Immunohistochemical Classification Research Articles

Clinicopathological differences between T-lymphoblastic leukemia/lymphoma, early T-precursor lymphoblastic leukemia/lymphoma, and mixed-phenotype acute leukemia with T lineage: An analysis of 41 adult cases

The histopathological diagnosis of T-lymphoblastic leukemia/lymphoma, NOS (T-ALL), is based on morphology and positivity for CD3 and TdT. Early T-precursor lymphoblastic leukemia/lymphoma (ETP-ALL) and mixed-phenotype acute leukemia (MPAL), T/M, and/or B rarely occur and are usually diagnosed using flow cytometry. Using only formalin-fixed paraffin-embedded tissue raises the risk of misdiagnosis due to underestimation. Immunostaining markers for T cell (CD1a, CD4, CD5, CD8), B cell (CD19, CD10, CD22, CD79a), and stem/myeloid-related cell (CD33, CD34, CD117, MPO, lysozyme) diagnosed 25 T-ALL cases (61%), 7 MPAL (17%), 6 ETP-ALL (15%), and 3 near ETP-ALL (7%), with subsequent analysis of their clinicopathological characteristics. Patients with MPAL had significantly poorer 2-year progression-free survival (14.3% vs. 60.4%, P = 0.012) and 5-year overall survival (28.6% vs. 65.9%, P = 0.011) than did those with T-ALL, whereas ETP-ALL and near ETP-ALL did not. Of the seven patients with MPAL, three were classified as T/B, two as T/M, and two as T/M/B. Because most MPALs (6/7) share the ETP-ALL phenotype, immunohistochemistry for CD19 and MPO should be performed to avoid misdiagnosing MPAL as ETP-ALL. All three patients with TdT-negative MPAL died of the disease. Four patients with MPO-positive MPAL relapsed during the early phase (1–9 months). Five patients received the ALL regimen, but two patients received acute myeloid leukemia and lymphoma regimens, respectively. In this study, MPAL exhibited a poorer prognosis compared to T-ALL, unlike ETP-ALL. Thus, immunohistochemical classification with multiple antibody panels is useful for accurate diagnosis and treatment.

Employing semi-quantitative methods to classify breast cancer markers (ER, PR, HER2, Ki67) expression, using the RT-qPCR based APIS Breast Cancer Subtyping Kit.

e13091 Background: Accurate determination of breast cancer (BC) biomarker status is crucial for guiding patient management decisions. Histopathologists commonly use semi-quantitative scoring systems to convert subjective observations of immunohistochemistry (IHC) marker expression into quantitative data, allowing a more detailed categorisation of marker status. The APIS Breast Cancer Subtyping Kit (BCSK) is an in vitro diagnostic test, detecting the relative expression of seven mRNA target genes ( ESR1, PGR, ERBB2, MKI67, CCNA2, PCNA, and KIF23) from invasive BC tissue. The APIS BCSK reports a positive/negative result for each biomarker alongside a molecular classification. Here, we demonstrate that by implementing additional RNA expression cut-off values, it is possible to generate a semi-quantitative result by further stratifying target expression into negative, low, medium, and high status using the APIS BCSK. Methods: 368 formalin-fixed paraffin-embedded (FFPE) samples of BC cases were used, obtained by core needle biopsy or resection. IHC scores were correlated with copy numbers as determined by an independently validated digital PCR (dPCR) assay and were used to set copy number cut-off values corresponding to IHC classification. The generated dPCR cut-off values were used to evaluate the corresponding ∆Ct values (RNA expression results derived from the APIS BCSK). For each target, three ΔCt cut-offs were established, facilitating classification into negative, low, medium, and high expression. Results: The semi-quantitative approach successfully classified target expression levels ( ESR1, PGR, ERBB2, MKI67). Overlap in ΔCt values was observed in central IHC categories for all targets, likely due to tumor heterogeneity and differing measurement methods for IHC and RT-qPCR. Despite these discrepancies, the derived cut-off values define a semi-quantitative scale for each target (Table). Conclusions: This study has established a methodology that utilises target copy number values and IHC protein quantification to establish ΔCt cut-off points. For each target we have defined four distinct groups as negative, low, medium, and high expression. This approach provides a semi-quantitative scale for evaluating each target with the APIS BCSK and serves as a valuable tool for classifying and understanding target expression levels. [Table: see text]

#2757 Clinical course and prognostic factors in amyloidosis

Abstract Background and Aims Early diagnosis and specific therapy are crucial for the prognosis of patients with systemic amyloidosis. The aim of this single-center retrospective study was to evaluate the clinical course and identify prognostic factors associated with survival in a large cohort of patients from the Regional Reference Center “Gemelli Isola” in Rome, Italy. Method A total of 244 patients with amyloidosis were studied, including 100 with AL (63 M, 37 F, mean age 64 ± 13 yrs), 18 with AA (3 M, 15 F, mean age 57 ± 21 yrs), 9 with amyloidosis due to myeloma (MM) (5 M, 4 F, mean age 65 ± 15 yrs), 69 with ATTR mutant (38 M, 31 F, mean age 63 ± 15 yrs), 33 with ATTR wild-type (31 M, 2 F, mean age of 70 ± 10 yrs) 2 with Apolipoprotein (1 M, 1 F, mean age 60 yrs), and 15 unclassified amyloidosis (AX) (9 M, 6 F, mean age 63 ± 9 yrs). We considered several parameters, comprising gender, age, type of amyloidosis, time interval between disease onset and diagnosis, clinical signs and symptoms at onset and diagnosis, biochemical parameters, score of renal involvement (RI) (absent, proteinuria, nephrotic syndrome, and eGFR < 60 ml/min), score of cardiac involvement (CI) (absent, hypertrophic cardiopathy, dilated cardiopathy, arrhythmias and heart failure) at onset, diagnosis, and follow-up (FU). Renal outcome was defined as renal replacement therapy (RRT) initiation. The diagnosis of amyloidosis was histologically confirmed in all patients by Congo red stain, immunohistochemical classification and specific diagnostic criteria. Data were collected at diagnosis and retrieved dating back to the clinical onset. The impact of treatment was evaluated, in terms of clinical course and prognosis. Results Regardless of the amyloidosis type, between the onset and diagnosis clinical signs and symptoms significantly worsened and the involvement of affected organs increased. The median time from disease onset to diagnosis was 12 months for AL, AX, and MM, and 36 and 37 months for AA and ATTR. RI increased in all patients, from 47% at onset to 65% at diagnosis, and 71% during FU (p < 0.001). In the AL subgroup an eGFR < 60 ml/min was identified in 16% of patients at onset, 37% at diagnosis and 51% at FU (p < 0.001). As expected, in the mutant ATTR group RI was identified in only a small proportion of patients and an eGFR < 60 ml/min was found only in 2% of patients at onset and at diagnosis and in 6% at FU. CI was observed in 77% of all patients at onset, 88% at diagnosis and 98% at FU (p < 0.001). In the AL subgroup, at onset there was a high prevalence of hypertrophic cardiopathy (65%), while at FU there was a significant increase in the prevalence of heart failure (51%) (p < 0.001). In the mutant ATTR group, at onset, hypertrophic cardiopathy was present in 80% of patients while, at FU, heart failure was present in 64%. The median global survival time was 8 years (IC 95%, 7-12 yrs). RRT was started in 28% of patients. Survival curves according to renal and cardiac scores are shown in Fig. 1. Mortality at 6 years was statistically significative in patients with RI (p < 0.005). Survival curve according to renal or cardiac vs RI and CI is shown in Fig. 2. The univariate analysis demonstrated that progressive cardiac impairment from onset to FU, the number of organs involved, serum creatinine, albumin and eGFR had a significant impact on survival (HR of 18.46, CI 95% = 2.57-132.79; HR = 1.33, CI 95% = 1.09-1.62; HR = 1.47, CI 95% = 1.05-2.05; HR = 0.37, CI 95% = 0.24-0.56; HR = 0.99, CI 95% = 0.98-0.99). The multivariate analysis was based on the ‘forward stepwise’ method to select the significant predictors of mortality. The number of organs involved, age at onset, CI at onset, albumin and progressive worsening of CI from onset to FU, significantly impacted on survival (HR = 1.30, CI 95% = 1.02-1.65, p = 0.031, HR = 1.03, CI 95% = 1.01-1.05; HR = 2.08, CI 95% = 1.20-3.60; HR = 0.33, CI 95% = 0.21-0.53; HR = 21.40, CI 95% = 2.95-155.14). Conclusion The diagnosis of amyloidosis is often delayed. Prognosis is significantly influenced by the degree of CI and RI, serum albumin, age at onset and the number of organs involved. Better diagnostic accuracy and the availability of novel therapies have however significantly improved survival rates.

Abstract PO1-19-09: Integrating gene signatures to guide HR+ MBC therapy in a diverse cohort (INSIGHT)

Abstract Background: Black women with breast cancer (BC) have a 40% higher mortality rate compared to Non-Hispanic White (NHW) women. Among women with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) BC, Black women have worse outcomes than NHW despite comparable systemic therapies. Gene expression profiling assays have been used in early-stage BC to provide prognostic and sometimes predictive information beyond standard immunohistochemical classifications. The 80-gene molecular subtype signature (BluePrint) with the 70-gene risk of distant recurrence signature (MammaPrint®) (Agendia, Irvine, CA) further classify HR+/HER2- BC into luminal A-type, luminal B-type, HER2-type, and basal-type tumors. Luminal B, HER2, and basal-type tumors (non-luminal A) are more aggressive with worse survival outcomes and are overrepresented among Black women. In the metastatic setting, the role of molecular subtype signatures in guiding therapeutic decisions has not yet been determined. We hypothesize that patients with metastatic HR+/HER2- non-luminal A-type BC post-progression on endocrine therapy +/- CKD4/6 inhibition will derive more benefit from chemotherapy than standard-of-care endocrine therapy in the second line setting. We also hypothesize that the impact of the intervention will be more pronounced in Black women compared to NHW women. Methods: This is a randomized phase II study that will evaluate the anti-tumor effect of capecitabine compared to physician’s choice endocrine therapy as second line therapy for adult ( >18 years) patients with non-Luminal A HR+/HER2- metastatic or unresectable locoregional invasive carcinoma (NCT 05693766). The study plans to enroll up to 62 patients. This trial enriches for racial/ethnic minority patients through collaborations with the University of Texas Southwestern and the University of Alabama at Birmingham, two health systems that serve a large minority population. Eligible patients who have received prior endocrine therapy with a CDK4/6 inhibitor will have their tumor tissue analyzed using the MammaPrint® and BluePrint assays. Patients with Luminal A tumors will receive standard of care and will be followed for survival only. Patients with non-Luminal A tumors will be randomized (1:1) to receive physician’s choice endocrine therapy versus capecitabine (dosed at 2000mg twice daily for 7 days on, 7 days off) with stratifications by molecular subtype and race. Patients will be evaluated for response every three cycles using the Response Evaluation Criteria in Solid Tumors. Therapy will be continued until evidence of disease progression or unacceptable major toxicity. The primary endpoint is progression free survival; secondary endpoints are overall response rate, clinical benefit rate, overall survival, and patient reported outcomes. The study will have 80% power to detect a minimal hazard ratio of 0.5 at one-sided significance level of 0.05. Cell-free DNA will be collected at baseline and at three times post-baseline to investigate potential genetic markers of disease response and resistance. Enrollment opened on June 1, 2023. We anticipate opening the trial at the external sites before the end of 2023. Citation Format: Moriah Forster, Jennifer Whisenant, Ben Park, Erica Stringer-Reasor, William Audeh, Andrea Menicucci, Fei Ye, Heather McArthur, Sonya Reid. Integrating gene signatures to guide HR+ MBC therapy in a diverse cohort (INSIGHT) [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 PO1-19-09.

Safety and efficacy of anlotinib combined with taxane and lobaplatin in neoadjuvant treatment of clinical stage II/III triple-negative breast cancer in China (the neoALTAL trial): a single-arm, phase 2 trial

Anlotinib is a new type of tyrosine kinase inhibitor that targets vascular endothelial growth factor receptors 1/2/3, platelet-derived growth factor receptors α/β, and fibroblast growth factor receptors 1-4 and c-Kit, with a broad spectrum of inhibitory effects on tumor angiogenesis and growth. It has been proven effective in HER2-negative metastatic breast cancer, but its efficacy in early-stage triple-negative breast cancer (TNBC) is unknown. This phase 2 study aims to evaluate the efficacy and safety of adding anlotinib to neoadjuvant chemotherapy in patients with TNBC. Patients with clinical stage II/III TNBC were treated with 5 cycles of anlotinib (12mg, d1-14, q3w) plus 6 cycles of taxanes (docetaxel 75 mg/m2 ,d1, q3w or nab-paclitaxel 125 mg/m2, d1 and d8, q3w) and lobaplatin (30 mg/m2, d1, q3w), followed by surgery. The primary endpoint was pathological complete response (pCR; ypT0/is ypN0) and the secondary endpoints include breast pCR (bpCR), axillary pCR (apCR), residual cancer burden (RCB), objective response rate (ORR), survival, and safety. Exploratory endpoints were efficacy biomarkers based on Fudan University Shanghai Cancer Center Immunohistochemical (FUSCC IHC) classification for TNBC and next-generation sequencing (NGS) of DNA from tumor tissue and blood samples of patients with 425-gene panel. This trial is registered with www.chictr.org.cn (ChiCTR2100043027). From Jan 2021 to Aug 2022, 48patients were assessed and 45 were enrolled. All patients received at least one dose of study treatment and underwent surgery. The median age was 48.5 years (SD: 8.7), 71% were nodal involved, and 20% had stage III. In the intention-to-treat population, 26 out of 45patients achieved pCR (57.8%; 90% CI, 44.5%-70.3%), and 39 achieved residual cancer burden class 0-I (86.7%; 95% CI, 73.2%-94.9%). The bpCR and apCR rate were 64.4% (29/45) and 71.9% (23/32), respectively. No recurrence or metastasis occurred during the short-term follow-up. Based on the FUSCC IHC-based subtypes, the pCR rates were 68.8% (11/16) for immunomodulatory subtype, 58.3% (7/12) for basal-like immune-suppressed subtype and 33.3% (4/12) for luminal androgen receptor subtype, respectively. NGS revealed that the pCR were 77% (10/13) and 50% (14/28) in MYC-amplified and wild-type patients, respectively, and 78% (7/9) and 53% (17/32) in gBRCA1/2-mutated and wild-type patients, respectively. The median follow-up time of the study was 14.9 months (95% CI: 13.5-16.3 months). There was no disease progression or death during neoadjuvant therapy. No deaths occurred during postoperative follow-up. In the safety population (N=45), Grade 3 or 4 treatment emergent adverse events occurred in 29patients (64%), and the most common events were neutropenia (38%), leukopenia (27%), thrombocytopenia (25%), anemia (13%), and hypertension (13%), respectively. The addition of anlotinib to neoadjuvant chemotherapy showed manageable toxicity and encouraging antitumor activity for patients with clinical stage II/III TNBC. Chongqing Talents Project, Chongqing Key Project of Technology Innovation and Application Development and Chongqing Outstanding Youth Natural Science Foundation.

Differences in Histological Subtypes of Invasive Lobular Breast Carcinoma According to Immunohistochemical Molecular Classification.

The aim of this study was to compare the differences between histological and molecular subtypes of invasive lobular carcinoma (ILC) of the breast, in order to be able to predict the behavior and prognosis of the disease, as well as to effectively determine therapy. This study included 263 cases of breast ILC diagnosed over a seven-year period. The diagnosis of invasive lobular carcinoma is based on the characteristic growth pattern and phenotype of cancer cells with the respective subtypes: classic, alveolar, solid, tubulolobular, pleomorphic and mixed lobular type. The examined cases were divided into five groups according to molecular classification based on the expression of ER, PR, HER2 and Ki67 immunohistochemical markers. It was found that the pleomorphic subtype occurred statistically significantly less often as the luminal A subtype compared to others (p = 0.00027), and the HER2-enriched subtype occurred statistically significantly more often in the pT4 stage (p = 0.024). The results of this study significantly singled out the luminal A subtype, and among them classic ILC, as the subtype with the most favorable expression ratio of the investigated predictive/prognostic immunohistochemical markers.

Prognostic significance of immunohistochemical classification utilizing biopsy specimens in patients with extensive-disease small-cell lung cancer treated with first-line chemotherapy and immune checkpoint inhibitors

PurposeAlthough immune checkpoint inhibitors (ICIs), together with cytotoxic chemotherapy (chemoimmunotherapy), have been adapted for the initial treatment of extensive-disease small-cell lung cancer (ED-SCLC), they have achieved limited success. In ED-SCLC, a subtype of SCLC, the expression of immune-related molecules and clinical data are not well understood in relation to ICI treatment efficiency.MethodsWe examined lung biopsy specimens from patients diagnosed with ED-SCLC treated with chemoimmunotherapy or chemotherapy. SCLC subtype, expression of HLA class I, and infiltration of CD8-positive cells were examined using immunohistochemistry (IHC). Subsequently, the association between clinical factors, IHC results, and progression-free survival or overall survival was assessed.ResultsMost of the cases showed the achaete-scute homolog 1 (ASCL1) subtype. Among the 75 SCLC cases, 29 expressed high levels of HLA class I, while 46 showed low levels or a negative result; 33 patients were characterized as CD8-high, whereas 42 were CD8-low. In the chemoimmunotherapy cohort, multivariate analysis revealed a correlation between CD8-high and improved survival. Specifically, patients in the CD8-high group of the chemoimmunotherapy cohort experienced enhanced survival compared to those in the chemotherapy cohort, which was attributed to ICI addition. IHC subtype analysis demonstrated a survival advantage in the SCLC-I and SCLC-A groups when ICI was combined with chemotherapy compared to chemotherapy alone.ConclusionOur study highlights the predictive value of IHC-classified subtypes and CD8-positive cell infiltration in estimating outcomes for patients with ED-SCLC treated with chemoimmunotherapy as a first-line therapy. These findings have practical implications for daily clinical assessments and treatment decisions.

Discrepancies Between Morphological and Immunohistochemical Classifications Are Associated With Prognoses and Subtypes of Lung Cancer.

Immunohistochemical (IHC) staining has been routinely used to distinguish adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of the lungs; however, it is challenging to obtain an accurate diagnosis, especially for cases with discrepancies between IHC and hematoxylin and eosin (H&E) staining results. This study aimed to clarify the clinicopathological characteristics of these discrepant cases. Tissue microarray specimens from 321 patients with ADC and SCC were used for H&E and IHC staining of thyroid transcription factor 1 (TTF-1), Napsin A, cytokeratin 5/6 (CK5/6), p40, and p63. The pathological diagnosis was made based on (1) H&E, (2) IHC, and (3) both H&E and IHC results. Discrepant cases were defined as those with different diagnoses based on the H&E and IHC results. A total of 32 (10%) discrepant cases were identified. ADC (3.9%) showed fewer discrepant cases than SCC (51%). Discrepant cases of ADC had a significantly higher proportion of poorly differentiated tumors and subtypes of solid and invasive mucinous ADC, and they also had shorter overall and disease-free survival than concordant cases. Solid and invasive mucinous ADC cases showed low positivity for TTF-1 (84% and 40%, respectively) and Napsin A (88% and 80%, respectively), and invasive mucinous ADC cases showed high positivity for CK5/6 (80%). The sensitivity and specificity of TTF-1+Napsin A for ADC were 91% and 83%, respectively, whereas those of CK5/6+p40 for SCC cases were 90% and 96%, respectively. Discrepant cases of ADC are associated with solid and invasive mucinous subtypes and shorter survival.

Subtyping of Breast Carcinoma According to ER/PR and HER2/neu Expression: A Cross-Sectional Study from Southern Part of Assam, India

Introduction: Globally, breast carcinoma is the most prevalent and lethal form of cancer in women. Breast cancer is no longer considered a single disease but a complex heterogeneous disease with multiple genetic and epigenetic alterations. The prognosis and management of the disease depend on histological stage, type, grade, tumour size, lymph node status, and the status of hormonal receptors like ER, PR, and Her2/ neu. Recently, more attention has been given to the molecular classification of breast cancer. Aim: To analyse and compare the clinicopathological characteristics of invasive breast cancer in the four breast carcinoma subtypes defined by the immunohistochemical expression of Estrogen Receptor (ER), Progesterone Receptor (PR), and Human Epidermal Growth Factor Receptor 2 (Her2/neu). Materials and Methods: The cross-sectional study was conducted on 64 primary invasive breast carcinoma cases diagnosed on mastectomy specimens between February 1, 2018, and July 30, 2022, in the Department of Pathology at the histopathology section of Silchar Medical College and Hospital, Silchar, Assam, India. Age and tumour characteristics (morphology, grade, stage, and size) and nodal disease status were included in the data for analysis. Immunohistochemical markers were analysed on the sections of these diagnosed cases. IBM Statistical Packages for Social Sciences (SPSS) software was used for data analysis. Qualitative data was presented as frequency and percentage, while quantitative data was presented as mean ±{Standard Deviation (SD)}. The Chi- square test was used to determine the statistical significance of hormonal receptors with the various clinicopathological features. A p-value of <0.05 was considered statistically significant. Results: In present study of 64 cases of invasive breast carcinoma, the mean age of patients was 51.95±12.72 years. Subtyping was performed based on hormonal receptors. Authors found that the Luminal A variety 33 (51.6%) was the most common hormonal subtype in present study, followed by the basal subtype 24 (37.5%). The Luminal A subtype was found to be predominant among others. The majority of the patients (59.4%) had stage-I tumours, and Ductal type carcinoma was the most common (57.8%). Histologically, most of the tumours were poorly differentiated (28, 43.8%), and most were sized ≤2 cm (41, 64.1%). Lymph nodes were not palpable in most of the patients (43, 67.2%). Subtype comparison with respect to age, stage, histological grade, type, size, and nodal status revealed statistically significant outcomes (p-value of <0.05). Conclusion: Classification based on Immunohistochemistry (IHC) provides prognostic and therapeutic information that cannot be obtained from either ER/PR or Her2/Neu status alone. The present study provides the incidence of different molecular subtypes in the southern region of Assam, and comparison among them with statistical correlation offers improved and crucial treatment guidance. IHC classification as a clinical tool for ER/PR and Her2/Neu testing is widely accessible, reasonably priced, based on immunophenotype/biologic phenotype categorisation of breast cancer, and is prognostic as well as partly predictive and needs to be practiced invariably.

Molecular Characterization of HER2-Low Invasive Breast Carcinoma by Quantitative RT-PCR Using Oncotype DX Assay.

HER2 immunohistochemistry (IHC) reproducibility is suboptimal for HER-low cases (IHC 1+ or 2+). The Yale cohort included 214 stages I-II estrogen receptor positive breast cancers with IHC scores 0, 1+, and 2+ and routine Oncotype DX Recurrence Score (RS) results. The Exact Sciences (ES) cohort included 9 57 624 patients who had an Oncotype DX RS assay that assigns HER2-negative, equivocal, or positive status based on HER2 mRNA levels. HER2 mRNA levels varied across IHC categories but with increasing medians of 9.10 (n = 89), 9.20 (n = 71), and 9.45 (n = 54) in IHC 0, 1+, and 2+, respectively. 22.4% of HER2-low (1+/2+) cancer had RS > 25. Over 98% of HER-low cancers were HER2-negative by Oncotype DX assignment. Cancers with higher mRNA levels exist within IHC 0 and low categories, most of the HER2-low patients by IHC have low RS indicating no benefit from current adjuvant chemotherapies.

High-throughput proteomics of breast cancer subtypes: Biological characterization and multiple candidate biomarker panels to patients' stratification

Background and aimsThe actual classification of breast tumors in subtypes represents an attempt to stratify patients into clinically cohesive groups, nevertheless, clinicians still lack reproducible and reliable protein biomarkers for breast cancer subtype discrimination. In this study, we aimed to access the differentially expressed proteins between these tumors and its biological implications, contributing to the subtype's biological and clinical characterization, and with protein panels for subtype discrimination. MethodsIn our study, we applied high-throughput mass spectrometry, bioinformatic, and machine learning approaches to investigate the proteome of different breast cancer subtypes. ResultsWe identified that each subtype depends on different protein expression patterns to sustain its malignancy, and also alterations in pathways and processes that can be associated with each subtype and its biological and clinical behaviors. Regarding subtype biomarkers, our panels achieved performances with at least 75% of sensibility and 92% of specificity. In the validation cohort, the panels obtained acceptable to outstanding performances (AUC = 0.740 to 1.00). ConclusionsIn general, our results expand the accuracy of breast cancer subtypes' proteomic landscape and improve the understanding of its biological heterogeneity. In addition, we identified potential protein biomarkers for the stratification of breast cancer patients, improving the repertoire of reliable protein biomarkers. SignificanceBreast cancer is the most diagnosed cancer type worldwide and the most lethal cancer in women. As a heterogeneous disease, breast cancer tumors can be classified into four major subtypes, each presenting particular molecular alterations, clinical behaviors, and treatment responses. Thus, a pivotal step in patient management and clinical decisions is accurately classifying breast tumor subtypes. Currently, this classification is made by the immunohistochemical detection of four classical markers (estrogen receptor, progesterone receptor, HER2 receptor, and the Ki-67 index); however, it is known that these markers alone do not fully discriminate the breast tumor subtypes. Also, the poor understanding of the molecular alterations of each subtype leads to a challenging decision-making process regarding treatment choice and prognostic determination. This study, through high-throughput label-free mass-spectrometry data acquisition and downstream bioinformatic analysis, advances in the proteomic discrimination of breast tumors and achieves an in-depth characterization of the subtype's proteomes. Here, we indicate how the variations in the subtype's proteome can influence the tumor's biological and clinical differences, highlighting the variation in the expression pattern of oncoproteins and tumor suppressor proteins between subtypes. Also, through our machine-learning approach, we propose multi-protein panels with the potential to discriminate the breast cancer subtypes. Our panels achieved high classification performance in our cohort and in the independent validation cohort, demonstrating their potential to improve the current tumor discrimination system as complements to the classical immunohistochemical classification.

Prognostic factors in Luminal B-like HER2-negative breast cancer tumors

BackgroundMolecular and genomic platforms can classify breast cancer intrinsic subtypeswith precision, however these are not widespread and immunohistochemical (IHC) classification is still used globally. This study aimed to evaluate the main clinical and pathologic prognostic factors for Luminal B-like HER2-negative breast cancer in our clinical setting. MethodsA retrospective review of early Luminal B-like HER2-negative breast cancer patients diagnosed in 2017 in our center was conducted. The main prognostic factors for relapse were evaluated, including patient's characteristics such as age, menopausal status, comorbidity index, personal and family history of breast cancer and obesity; tumor features such as size, histology and grade, oestrogen and progesterone receptor (PgR) status, HER2 status, Ki67 index and nodal involvement; and the given treatment. Cancer relapse during five years of follow-upwas considered the main outcome. ResultsFifty-six patients with early Luminal B-like HER2-negative breast cancer were included. Seven patients relapsed within five years of follow-up. Lymph node involvement at diagnosis and postoperatively were significantly associated with relapse (24,5% vs 71,43%p = 0.022; 38,8% vs 83,3%p = 0.004, respectively),although the number of pathologic positive lymph nodes was not associated with relapse occurrence (mean 1.5 in no-relapse group vs 0.8 in relapse group; p = 0.308).Other possible risk factors such as young age, premenopausal status, self-history of breast cancer, tumor size, histologic grade, PgR, or Ki 67 were not significantly associated with relapse. Additionally, the distribution of the number of positive nodes among relapse and no relapse groups(2,1 vs 1,8; p = 0.082) was not significant. ConclusionsLymph node involvement was the only prognostic factor in Luminal B-like HER2-negative breast cancer identified in this study, independently of the number of affected nodes.

Tumor-Infiltrating CD8-Positive T-Cells Associated with MMR and p53 Protein Expression Can Stratify Endometrial Carcinoma for Prognosis.

Inspired by the molecular classification of endometrial carcinoma (EC) proposed by The Cancer Genome Atlas Research Network (TCGA), we investigated tumor-infiltrating CD8-positive T-cell as well as DNA mismatch repair (MMR) protein and p53 protein expression, and we developed a new classification system for ECs to predict patients' prognosis using immunohistochemical methods. The study included 128 patients with ECs who underwent surgery. Paraffin-embedded tissue sections of the tumor were stained using antibodies against MMR protein, p53, and CD8. Cases were stratified into four classes by a sequential algorithm. An immunohistochemical classification system for ECs (ICEC) was created, including HCD8, MMR-D, LCD8, and p53 LCD8. In ICEC, 16 cases (12.5%), 27 cases (21.09%), 67 cases (52.34%), and 18 cases (14.06%) belonged to HCD8, MMR-D, LCD8, and p53 LCD8, respectively. ICEC did not show any correlation with clinical stage, lymphovascular space invasion, or lymph node metastasis. However, the p53 LCD8 class contained a significantly higher proportion of G3 ECs and serous carcinoma (p < 0.0001). ICEC showed prognostic significance in overall survival (OS) (p < 0.0001) and disease-free survival (DFS) (p < 0.0001). The class of p53 LCD8 showed the worst prognosis among the classes. ICEC classification is useful in predicting the prognosis of ECs.

Immune infiltrates in metastatic uveal melanoma liver metastases and associations with clinical outcomes.

9538 Background: Uveal melanoma has a poor prognosis following metastasis to the liver. Immune checkpoint inhibitors (ICIs) are often utilized in patients with metastatic uveal melanoma (MUM); however, ICIs in MUM have low response rates. We studied the association of immune infiltrates in MUM liver metastases with survival outcomes for all patients, in addition to evaluating if immune infiltrates improve survival for patients treated with ICIs. Methods: We included MUM patients with liver metastases who had pre-treatment liver biopsies available at Mayo Clinic Rochester. Demographics, disease characteristics, treatments, and clinical outcomes were obtained from the electronic medical record. Core biopsies of liver metastases were reviewed for the presence of lymphocytes at interface of the tumor and liver parenchyma, and for tumor-infiltrating lymphocytes (TILs). If TILs were present, then additional immunohistochemistry (IHC) staining was performed for CD3, CD4, CD8, PD1, CD25, and FoxP3. Overall survival (OS) was estimated with the Kaplan-Meier method and compared between groups with Cox proportional hazards regression. Median OS and hazard ratios (HR) were calculated and p-values (p) &lt;0.05 were considered statistically significant. Results: 140 patients with MUM who had pre-treatment liver biopsies were identified. 122 biopsies were evaluable for the presence of lymphocytes, either at the tumor-liver interface, or for TILs. 80/122 (65.6%) biopsies showed presence of any lymphocyte and 27/80 had TILs. 27 biopsies had sufficient tissue for further IHC classification. CD8+ T cells were identified in 27/27 samples, T cell PD1 positivity ≥1% in 4/27 samples, and regulatory T cell markers (CD25 and/or FoxP3) in 18/27 samples. Median OS for the entire cohort was 16 months (mo). Patients still alive at the time of data analysis had at least one year of follow-up available (range 13-124 mo). Univariate analyses (UVA) showed no significant improvement in OS based on the presence of any lymphocytes (HR 1.01, p 0.98), lymphocytes at the tumor-liver interface (HR 1.10, p 0.64), TILs (HR 0.78, p 0.27), &gt;50% CD8+ T cells (HR 0.88, p 0.77), or absence of FoxP3+ T cells (HR 0.68, p 0.40). In the 69 patients treated with ICIs (pembrolizumab, nivolumab, or ipilimumab plus nivolumab), neither the presence of any lymphocytes (HR 1.19, p 0.56) nor TILs (HR 0.68, p 0.24) were associated with an OS benefit. Conclusions: We present a large cohort of patients with MUM where we characterized immune infiltrates in liver metastases and evaluated clinical outcomes. OS did not significantly improve with the presence of any lymphocytes, TILs, CD8+ T cells, or with the absence of FoxP3+ T cells. These results were consistent across all patients whether they were treated with ICIs or not. Our findings suggest that lymphocytes in MUM are functionally dormant and highlights the need for novel immune-activating treatment strategies.

Programmed Death-Ligand 1 Copy Number Alteration as an Adjunct Biomarker of Response to Immunotherapy in Advanced NSCLC

IntroductionThis study aimed to evaluate the value of programmed death-ligand 1 (PD-L1) copy number (CN) alteration as an additional biomarker to standard immunohistochemistry (IHC) in predicting response to immune checkpoint inhibitor (ICI) therapy in advanced NSCLC. MethodsBefore ICI monotherapy, tumor PD-L1 CN alteration (gain, neutral, or loss) was called using whole-exome sequencing data and compared with IHC results (tumor proportion score ≥50, 1–49, or 0). Progression-free survival (PFS) and overall survival were correlated with both biomarkers. In addition, the impact of CN alteration was further evaluated in two independent cohorts using next-generation sequencing panel. ResultsA total of 291 patients with advanced-stage NSCLC met the study inclusion criteria. Although the IHC classification distinguished the best responsive group (tumor proportion score ≥ 50), the CN-based classification distinguished the worst responsive group (CN loss) from the others (PFS, p = 0.020; overall survival, p = 0.004). After adjusting for IHC results, CN loss was an independent risk factor for progression (adjusted hazard ratio = 1.32, 95% confidence interval: 1.00–1.73, p = 0.049) and death (adjusted hazard ratio = 1.39, 95% confidence interval: 1.05–1.85, p = 0.022). A risk classification system was developed on the basis of IHC and CN profiles, which outperformed the conventional IHC system. In the validation cohorts, CN loss determined by next-generation sequencing panel was independently associated with worse PFS after ICI treatment, revealing its practical value. ConclusionsThis is the first study to directly compare CN alterations with IHC results and survival outcomes after anti–PD-(L)1 therapy. Tumor PD-L1 CN loss can serve as an adjunct biomarker to predict the lack of response. Prospective studies are required to further validate this biomarker.

Abstract P2-23-14: Molecular characterization of HER2-low invasive breast carcinoma by quantitative RT-PCR using Oncotype DX®

Abstract Background: The DESTINY-Breast04 clinical trial demonstrated the superiority of trastuzumab deruxtecan (T-DXd) versus physician’s choice chemotherapy in HER2-low [immunohistochemistry (IHC) staining score 1+ or 2+ with no gene amplification by in-situ hybridization] metastatic breast cancer. The reproducibility of HER2 IHC category 0 versus 1+ scoring is poor in community practice. 89% of the DESTINY-04 participants were hormone receptor positive (HR+). In HR+ breast cancers, the Oncotype DX® (ODX) assay is widely used to estimate risk of recurrence and guide adjuvant chemotherapy selection. It also provides standardized quantification of HER2 mRNA expression by RT-PCR. Prior studies have demonstrated a high degree of overall concordance between central IHC and RT-PCR using ODX in HER2+ and HER2- cases. Here, we (i) compare HER2 mRNA levels quantified by ODX in HER2 IHC 0, 1+ and 2+ invasive breast carcinomas (IBC), (ii) compare the Recurrence Score® (RS) distribution across these three IHC categories and (iii) describe RS distribution and proliferation score in HER2-low IBC. Methods: 212 patients with HER2 IHC 0, 1+ and 2+ who were negative for HER2 gene amplification by FISH and had RS results were identified in Yale Department of Pathology archives. All US samples submitted for IBC ODX testing between 2005 to 2021 were reviewed in the Exact Sciences database. RS, quantitative HER2 mRNA expression, and proliferation scores were examined. IHC results were not available. Based on quantitative RT-PCR measures of HER2 expression, cases were assigned to: HER2 positive ≥11.5, equivocal ≥10.7 to &amp;lt; 11.5, and negative &amp;lt; 10.7 (1 unit increment is equivalent to approximately 2-fold change). Results: In the Yale cohort, 42%, 33%, and 25% of cases were IHC 0, 1+ and 2+, respectively. There was no difference in age or tumor grade by IHC category. HER2 mRNA levels increased across IHC categories (means 9.05, 9.16, 9.39, respectively), but in group-wise comparisons, only the IHC 0 compared to IHC 2+ reached statistical significance (Mann-Whitney test, p=0.0014). The RS scores were also modestly, but significantly higher in IHC 2+ compared to IHC 0 cases (mean 19.5 vs 14.51, Mann-Whitney p=0.034). Among IHC 0 and 1+ cases, 14% had RS &amp;gt;25, and among IHC 2+ cases, 32% had RS &amp;gt;25. All IHC 0 and 1+ cases were HER2 negative by RT-PCR. Of the HER2 2+ cases, there was one HER2 positive and one HER2 equivocal by RT-PCR. There was substantial variation in HER2 expression by RT-PCR in all IHC groups. In the Exact Sciences cohort, a total of 957,624 samples were analyzed. 0.8% of samples were HER2 positive, 1.2% were equivocal, and 98% were negative by RT-PCR. There was a wide range of RS results. Of the HER2 positive cases, 94.7% had RS &amp;gt;25. Among the HER2 equivocal cases 39.1% had RS &amp;gt;25, and among the HER2 negative cases, 15.5% had RS &amp;gt;25. Conclusions: HER2 IHC 0 and HER2 low IBC have a broad and overlapping range of HER2 expression by RT-PCR. Whether RT-PCR based HER2 expression predicts benefit from T-DXd is yet to be determined. Most HER2 low cases have RS ≤25, indicating no or limited benefit from adjuvant chemotherapy. Further studies are required to determine if patients with HER2 low IBC and RS ≤25, particularly with high anatomical risk, could benefit from adjuvant T-DXd. Table 1. Clinicopathologic and molecular characteristics by HER2 IHC group Table 2. Quantitative gene expression by HER2 IHC group Citation Format: Mariya Rozenblit, Hao-Kuen Lin, Nhu Thuy Can, Cynthia A. Flannery, Jess Hoag, Alekhya Akkunuri, Helen Bailey, Frederick Baehner, Lajos Pusztai. Molecular characterization of HER2-low invasive breast carcinoma by quantitative RT-PCR using Oncotype DX® [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-14.

Abstract HER2-10: HER2-10 Dynamics of HER2-low expression in triple-negative breast cancer

Abstract Background: With the development of novel antibody drug conjugates (ADC), it is increasingly important to understand the changes that occur in cell-surface targets over time from early-stage to metastatic breast cancer. Discordance in HER2 expression per immunohistochemistry (IHC) has been reported between primary and metastatic tumors in patients (pts) with HER2-negative breast cancer defined per ASCO/CAP guidelines, with both gain and loss of expression described. Representation of HR-negative (TNBC) tumors has been limited in these studies, and HER2 status at multiple time points in TNBC has not been described. Here we report changes in HER2 IHC in patients diagnosed with TNBC, using a collection of matched tumor samples over time. Methods: Pts were identified from two sources: 1) an institutional database including all consecutive pts who underwent surgery for stage I-III breast cancer at Dana-Farber/Brigham Cancer Center between 2015-2018, and 2) a prospective research biopsy protocol for pts with known or suspected metastatic breast cancer. Pts were included in the present analysis if they received neoadjuvant chemotherapy (NAC) for stage I-III TNBC (eTNBC), or if they were diagnosed with any stage TNBC and ultimately developed metastatic TNBC (mTNBC). Clinical pathology records were reviewed for HER2 IHC results of samples collected at: initial diagnosis (DX); residual disease (RD) post-NAC, if applicable; and at recurrence (M). HER2 IHC was classified as HER2-0 if HER2 IHC 0, and HER2-low if 1+ or 2+ (and ISH non-amplified). For matched comparisons, if IHC was performed in more than one DX sample (e.g., breast, node), only the breast was considered; if more than one M sample, only the first M biopsy with available IHC was considered. Results: Among 110 pts in this cohort, 101 were initially diagnosed with eTNBC (79 received NAC; 22 underwent surgery as first intervention) and 9 with de novo mTNBC. Median age was 48.7 years (range 19.8-71.6). Among all pts, a total of 292 samples (136 DX, 53 RD, 103 M) had available HER2 IHC scores. When restricting to one sample per time point, HER2-low prevalence was 49/102 (48.0%) in DX breast tumors, 21/53 (39.6%) in RD, and 13/58 (22.4%) in first M samples (with all remaining samples HER2-0, except one HER2 3+ sample). In eTNBC pts, HER2 IHC scores were available for 50 paired DX and RD, and 48 paired DX and M samples. Among 50 pts with paired DX and RD, HER2 IHC was discordant in 56% (28/50) (Table 1). Of the 21 HER2-0 DX tumors, 23.8% (5/21) became HER2-low at RD. Of the 29 HER2-low DX tumors, 51.7% (15/29) became HER2-0 at RD. Among 48 eTNBC pts who recurred and had paired DX and M samples, HER2 IHC was discordant in 50% (24/48) (Table 1). Change from HER2-0 to low was 12.5% (3/24), and from HER2-low to HER2-0 was 66.7% (16/24). Among 9 de novo mTNBC pts, 5 had HER2 IHC available in paired DX breast and M prior to starting therapy; 3 were concordant (IHC 0, n=2; IHC 1+, n=1), one had IHC 0 in DX breast and IHC 2+ in M (liver), one had IHC 1+ in DX breast and IHC 0 in M (node). Conclusions: HER2 IHC classification was discordant in about half of the TNBC cases we examined, with more frequent rates of conversion from HER2-low to HER2-0 in both paired DX/RD post-NAC, and paired DX/M samples. Additional analyses will be presented exploring HER2 IHC changes among multiple metastases per patient. Genomic and molecular analysis, including whole exome sequencing, RNA sequencing, and methylation profiling, are underway in these samples to further elucidate HER2 evolutionary dynamics. Citation Format: Ana C. Garrido-Castro, Lan D. Ngo, Edward T. Richardson, Allison Frangieh, Ayesha Mohammed-Abreu, Melissa E. Hughes, Jorge Gomez Tejada Zanudo, John Navarro, Paolo Tarantino, Elizabeth A. Mittendorf, Sara Tolaney, Tari King, Eric Winer, Nancy U. Lin, Nikhil Wagle. HER2-10 Dynamics of HER2-low expression in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-10.

The Lund taxonomy for bladder cancer classification - from gene expression clustering to cancer cell molecular phenotypes, and back again.

Treatment of bladder cancer patients depends on precise diagnosis. Molecular subtyping by gene expression profiling may contribute substantially to subclassification of bladder cancer. Several classification systems have been proposed. Most of these base their classification on whole biopsy features, and molecular subtypes are therefore often defined by a combination of features from the cancer cells as well as infiltrating noncancer cells. This makes the link to what is seen at the cancer cell level unclear. The aim of the Lund taxonomy (LundTax) has been to align gene expression-level classification with immunohistochemical classification to identify cancer cell phenotypes independent of infiltration and proliferation. A systematic approach was used in which gene expression clusters were validated and adjusted by immunohistochemistry using markers expressed only by the cancer cells. This review provides a rationale for defining molecular subtypes and a step-by-step description of the development of the LundTax with motivations for each modification and extension. As the cancer cell phenotype defined by gene expression profiling corresponds with the immunohistochemistry of cancer cells, the LundTax represents a harmonization of the gene expression and immunohistochemical levels. Furthermore, the classification system is independent of pathological stage and is, thus, applicable to all urothelial carcinomas. A unified classification system relevant for both the molecular biologist and pathologist will facilitate systematization of current treatment practices, as well as the development of new treatments. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Effect of the MiR-99b and MiR-135b on peritoneal carcinomatosis and liver metastasis in colorectal cancer

AimThis study aimed to evaluate the expression levels of miR-99b and miR-135b in peritoneal carcinoma and liver metastases associated with Colorectal Cancer (CRC), assess their association with the intracellular signaling pathway proteins Kirsten Rat Sarcoma Virus (KRAS) and Akt, and investigate their effects on survival. Materials and methodsChanges in the KRAS gene and Akt proteins, expression levels of miR-99b and miR-135b, and factors affecting survival were compared between colorectal cancer-associated peritoneal carcinomatosis and liver metastasis. ResultsThe expression levels of miR-99b and miR-135b and the immunohistochemical grade classification score of Akt were higher in colorectal cancer, peritoneal carcinomatosis, and liver metastasis than in normal tissues (p < 0.05). MiR-99b expression was highest in CRC, whereas miR-135b expression was highest in peritoneal carcinomatosis (p < 0.05). The expression level of miR-99b decreased and that of miR-135b increased in peritoneal and liver metastases compared with that in the tumor tissue. MiR-99b, Akt, and recurrence were risk factors that affected the overall survival rate in the model of clinical predictions (p = 0.045, p = 0.006, and p = 0.012, respectively). ConclusionWhile the expression of miR-99b was highest in the primary tumor, its decrease in liver metastasis and peritoneal carcinomatosis suggests that miR-99b has a protective effect against liver metastasis and peritoneal carcinomatosis. However, the detection of miR-135b expression was highest in peritoneal carcinomatosis and liver metastasis compared with that in the colorectal cancer tissues suggesting that it facilitates peritoneal carcinomatosis and liver metastasis. Furthermore, miR-99b, KRAS mutations, and Akt are risk factors for the overall survival of colorectal cancer.

Interobserver Variation in the Assessment of Immunohistochemistry Expression Levels in HER2-Negative Breast Cancer: Can We Improve the Identification of Low Levels of HER2 Expression by Adjusting the Criteria? An International Interobserver Study

The classification of human epidermal growth factor receptor 2 (HER2) expression is optimized to detect HER2-amplified breast cancer (BC). However, novel HER2-targeting agents are also effective for BCs with low levels of HER2. This raises the question whether the current guidelines for HER2 testing are sufficiently reproducible to identify HER2-low BC. The aim of this multicenter international study was to assess the interobserver agreement of specific HER2 immunohistochemistry scores in cases with negative HER2 results (0, 1+, or 2+/in situ hybridization negative) according to the current American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Furthermore, we evaluated whether the agreement improved by redefining immunohistochemistry (IHC) scoring criteria or by adding fluorescent in situ hybridization (FISH).We conducted a 2-round study of 105 nonamplified BCs. During the first assessment, 16 pathologists used the latest version of the ASCO/CAP guidelines. After a consensus meeting, the same pathologists scored the same digital slides using modified IHC scoring criteria based on the 2007 ASCO/CAP guidelines, and an extra “ultralow” category was added.Overall, the interobserver agreement was limited (4.7% of cases with 100% agreement) in the first round, but this was improved by clustering IHC categories. In the second round, the highest reproducibility was observed when comparing IHC 0 with the ultralow/1+/2+ grouped cluster (74.3% of cases with 100% agreement). The FISH results were not statistically different between HER2-0 and HER2-low cases, regardless of the IHC criteria used.In conclusion, our study suggests that the modified 2007 ASCO/CAP criteria were more reproducible in distinguishing HER2-0 from HER2-low cases than the 2018 ASCO/CAP criteria. However, the reproducibility was still moderate, which was not improved by adding FISH. This could lead to a suboptimal selection of patients eligible for novel HER2-targeting agents. If the threshold between HER2 IHC 0 and 1+ is to be clinically actionable, there is a need for clearer, more reproducible IHC definitions, training, and/or development of more accurate methods to detect this subtle difference in protein expression levels.