using CNSide. There were no new neurological symptoms. Despite MRI brain findings, she was continued on T-DXd given overall favorable clinical, radiological and CSF data. After 7th cycle of T-DXd, repeat CSF cytology continued to remain negative, CNSide CSF tumor cell count decreased further to 6.27 cells/ml, MRI brain showed interval resolution of prior leptomeningeal enhancement, CT scan showed further decrease in size of the adrenal mass (baseline 5.4cm to 3cm), stable rib sclerotic lesion and no additional metastatic lesion. To date, she has undergone eight cycles of T-DXd which has effectively controlled her disease for a period of seven months. She has not needed VP shunt revision and continues to do well. To our knowledge, this is the first case demonstrating the activity of T-DXd against CSF tumor cells where a small subset exhibited HER2 amplification. This case also highlights the benefits of using advanced and highly sensitive CSF assays to diagnose and surveil leptomeningeal disease and to assess HER2 signaling. This case is well-timed especially in the era of next-generation, CNS penetrating, potent HER2-directed antibody-drug conjugate like T-DXd which can mount antitumor activity even with minimal HER2 expression in tumors. Citation Format: Sarah Stanford, Zeni Kharel, Lauryn Hemminger, Tyler Schmidt, Sara Hardy, Jason Zittel, Nimish Mohile, Ajay Dhakal. Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-11.

Abstract PO5-24-03: Breast cancer intrinsic subtypes predict outcomes in primary and metastatic samples

Abstract Background: The prognostic and predictive value of the PAM50 intrinsic subtypes, namely Luminal A, Luminal B, Her2, and Basal-like subtypes, is well-studied in primary as well as metastatic breast cancer settings. Prosigna has emerged as a rapid PAM50 subtype predictor based on the NanoString nCounter assay. However, assay reproducibility across various RNASeq or qRT-PCR platforms can be challenging, especially when applying the predictor on metastatic breast cancer tumors. Here, we used SpinAdapt to create an intrinsic subtype predictor that works on RNA sequencing data, and validates on multiple tumor-sites. We evaluate real-world outcomes for our intrinsic subtype predictions across various immunohistochemical (IHC) labels and metastatic sites. Methods: We trained the subtype predictor on a cohort of 2,497 breast cancer patients using the PAM50 genes, profiled using Nanostring RNA nCounter assay (GSE148426). Approximately 5,423 de-identified records of breast cancer patients sequenced using whole-exome capture RNA-seq were included in our reference dataset. The reference dataset contained samples collected from various sites including breast (n=2440), liver (n=936), lymph node (n=577), lung (n=540), and bone (n=304). For subtype classification, we first batch-corrected the external dataset to Tempus RNA-seq reference dataset using SpinAdapt, then trained a Support-Vector Classifier (SVC) on the corrected data. A 10-fold CV experiment was performed on the corrected dataset to analytically validate the intrinsic subtype predictions. We retrospectively analyzed 7,021 de-identified breast cancer patients with known hormone receptor (HR) or HER2 status and a matched RNASeq sample. The concordance between HR/HER2 status and PAM50 prediction was analyzed, and these patients were excluded from training. Real-world overall survival (rwOS) was evaluated from the time of first diagnosis. The outcomes across intrinsic subtypes were further assessed according to tumor collection site and HR/HER2 IHC status. Results: The 10-fold CV experiment on the Tempus-adapted GSE148426 dataset achieved F-1 scores of 0.97, 0.86, 0.94, and 0.87 on Basal, HER2-like, Luminal A, and Luminal B PAM50 subtypes, respectively. On the Tempus evaluation dataset, 85.3% of HR+/HER2- patients (n=4,366), 65.4% of HR-/HER2+ patients (n=240), and 75.4% of HR-/HER2- patients (n=1,930) were classified as Luminal, HER2-like, and Basal, respectively. Evaluating outcomes on Tempus patients for each PAM50 group, the rwOS for the basal group was significantly shorter than patients not predicted to be basal (n=5,845, p< 2e-90). The rwOS for the predicted PAM50 basal patients remained significantly shorter than non-basal patients even when stratified by site of metastasis: breast (n=2,405; p< 1e-27), lymph node (n=577, p< 1e-7), liver (n=936; p< 1e-25), lung (n=540, p< 1e-13), and bone (n=304, p< 1e-3). Interestingly, within both HR+/HER2- (n=3,653) and HR-/HER2- (n=1,664) IHC cohorts with available outcomes data, the predicted PAM50 basal subtype could further stratify each of these populations with basal-subtype showing significantly worse prognosis than the non-basal subtype (p< 1e-27 and p< 1e-7, respectively). Conclusions: We retrospectively analyzed Tempus multimodal RWD to validate an in-house breast intrinsic subtype predictor that is agnostic to the site of metastasis. The prognostic value of the basal subtype was significant for breast cancer patients across various sites of metastasis including lymph node, liver, lung, and bones and IHC groups. For patients in each of the HR+/HER2- and triple negative IHC groups, the intrinsic molecular subtypes provided an additional level of prognostic detail with statistical significance. These data emphasize the importance of combining molecular subtypes with IHC-based diagnostics to fully characterize clinically relevant subpopulations and risk. Citation Format: Talal Ahmed, Mark Carty, Kaveri Nadhamuni, Raphael Pelossof. Breast cancer intrinsic subtypes predict outcomes in primary and metastatic samples [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-03.

Abstract 6546: Spatial immunology landscapes that correlate with survival in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by negative immunohistochemistry staining for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Treatment options for TNBCs are limited due to lack of tumor-specific targeted therapies, resulting in poor prognosis and patient survival. This is exacerbated by lack of prognostic markers of response. While current studies suggest tumor immune interactions are important, current immune activity quantifications, such as scores quantifying abundance of tumor-infiltrating lymphocytes (TILs), are not sufficient to define tumor immune status. We used imaging mass cytometry (IMC) to measure 33 markers on primary tumors of 100 patients undergone chemotherapy with long-term follow-up to explore the tumor immune microenvironment of TNBCs. Concordant with previous studies, a higher ratio of T-cells was associated with response to chemotherapy and longer overall survival (adjusted p-value<0.05). Moreover, using Kmeans clustering we identified 13 distinct cellular neighborhoods, reflective of the heterogenous nature of TNBCs. Among others, we identified a neighborhood, characterized by low CD14+ expression, low cytokeratin, high vimentin expression and high CD45RO, that contributes highest impact on correlation with overall survival (adjusted p-value<0.01). These results suggest an important role for memory T cells in the stroma. Thus, resolving spatial organization of the tissue is important to understanding patient response to therapy in TNBCs. Citation Format: Ali Foroughi pour, Jan Martinek, Te-Chia Wu, Santhosh Sivajothi, Zichao Liu, Jie Zhou, David Rimm, Paul Robson, Katherine Bates, Karolina Palucka, Jeffrey H. Chuang. Spatial immunology landscapes that correlate with survival in triple negative breast 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 6546.

CytoLyt fixation impedes insulinoma-associated protein 1 (INSM1) immunoreactivity compared to formalin fixation

Insulinoma-associated protein 1 (INSM1) is an immunohistochemical marker commonly used to confirm cytomorphological concordant neuroendocrine tumors/carcinomas (NETs/NECs), demonstrating high utility in small samples. Previous reports have suggested comparable INSM1 staining in CytoLyt-fixed cell blocks and formalin-fixed surgical pathology specimens. This study aimed to assess INSM1 immunoreactivity using both fixation methods and investigate potential factors contributing to its variable expression. A retrospective query was performed (03/31/21-05/31/22) for NET/NEC cases that had both formalin- and CytoLyt-fixed cell blocks. We collected clinical data and reporting of immunostains for each case. INSM1 staining was evaluated in both fixation methods, and reported as positive, negative, or equivocal. Equivocal INSM1 staining was further scored as a percentage of 1%-100% and intensity of weak (faint staining), moderate (darker staining), and strong (dense staining). Our search identified 20 cases from diverse body sites, including mediastinal lymph nodes (40%), pancreas (35%), lung (20%), and porta hepatis lymph nodes (5%). All cases exhibited a widespread positivity (over 90%) in formalin-fixed cell blocks. In contrast, CytoLyt fixed cells showed a negative stain in 65% of cases and 30% exhibited an equivocal positivity. While INSM1 is previously reported as a sensitive (75%-100%) and specific (82.7%-100%) marker for NET/NECs, our study found a reduced immunohistochemical staining in CytoLyt-fixed cell blocks. Consequently, false negative INSM1 immunohistochemical results in CytoLyt-fixed cell block material may pose a pitfall in the diagnosis of NET/NEC.

Prognostic value of O6-methylguanine-DNA methyltransferase (MGMT) status in pancreatic neuroendrocrine tumors with capecitabine and temozolomide (CAPTEM).

598 Background: Loss of MGMT expression has been suggested as a predictor of response to alkylating agents in pancreatic neuroendocrine tumors (pNET), however, its determination is controversial. We aim to analyze the prognostic value of MGMT status assessed by immunohistochemistry (IHC) or pyrosequencing (PSQ) in patients with pNET receiving CAPTEM. Methods: Retrospective and unicentric analysis in patients (p) with pNET treated with CAPTEM from April 2008 to February 2023. MGMTdeficiency was determined by IHC and MGMT promoter methylation by PSQ. We analyzed patients’ characteristics, progression-free survival (PFS) and overall survival (OS) according to MGMT status. Results: 20p were included. Age (median): 62 [46-77]. Males (45%). Tumor grade (G): G1 (15%), G2 (55%) and G3 (20%). MGMT deficiency was detected in 12p (60%) and promoter methylation in 10p (50%). Results by IHC were consistent with PSQ in 10p (50%), with mismatches in 4p (methylated MGMT and positive IHC) and 6p (unmethylated MGMT and negative IHC). We reported a higher response rate in MGMT-deficient and MGMT-methylated p (Table) . In MGMT-deficient p we observed significantly better PFS (88m vs 11.1m; p=0.02, HR=0.21) and OS (141.9m vs 35.8m; p=0.03, HR=0.20). Also, in MGMT-methylated p we detected better PFS (88m vs 17.7m; p=0.20, HR=0.44) and OS (188.7m vs 141.9m; p=0.11, HR=0.28). A subanalysis was performed based on G, showing better PFS (88m vs 6.2m; p=0.03, HR=0.11) and OS (188.7m vs 35.8m; p<0.01, HR=0.06) in those with G2 vs G3. There was a strong negative correlation between G and MGMT by PSQ; the higher G, the lower probability of promoter methylation r(16)= -0.62, p<0.01. Conclusions: Our results support MGMT status, deficiency by IHC or promoter methylation by PSQ, as a potential prognostic biomarker in pNET treated with alkylating agents. However, it is necessary to evaluate its prognostic value in prospective studies. [Table: see text]

HER2 amplification in cases with low HER2 expression by immunohistochemistry (IHC): A Single Institutional Experience

Abstract Introduction/Objective HER2 testing in invasive breast carcinoma (IBC) is the standard of care to determine patient eligibility for HER2 directed therapy with trasuzumab. Initial testing of tissue by HER2 immunohistochemistry (IHC) can be positive, negative, or equivocal. In equivocal cases as well as negative cases in our institution HER2 FISH is also performed. According to ASCO/CAP guidelines a subset of equivocal and negative IHC cases are considered HER2 positive if they meet criteria for increased HER2/CEP17 ratio and HER2 copy number by FISH, allowing more patients to be treated with HER2 directed therapies. HER2 grouping per ASCO is as follows; Group 1: HER2 copy number ≥4 signals/cell, Group 2: <4.0 signals/cell, Group 3: ≥6 signals/cell, Group 4: HER2 copy number ≥4 and <6 signals/cell, Group 5: HER2 copy number <4 signals/cell Methods/Case Report All cases of IBC resection with HER2 IHC and FISH results from Jan 2018-Dec 2021 were included in this study. All cases positive for HER2 by IHC alone (3+ staining) were excluded. The ASCO/CAP 2018 guidelines for HER2 testing in breast cancer were used to evaluate HER2 positivity by FISH and the relation to IHC scores Results (if a Case Study enter NA) A total of 2630 IBC cases with both HER2 IHC and FISH testing were included. Of these, 131 (5.0%) were in FISH group 1, 17 (0.7%) in group 2, 11 (0.4%) in group 3, 161 (6.1%) in group 4, and 2310 (87.8%) in group 5. A total of 142 (5.4%) cases that were HER2 equivocal or negative by IHC were HER2 positive by FISH. Specifically, 0.3% of cases with an IHC score of 0, 1.4% with a score of 1+, and 14.6% of cases with a score of 2+ were positive by FISH. Of the 14.6% IHC 2+ cases that were FISH positive 13.3% were in FISH group 1 (115 cases) and 1.3% (11 cases) in FISH group 3. Of FISH positive cases 1.4% had an IHC score of 0, 9.9% had an IHC score of 1 and 88.7% had a score of 2 Conclusion At our institution 14.6% of IBC with equivocal HER2 staining on IHC, 1.4% of cases with negative (1+) staining and 0.3% of cases with negative (0) were HER2 FISH positive. Since HER2 FISH is not reflexively performed on cases HER2 negative by IHC, this additional 1.7% will qualify for HER2 targeted therapy Interpretation of low HER2 expression by IHC by traditional IHC assay may miss a small percentage of cases eligible for HER2 directed therapy. Accurate interpretation of HER2 low by IHC is critical as recent clinical trials have shown that low HER2 expression with IHC scores of 1+ or 2+ without gene amplification by FISH could benefit from Trastuzumab Deruxtecan

Immunohistochemical Tumor Expression Predicts Response to CD3xCD20 Bispecific Antibodies in Patients with Relapsed/Refractory B Cell Lymphoma

Introduction Several studies show that CD3xCD20 bispecific antibodies show promising results in relapsed or refractory B-cell lymphomas and may have a milder adverse effect profile compared to chimeric antigen receptor T cell therapy (CAR-T). Tumor loss of antibody target (i.e. CD19 or PD1) have shown to negatively affect treatment outcomes in CD19-CAR-T cell therapy in DLBCL, and anti-PD1 therapy in Hodgkin lymphoma. It has, to our knowledge, not been reported how loss of CD20 expression and other immunohistochemical (IHC) expression patterns affects treatment outcomes in patients treated with CD3xCD20 antibodies. Methods We collected data on all patients in Denmark who received CD3xCD20 bispecific antibodies from 2017 to 2023. Electronic health records were assessed for general baseline characteristics and response according to Lugano criteria. Pathology reports on pre- and post-CD3xCD20 therapy lymphoma samples were collected and assessed on IHC analysis of CD20, Ki67, CD10, Bcl2, Bcl6, MUM1, germinal center B-cell (GCB) subtype and non-germinal center B-cell subtypes (non-GCB) according to Hans algorithm, as well as reports on cytogenetic FISH analysis regarding MYC with BCL2 and/or BCL6 rearrangements. To determine whether an expression was weak, negative, or strong the assessment of the original pathology reports was used. Results Pre-treatment IHC assessments were available in 122 patients. These were distributed across 81 patients with DLBCL, 32 with FL and 9 with other lymphoid neoplasms (MZL, HL, CLL). Of these, 109 had a strong CD20 expression, 10 had a weak CD20 expression, while 3 had no expression of CD20. Patients with a weak CD20 expression before treatment had an overall survival (OS) rate after 12 months at 40% (95% CI, 9.5-70) compared to 69% in patients with strong CD20 expression (95% CI, 45-61) (Figure 1, p=0.08). In a univariable Cox regression, we found that negative CD20 expression and higher Ki67 was significantly associated with inferior OS (Table 1, respectively HR=2.11 and HR=1.02 (per %-change in Ki67)). Worse performance status and a higher number of previous lines of treatment were also significantly associated with inferior overall survival (respectively HR=1.85 and HR=1.20). Surprisingly, BCL6 rearrangement was significantly associated with superior OS (HR=0.46). In a multivariable cox regression using OS as the endpoint, weak and negative IHC expression of CD20 was significantly associated with inferior OS (respectively HR=3.29, 95% CI 1.32-8.23, p=0.011 and HR=6.79 95% CI 1.25-36.76, p=0.026). Positive BCL6 compared to negative was associated with increased OS (HR =0.48, 95% CI 0.24 - 0.94, p=0.032). Analyzing only patients with DLBCL (n=81), weak and negative IHC expression of CD20 were the only factors to remain significantly associated with inferior overall survival (respectively HR=3.01, 95% CI 1.10-8.28, p=0.032 and HR=7.26, 95% CI 1.35-43.35, p=0.026). BCL6 rearrangement was associated with an increased overall survival, however only borderline statistically significant (HR=0.53, 95% CI 0.26-1.08, p=0.082). With regards to treatment response weak CD20 expression, worse performance status, higher Ki67 and a higher number of prior lines of treatment was in univariable analysis significantly associated with not being in a complete remission (CR) (respectively HR=2.28, HR=1.73, HR=1.01(per %-change in Ki67), HR=1.23 (per prior line of treatment). In a multivariable cox regression only weak and negative CD20 expression remained significantly associated with not being in CR (respectively HR=2.40, 95% CI 1.1-5.25, p=0.028 and HR=5.7 95% CI 1.14-28.58, p=0.034). Of the 48 patients who had biopsy-confirmed relapse, 29 had a weak or negative IHC CD20 expression (24 with a negative expression and 5 with a weak expression). Of these 29 patients, 24 had a strong pre-treatment IHC CD20 expression. Conclusions To our knowledge, we here present the first long-term analysis of the prognostic effect of tumor immunohistochemistry properties in patients treated with CD3xCD20 bispecific antibodies. We find that CD20 expression before treatment is an important prognostic factor, as patients with lymphomas with weak CD20 expression yield significantly poorer responses and decreased overall survival. Additionally, we find that BCL6 rearrangement is associated with superior overall survival in patients treated with CD3XCD20 bispecific antibodies.

Are There More HER2 FISH in the Sea? An Institution’s Experience in Identifying HER2 Positivity Using Fluorescent In Situ Hybridization in Patients with HER2 Negative Immunohistochemistry

BackgroundApproximately 20% of breast cancers express HER2-positive receptors in the USA. HER2 receptor immunohistochemistry (IHC) staining with equivocal (2+) results commonly undergoes fluorescence in-situ hybridization (FISH) for further classification. Current guidelines do not recommend routine FISH testing in IHC-negative (0 or 1+) cases. This study investigates an institution that performs both IHC and FISH testing on all cases to identify the true HER2-positive rate.Patients and MethodsA retrospective chart review from 2015 to 2021 was conducted at an institution where both HER2 IHC and FISH testing were performed at the time of diagnosis for all invasive breast cancers. The rate of true HER2-positive patients was determined, and patient and tumor characteristics were further explored.ResultsA total of 1835 invasive breast cancer cases were primarily treated at this institution. A total of 289 cases were HER2 positive on IHC and FISH testing (15.7%). An additional 38 cases were identified as HER2 negative on IHC, but reclassified as HER2 positive on reflex FISH testing. Total HER2 positive cases increased from 289 (15.7%) to 327 cases (17.8%) with reflex FISH testing.ConclusionsThe additional HER2-positive cases after completing FISH testing on IHC-negative tumors suggests there may be a role for routine FISH testing in addition to standard IHC staining to determine HER2 status for breast cancer. The ethical, prognostic and even benefits of a correct diagnosis outweigh the added expense of FISH testing.

FRI328 Granulomatous Hypophysitis With Magnificent Response To Glucocorticoids

Abstract Disclosure: M. Rodriguez Caballero: None. F. Romero: None. C. Escurra: None. R. Rodriguez: None. R. Rolon: None. E. Valinotti: None. A. Ayala: None. Granulomatous hypophysitis corresponds to 1% of sellar lesions. It maybe primary or secondary to tuberculosis, Pneumocystis carinii, Toxoplasma gondii, Cytomegalovirus or parasitic infections. Non-infectious etiologies include sarcoidosis, vasculitis, dendritic cell disorders, ruptured Rathke cysts or Crohn's disease. A 59-year-old immunocompetent woman was evaluated for decreased visual acuity, diplopia, and palsy of the 6th cranial nerve. Asellar MRI confirmed an 18 x 14 x 12 mm pituitary lesion with chiasmatic compression and visual field involvement. Hormonal evaluation revealed a TSH 0.03 mIU/L, FT4 0.44 ng/dL, random of GH 5.64 ng/mL, FSH 55.2 IU/mL, LH33.1 mIU/mL, and cortisol of 16 ng/dl. There was no evidence of diabetes insipidus. Transsphenoidal resection resulted in visual field improvement with a noticeable residual tumor of 12 x 9 x5 mm within the sellar space on the postoperative MRI. The patient developed panhypopituitarism. Pathology revealed non-caseous granulomatosis, with histiocytic aggregates of giant cells forming granulomas, with negative immunohistochemistry for mycobacteria. Tuberculin test, VDRL, blood cultures, ANA, Anti-DNA, ANCA C and P were negative. Tomography images and PET scan ruled out systemic involvement. The patient was treated with high doses of glucocorticoids under a presumptive diagnosis of isolated pituitary sarcoidosis vs. primary hypophysitis, resulting in complete disappearance of the lesion. Conclusion: Granulomatous hypophysitis is the second most common cause of chronic inflammation of the pituitary gland mimicking pituitary adenomas. Surgical removal and glucocorticoids are often utilized although there is no consensus regarding the treatment. Our case illustrates the importance of considering this diagnosis when confronted with incidental sellar lesions. Pharmacotherapy resulted in symptomatic improvement and complete involution of the pituitary lesion confirming the utility of empiric high-dose glucocorticoid as a therapeutic strategy for granulomatous hypophysitis. Presentation: Friday, June 16, 2023

An observational cohort study of histological screening for BK polyomavirus nephropathy following viral replication in plasma

Systematic screening for BKPyV-DNAemia has been advocated to aid prevention and treatment of polyomavirus associated nephropathy (PyVAN), an important cause of kidney graft failure. The added value of performing a biopsy at time of BKPyV-DNAemia, to distinguish presumptive PyVAN (negative SV40 immunohistochemistry) and proven PyVAN (positive SV40) has not been established. Therefore, we studied an unselected cohort of 950 transplantations, performed between 2008-2017. BKPyV-DNAemia was detected in 250 (26.3%) transplant recipients, and positive SV40 in 91 cases (9.6%). Among 209 patients with a concurrent biopsy at time of first BKPyV-DNAemia, 60 (28.7%) biopsies were SV40 positive. Plasma viral load showed high diagnostic value for concurrent SV40 positivity (ROC-AUC 0.950, 95% confidence interval 0.916-0.978) and the semiquantitatively scored percentage of tubules with evidence of polyomavirus replication (pvl score) (0.979, 0.968-0.988). SV40 positivity was highly unlikely when plasma viral load is below 4 log10 copies/ml (negative predictive value 0.989, 0.979-0.994). In SV40 positive patients, higher plasma BKPyV-DNA load and higher pvl scores were associated with slower viral clearance from the blood (hazard ratio 0.712, 95% confidence interval 0.604-0.839, and 0.327, 0.161-0.668, respectively), whereas the dichotomy positivity/negativity of SV40 immunohistochemistry did not predict viral clearance. Although the pvl score offers some prognostic value for viral clearance on top of plasma viral load, the latter provided good guidance for when a biopsy was unnecessary to exclude PyVAN. Thus, the distinction between presumptive and proven PyVAN, based on SV40 immunohistochemistry, has limited clinical value. Hence, management of BKPyV-DNAemia and immunosuppression reduction should be weighed against the risk of occurrence of rejection, or exacerbation of rejection observed concomitantly.

Composite Biomarker Image for Advanced Visualization in Histopathology.

Immunohistochemistry (IHC) biomarkers are essential tools for reliable cancer diagnosis and subtyping. It requires cross-staining comparison among Whole Slide Images (WSIs) of IHCs and hematoxylin and eosin (H&E) slides. Currently, pathologists examine the visually co-localized areas across IHC and H&E glass slides for a final diagnosis, which is a tedious and challenging task. Moreover, visually inspecting different IHC slides back and forth to analyze local co-expressions is inherently subjective and prone to error, even when carried out by experienced pathologists. Relying on digital pathology, we propose "Composite Biomarker Image" (CBI) in this work. CBI is a single image that can be composed using different filtered IHC biomarker images for better visualization. We present a CBI image produced in two steps by the proposed solution for better visualization and hence more efficient clinical workflow. In the first step, IHC biomarker images are aligned with the H&E images using one coordinate system and orientation. In the second step, the positive or negative IHC regions from each biomarker image (based on the pathologists' recommendation) are filtered and combined into one image using a fuzzy inference system. For evaluation, the resulting CBI images, from the proposed system, were evaluated qualitatively by the expert pathologists. The CBI concept helps the pathologists to identify the suspected target tissues more easily, which could be further assessed by examining the actual WSIs at the same suspected regions.

In-depth analysis of immunohistochemistry concordance in biopsy-resection pairs of bronchial carcinoids

Primary diagnosis of bronchial carcinoids (BC) is always made on biopsies and additional immunohistochemistry (IHC) is often necessary. In the present study we investigated the concordance of common diagnostic (synaptophysin, chromogranin, CD56 and INSM-1) and potential prognostic (OTP, CD44, Rb and p16) IHC markers between the preoperative biopsies and resections of in total 64 BCs, 26 typical (41 %) and 38 atypical (59 %) carcinoid tumors. Synaptophysin and chromogranin had 100 % concordance in all resected carcinoids and paired diagnostic biopsies. Synaptophysin was not affected by variable expression in biopsies compared to chromogranin, CD56 and INSM-1. Notably, INSM-1 IHC was false negative in 8 % of biopsies. Of the novel and potential prognostic markers, only CD44 showed 100 % concordance between biopsies and resections, while OTP showed two (4 %) false negative results in paired biopsies. While Rb IHC was false negative in 8 % of biopsies, no strong and diffuse pattern of p16 expression was observed. In this study, most false negative IHC results (85 %, 22/26) were observed in small flexible biopsies. Taken together, our data suggest excellent concordance of synaptophysin and CD44 on the preoperative biopsy samples, while other neuroendocrine markers, Rb and OTP should be interpreted with caution, especially in small biopsies.

Mammary myofibroblastoma: a clinicopathological analysis of fifteen cases

Objective: To investigate the clinicopathological features, diagnosis and differential diagnosis of breast myofibroblastoma. Methods: The clinicopathological data and prognostic information of 15 patients with breast myofibroblastoma diagnosed at the Department of Pathology of the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China from 2014 to 2022 were collected. Their clinical characteristics, histological subtypes, immunophenotypes and molecular characteristics were analyzed. Results: There were 12 female and 3 male patients, ranging in age from 18 to 78 years, with a median and average age of 52 years. There were 6 cases in the left breast and 9 cases in the right breast, including 12 cases in outer upper quadrant, 2 cases in inner upper quadrant and 1 case in outer lower quadrant. Most of the cases showed a well-defined nodule grossly, including pushing growth under the microscope in 13 cases, being completely separated from the surrounding breast tissue in 1 case, and infiltrating growth in 1 case. Among them, 12 cases were classic subtype and composed of occasional spindle cells with varying intervals of collagen fiber bundles; eight cases had a small amount of fat; one case had focal cartilage differentiation; one case was epithelioid subtype, in which epithelioid tumor cells were scattered in single filing or small clusters; one case was schwannoma-like subtype, and the tumor cells were arranged in a significant palisade shape, resembling schwannoma, and one case was invasive leiomyoma-like subtype, in which the tumor cells had eosinophilic cytoplasm and were arranged in bundles, and infiltrating into the surrounding mammary lobules like leiomyoma. Immunohistochemical studies showed that the tumor cells expressed desmin (14/15) and CD34 (14/15), as well as ER (15/15) and PR (15/15). Three cases with histologic subtypes of epithelioid subtype, schwannoma-like subtype and infiltrating leiomyoma-like subtype showed RB1 negative immunohistochemistry. Then FISH was performed to detect RB1/13q14 gene deletion, and identified RB1 gene deletion in all three cases. Fifteen cases were followed up for 2-100 months, and no recurrence was noted. Conclusions: Myofibroblastoma is a rare benign mesenchymal tumor of the breast. In addition to the classic type, there are many histological variants, among which the epithelioid subtype is easily confused with invasive lobular carcinoma. The schwannoma-like subtype is similar to schwannoma, while the invasive subtype is easily misdiagnosed as fibromatosis-like or spindle cell metaplastic carcinoma. Therefore, it is important to recognize the various histological subtypes and clinicopathological features of the tumor for making correct pathological diagnosis and rational clinical treatment.

Apoptosis, Crypt Dropout, and Equivocal Immunohistochemical Staining May Indicate Cytomegalovirus Infection in Inflammatory Bowel Disease Patients.

Cytomegalovirus (CMV) colitis superimposed on inflammatory bowel disease (IBD) can be challenging to diagnose. This study aimed to determine what histologic clues and immunohistochemistry (IHC) utilization practices, if any, can help diagnose CMV superinfection in IBD. Colon biopsies were reviewed from all patients with CMV colitis with and without IBD between 2010 and 2021 at one institution, along with a separate cohort of IBD patients with negative CMV IHC. Biopsies were assessed for histologic features of activity and chronicity, phlebitis, fibrin thrombi, basal crypt apoptosis, CMV viral cytopathic effect (VCE), and CMV IHC positivity. Features between groups were compared, with statistical significance set at P -value <0.05. The study included 251 biopsies from 143 cases (21 CMV-only, 44 CMV+IBD, 78 IBD-only). Compared with the IBD-only group, the CMV+IBD group was more likely to show apoptotic bodies (83% vs. 64%, P =0.035) and crypt dropout (75% vs. 55%, P =0.045). CMV was detected by IHC in 18 CMV+IBD cases without VCE on hematoxylin and eosin (41%). In the 23 CMV+IBD cases where IHC was performed on all concurrent biopsies, IHC was positive in at least 1 biopsy in 22 cases. Six individual CMV+IBD biopsies with no VCE on hematoxylin and eosin demonstrated equivocal IHC staining. Of these, 5 had evidence of CMV infection. IBD patients with superimposed CMV infection are more likely to demonstrate apoptotic bodies and crypt dropout compared with their noninfected counterparts. Equivocal IHC staining for CMV may indicate true infection in IBD patients, and staining multiple biopsies from the same accession can improve CMV detection.

Is Docetaxel the Main Therapy for Lung Metastasis in Granular Cell Tumors?

Introduction: A granular cell tumor is a rare neoplasm originating from mesenchymal tissue. Malignant granular cell tumors have a poor prognosis. The lung is one of the target organs for metastatic malignant granular cell tumors. The number of rare cases makes managing metastatic malignant granular cell tumors unknown. Case: A 19-year-old male complained of shortness of breath for two months. A liver nodule biopsy revealed poorly differentiated carcinoma metastases. Histopathology of the left manus and frontal masses found granular cell tumors—negative immunohistochemistry on S-100 and myogenin. Ki-67 obtained a value of 5%. An X-ray and contrast multislice computerized tomography (MSCT) scan showed multiple metastatic nodules. Right bronchial biopsy forceps revealed a metastatic granular cell tumor. The patient was given docetaxel for six cycles. During the post-administration of docetaxel, the patient experienced clinical and radiological improvement. Conclusion: Until 2016, only three case reports of S-100 negative granular cell tumors existed. A negative S-100 test does not rule out the diagnosis of granular cell tumors. Malignant lesions are more common in males, as in this case. The number of rare cases means that management cannot be determined. Reports of similar cases show patients surviving up to 11 years with docetaxel administration. The patient in this study has survived for one year and is complaint-free.

Identification of MGMT Downregulation Induced by miRNA in Glioblastoma and Possible Effect on Temozolomide Sensitivity.

Glioblastoma multiforme (GBM) remains one of the tumors with the worst prognosis. In recent years, a better overall survival (OS) has been described in cases subjected to Gross Total Resection (GTR) that were presenting hypermethylation of Methylguanine-DNA methyltransferase (MGMT) promoter. Recently, also the expression of specific miRNAs involved in MGMT silencing has been related to survival. In this study, we evaluate MGMT expression by immunohistochemistry (IHC), MGMT promoter methylation and miRNA expression in 112 GBMs and correlate the data to patients' clinical outcomes. Statistical analyses demonstrate a significant association between positive MGMT IHC and the expression of miR-181c, miR-195, miR-648 and miR-767.3p between unmethylated cases and the low expression of miR-181d and miR-648 and between methylated cases and the low expression of miR-196b. Addressing the concerns of clinical associations, a better OS has been described in presence of negative MGMT IHC, in methylated patients and in the cases with miR-21, miR-196b overexpression or miR-767.3 downregulation. In addition, a better progression-free survival (PFS) is associated with MGMT methylation and GTR but not with MGMT IHC and miRNA expression. In conclusion, our data reinforce the clinical relevance of miRNA expression as an additional marker to predict efficacy of chemoradiation in GBM.