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We compared the performance of two commonly-used HER2 immunohistochemistry (IHC) assays in uterine serous carcinomas (USC), correlating with HER2 gene amplification by fluorescence in-situ hybridization (FISH). Sixty-five USCs were stained by both HercepTest™ and PATHWAY 4B5 assays. FISH was performed by HER2 IQFISH pharmDx. Consensus HER2 IHC scoring was performed, and HER2 testing results were evaluated using USC-specific criteria. Complete concordance between HercepTest and 4B5 assays was achieved in 44/65 tumors (68%). The overall HER2 IHC/FISH concordance was 94% (45/48) by HercepTest and 91% (42/46) by 4B5. All HER2 IHC 3+ cases with HercepTest (n = 6) and 4B5 (n = 4) were gene-amplified, corresponding to specificities of 100%. For cases with IHC 2+, 41% (7/17) by HercepTest and 42% (8/19) by 4B5 had HER2 gene amplification. The sensitivity for HercepTest and 4B5 were 38% and 25%, respectively, at a cut-off of IHC 3+ (P = 0.50), and were 81% and 75%, respectively, at a cut-off of IHC 2+ (P > 0.99). Among HER2 IHC 0-1+ cases, 3/42 cases by HercepTest and 4/42 cases by 4B5 showed amplified FISH results, corresponding to overall false negative rates of 19% for HercepTest and 25% for 4B5. By using USC-specific IHC scoring criteria, both HercepTest and 4B5 assays showed high specificities (100%) for HER2 gene amplification in IHC 3+ cases, high IHC/FISH concordance, and comparable sensitivity for detecting HER2 gene amplification. The notable false negative rates using IHC 2+ as a cut-off for reflexing FISH analysis may warrant consideration for performing FISH in IHC 1+ cases until more data become available., Competing Interests: Declaration of competing interest HZ serves on the Within3 Pathologist Virtual Advisory Board of AstraZeneca. DGH serves on the Advisory Board and Speaks Bureau of AstraZeneca. There is no conflict of interest associated with this publication. All other authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Multigene genomic profiling has become the standard of care in the clinical risk-assessment and risk-stratification of ER + , HER2 - breast cancer (BC) patients, with Oncotype DX ® (ODX) emerging as the genomic profile test with the most support from the international community. The current state of the health care economy demands that cost-efficiency and access to testing must be considered when evaluating the clinical utility of multigene profile tests such as ODX. Several studies have suggested that certain lower risk patients can be identified more cost-efficiently than simply reflexing all ER + , HER2 - BC patients to ODX testing. The Magee equations TM use standard histopathologic data in a set of multivariable models to estimate the ODX recurrence score. Our group published the first outcome data in 2019 on the Magee equations TM , using a modification of the Magee equations TM combined with an algorithmic approach-the Rochester Modified Magee algorithm (RoMMa). There has since been limited published outcome data on the Magee equations TM . We present additional outcome data, with considerations of the TAILORx risk-stratification recommendations., Methods: 355 patients with an ODX recurrence score, and at least five years of follow-up or a BC recurrence were included in the study. All patients received either Tamoxifen or an aromatase inhibitor. None of the patients received adjuvant systemic chemotherapy., Results: There was no significant difference in the risk of recurrence in similar risk categories (very low risk, low risk, and high risk) between the average Modified Magee score and ODX recurrence score with the chi-square test of independence ( p > 0.05) or log-rank test ( p > 0.05). Using the RoMMa, we estimate that at least 17% of individuals can safely avoid ODX testing., Conclusion: Our study further reinforces that BC patients can be confidently stratified into lower and higher-risk recurrence groups using the Magee equations TM . The RoMMa can be helpful in the initial clinical risk-assessment and risk-stratification of BC patients, providing increased opportunities for cost savings in the health care system, and for clinical risk-assessment and risk-stratification in less-developed geographies where multigene testing might not be available.

Objectives: We compared the clinicopathologic features, clinical management, and outcomes of human epidermal growth factor receptor 2 (HER2)-expressing and nonexpressing microinvasive breast carcinomas (MiBC) to explore the significance of HER2 in MiBC., Methods: Clinicopathologic and follow-up information of cases with final diagnosis of MiBC with known HER2 status between 2007 and 2019 were analyzed., Results: Nineteen (41.3%) HER2-positive (HER2+) and 27 (58.7%) HER2-negative (HER2-) MiBCs were identified. HER2 positivity was likely to be associated with high nuclear grade, presence of tumor-infiltrating lymphocytes, hormonal receptor negativity, and increased Ki-67 in both microinvasive and associated in situ carcinomas. Nodal metastases were found in 2 ER+/HER2- cases (5.3%). One HER2+ case was found to have isolated tumor cells in the axillary node. The majority of patients with HER2+ MiBCs (76.5%) did not receive HER2-targeted therapy. All patients with available follow-up were alive without recurrence or distant metastasis, with a median follow-up of 38 months., Conclusions: Similar to the larger size of invasive breast carcinomas, HER2 positivity is associated with high-grade morphologic features in MiBCs. However, HER2 overexpression in MiBCs does not appear to be associated with nodal metastasis or worse outcome in our study cohort. The role of HER2-targeted therapy in this clinical setting merits additional study., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We investigate L1 cell adhesion molecule (L1CAM) expression in estrogen receptor (ER)-positive/human epidermal growth factor receptor (HER2)-negative breast carcinomas. The finding of a potential correlation between high L1CAM expression and recurrent/metastatic disease in luminal A and B breast carcinomas may be helpful for risk stratification and open opportunities for targeted therapies. 304 cases comprising 152 cases of ER-positive, progesterone receptor (PR)-positive/negative, and HER2-negative recurrent/metastatic breast carcinomas and 152 nonrecurrent controls were included. ER, PR, HER-2, Ki-67 status, Nottingham grade, tumor size, tumor stage, number of foci, lymph node status, lymphovascular invasion, phenotype, laterality, age at diagnosis and first distant or local recurrence were recorded. L1CAM positive cases showed increased specificity for recurrence and these patients were significantly younger than L1CAM negative ones. Compared with L1CAM negative recurrent cases, L1CAM positive ones had a noticeably higher Ki-67, tended to be larger and recurred sooner. All L1CAM positive recurrent/metastatic cases were of the luminal B subtype compared with 67.3% of the L1CAM negative cases. L1CAM is highly specific for recurrence in a subset of breast cancer patients and may be associated with more aggressive behavior, particularly in luminal B breast cancers with higher Ki-67 expression. Further investigation about the prognostic value of L1CAM is warranted., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Magee Equations™ (ME) are multivariable models that can estimate oncotype DX ® recurrence score. One of the equations, Magee Equation 3 (ME3) which utilizes only semi-quantitative receptor results has been shown to provide chemopredictive value in the neoadjuvant setting in a single institutional study. This multi-institutional study (seven institutions contributed cases) was undertaken to examine the validity of ME3 in predicting response to neoadjuvant chemotherapy in estrogen receptor positive, HER2-negative breast cancers. Stage IV cases were excluded. The primary endpoint was the pathologic complete response (pCR) rate in different categories of ME3 scores calculated based on receptor results in the pre-therapy core biopsy. A total of 166 cases met the inclusion criteria. The patient age ranged from 24 to 83 years (median 53 years). The average pre-therapy tumor size was 3.9 cm, and axillary lymph nodes were confirmed positive by pre-therapy core biopsy in 85 of 166 cases (51%). The pCR rate according to ME3 scores was 0% (0 of 64) in ME3 < 18, 0% (0 of 46) in ME3 18-25, 14% (3 of 21) in ME3 > 25 to <31, and 40% (14 of 35) in ME3 score 31 or higher (p value: <0.0001). There were no distant recurrences and no deaths in the 17 patients with pCR. In the remaining 149 cases with residual disease, ME3 score of >25 was significantly associated with shorter distant recurrence-free survival and showed a trend for shorter breast cancer-specific survival. The results of this multi-institutional study are similar to previously published data from a single institution (PMID: 28548119) and confirm the chemo-predictive value of ME3 in the neoadjuvant setting. In addition, ME3 may provide prognostic information in patients with residual disease which should be further evaluated.

Purpose of Review: The human epidermal growth factor receptor 2 (HER2) is an important prognostic and predictive biomarker in the breast cancer. The American Society of Clinical Oncology/College of American Pathology (ASCO/CAP) has published HER2 testing guidelines in breast cancer. We herein reviewed the HER2 testing guidelines in breast cancer with a focus on the application of the current guidelines., Recent Findings: The continual investigation of HER2 testing in breast cancer has resulted in updates in the HER2 testing guidelines. The current guidelines focus on the uncommon clinical scenarios and emphasize the coordination between immunohistochemistry and in situ hybridization results, in an effort to improve clarity and accuracy. The ASCO/CAP guidelines provide valuable recommendations to ensure the accurate evaluation of HER2 status in breast cancer patients through standardization. Additional studies, particularly those with long-term outcome data are still needed to validate the guideline recommendations, especially the uncommon cases.

Objectives: To investigate human epidermal growth factor receptor 2 (HER2)-positive nonpleomorphic invasive lobular carcinoma (ILC), which has rarely been addressed., Methods: Clinicopathologic characteristics and follow-up of HER2-positive nonpleomorphic ILCs were collected and compared to those of HER2-negative counterparts., Results: Twenty-one cases of HER2-positive nonpleomorphic ILCs were identified, 6.3% of the study population. Compared to HER2-negative nonpleomorphic ILC, patients with HER2 positivity were older (P < .05), likely to be hormonal receptor negative (P < .01), and had higher histologic grade and angiolymphatic invasion (P < .01). HER2 positivity in nonpleomorphic ILCs was associated with higher recurrence/metastasis with hazard ratio of 2.03 (P < .05). No patient who received neoadjuvant therapy achieved pathologic complete response, and HER2-targeted therapy tended to reduce recurrence/metastasis in patients with HER2-positive nonpleomorphic ILC., Conclusions: Our results highlight the existence of HER2 positivity in nonpleomorphic ILCs and reinforce that HER2 is associated with worse prognosis in nonpleomorphic ILC., (© American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Spindle cell neoplasms of the gastrointestinal (GI) tract constitute a wide group of lesions that may raise diagnostic difficulties on hematoxylin-eosin-stained slides. Appropriate endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) technique with sufficient cell block material for immunohistochemistry (IHC) can lead to accurate diagnosis., Methods: This is a retrospective study of 29 cases obtained from our institution's records over a five-year period (2011-2015). Cytomorphology, histology (when available), IHC, FNA procedure details, imaging characteristics, and clinical history were reviewed. Rapid onsite evaluation (ROSE) was used in all cases. Cytologic samples were correlated with surgical pathology resection specimens when available., Results: Eighteen GI stromal tumors, six leiomyomas, two schwannomas, and one granular cell tumor were analyzed; two cases were not amenable for a definitive diagnosis: one showed fragments of smooth muscle not otherwise specified (smooth muscle vs. leiomyoma) and the other one was insufficient for diagnosis. Locations included stomach, esophagus, duodenum, and colon. EUS-FNA was performed with different gauge needles. Total number of passes ranged between two and nine. We found no evidence that larger-sized needles are superior in procuring adequate lesional tissue. Cell block material was stained with various antibodies. Fourteen surgical resection specimens available showed 100% correlation between cytology and histology. None of the neoplasms recurred until now; one patient succumbed to known esophageal squamous cell carcinoma., Conclusion: FNA is a pivotal and inexpensive method for rapid evaluation of GI spindle cell tumors and should be used widely in the attempt to avoid unnecessary surgery. Size of needle used for EUS-FNA does not seem to influence the yield of lesional tissue; rather, ROSE can guide the number of passes and subsequently lead to an adequate cell block., (© 2018 Wiley Periodicals, Inc.)

Male NZW/BXSB.Yaa (W/B) mice express two copies of TLR7 and develop pathogenic autoantibodies, whereas females with only one copy of TLR7 have attenuated disease. Our goal was to analyze the regulation of the autoantibody response in male and female W/B mice bearing the autoreactive site-directed H chain transgene 3H9. Serum anti-dsDNA Abs appeared in males at 12 wk, and most had high-titer IgG anti-dsDNA and anti-cardiolipin Abs and developed >300 mg/dl proteinuria by 8 mo. Females had only low-titer IgG anti-cardiolipin Abs, and none developed proteinuria by 1 y. Males had a smaller marginal zone than females with a repertoire that was distinct from the follicular repertoire, indicating that the loss of marginal zone B cells was not due to diversion to the follicular compartment. Vk5-43 and Vk5-48, which were rare in the naive repertoire, were markedly overrepresented in the germinal center repertoire of both males and females, but the VJ junctions differed between males and females with higher-affinity autoreactive B cells being selected into the germinal centers of males. Administration of IFN-α to females induced anti-cardiolipin and anti-DNA autoantibodies and proteinuria and was associated with a male pattern of junctional diversity in Vk5-43 and Vk5-48. Our studies are consistent with the hypothesis that presence of the Yaa locus, which includes an extra copy of Tlr7, or administration of exogenous IFN-α relaxes the stringency for selection in the germinal centers resulting in increased autoreactivity of the Ag-driven B cell repertoire.

Lingual adenocarcinomas (ADC), either primary or metastatic to the tongue are extraordinarily rare neoplasms. Primary lingual adenocarcinomas are primarily of minor salivary gland origin. Two cases of primary colonic-type adenocarcinomas of the base of the tongue were recently reported for the first time in the English literature. We present an additional case of lingual intestinal-type adenocarcinoma with mucinous features that occurred in association with cervical node metastasis and discuss the clinicopathologic features and histogenetic aspects of this rare entity.

BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint.

Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP84-102 epitope to HLA-DR2 and either incorporate or lack a TCRzeta signaling domain. The Ag-MHC domain serves as a bait, binding the TCR of MBP-specific target cells. The zeta signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic (alpha, beta, green fluorescent protein) retroviral expression system. MBP-DR2-zeta-, but not MBP-DR2, modified CTL were specifically stimulated by cognate MBP-specific T cells, proliferating, producing cytokine, and killing the MBP-specific target cells. The receptor-modified therapeutic cells were active in vivo as well, eliminating Ag-specific T cells in a humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP84-102/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-zeta chimeric receptors as a potential therapeutic in MS.

Recent preclinical and clinical trials have demonstrated the therapeutic potential of T lymphocytes redirected with genetically engineered T-cell receptor (TCR) surrogates against infected, cancerous, or autoreactive cells. These surrogate TCRs link a ligand-recognition domain to signaling regions from the TCR. We previously compared the function of surrogate TCRs that include TCR or TCR and CD28 signaling regions. We found that primary murine T cells modified to specifically target Kb-restricted CD8+ T cells using either Kb-zeta or Kb-CD28-zeta receptors had similar functional activities, although the CD28-zeta receptor showed a 2-fold to 4-fold decreased expression. We have now identified a previously unrecognized dileucine motif in the murine CD28 signaling domain that accounts for this reduced expression. Inactivation of this motif increased chimeric receptor surface expression 2- to 5-fold. T cells expressing the dileucine-mutated CD28-zeta chimeric receptor demonstrated enhanced proliferation, cytokine production, and cytolytic activities. Further, cells expressing this dileucine-mutated receptor were highly effective in eliminating antigen-specific CD8+ T lymphocytes in vivo. These results therefore identify a critical motif limiting the function of receptor-modified T lymphocytes, demonstrate that inactivation of this motif enhances chimeric receptor function, and illustrate a potential novel application of receptor-modified T lymphocytes in the induction of immune tolerance.