M2 tumor-associated macrophages play important role in predicting response to neoadjuvant chemotherapy in triple-negative breast carcinoma.

PURPOSE: Two types of macrophages are present in tumor microenvironment. M1 macrophages exhibit potent anti-tumor properties, while M2 macrophages play the pro-tumoral roles. The presence of M2 macrophages is associated with worsened overall survival in triple-negative breast carcinoma (TNBC) patients. However, the relationship between M2 macrophages and response to neoadjuvant chemotherapy (NAC) is unknown. METHODS: M2 macrophages were investigated on biopsy whole sections from 66 TNBCs treated with NAC by CD163 together with other immune checkpoint markers (PD1, PD-L1 and CD8) using a multi-color immunohistochemical multiplex assay. RESULTS: Incomplete response was significantly associated with older age, lower PD-L1 expression (tumor and stroma), lower levels of CD8-positive TILs in stroma, but higher level of CD163-positive macrophages, with the level of CD163-positive M2 macrophages in peritumoral area as the strongest factor. CONCLUSIONS: Our data have demonstrated that the level of CD163-positive M2 macrophages was significantly higher in TNBC patients with incomplete response than patients with complete response, suggesting M2 macrophages' important role in predicting TNBC patients' response to NAC.

HER2 intratumoral heterogeneity is independently associated with distal metastasis and overall survival in HER2-positive breast carcinomas.

PURPOSE: Human epidermal growth factor receptor 2 (HER2) intratumoral heterogeneity (ITH) occurs in a subset of breast cancers. Our recent study revealed it as an independent predictive factor for the response to anti-HER2 neoadjuvant therapy. In this study, we aimed to investigate its association with distal metastasis. METHODS: HER2 ITH was assessed using HER2 gene protein assay (GPA) on whole tissue sections of pretreatment biopsies from a cohort of 158 HER2-positive invasive breast carcinomas and correlated with patients' clinical follow-up outcomes along with other clinicopathologic characteristics. RESULTS: Fifty-seven cases (36%) showed HER2 ITH including 19 with genetic, 8 with both genetic and non-genetic, and 30 with non-genetic ITH. Multivariate analysis demonstrated larger tumor size, positive resected lymph node(s), negative PR, and the presence of HER2 ITH were independently associated with distal metastasis. Additionally, multivariate analysis demonstrated larger tumor size and the presence of HER2 ITH were the only independent factors associated with decreased overall survival (death). CONCLUSION: The presence of HER2 ITH is an independent factor associated with poor overall survival and increased distal metastasis in HER2-positive breast cancer patients.

Exploratory analysis of immune checkpoint receptor expression by circulating T cells and tumor specimens in patients receiving neo-adjuvant chemotherapy for operable breast cancer.

BACKGROUND: While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun testing in patients with breast cancer (BC), the effects of chemotherapy on ICP expression in circulating T cells and within the tumor microenvironment are still unclear. This information could help with the design of future clinical trials by permitting the selection of the most appropriate ICP inhibitors for incorporation into NAC. METHODS: Peripheral blood samples and/or tumor specimens before and after NAC were obtained from 24 women with operable BC. The expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured using flow cytometry. Furthermore, using multi-color immunohistochemistry (IHC), the expression of immune checkpoint molecules by stromal tumor-infiltrating lymphocytes (TILs), CD8+ T cells, and tumor cells was determined before and after NAC. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors were determined by a paired Student's t-test. RESULTS: This analysis showed decreased ICP expression by circulating CD4+ T cells after NAC, including significant decreases in CTLA4, Lag3, OX40, and PD-1 (all p values < 0.01). In comparison, circulating CD8+ T cells showed a significant increase in CTLA4, Lag3, and OX40 (all p values < 0.01). Within tumor samples, TILs, CD8+ T cells, and PD-L1/PD-1 expression decreased after NAC. Additionally, fewer tumor specimens were considered to be PD-L1/PD-1 positive post-NAC as compared to pre-NAC biopsy samples using a cutoff of 1% expression. CONCLUSIONS: This work revealed that NAC treatment can substantially downregulate CD4+ and upregulate CD8+ T cell ICP expression as well as deplete the amount of TILs and CD8+ T cells found in breast tumor samples. These findings provide a starting point to study the biological significance of these changes in BC patients. TRIAL REGISTRATION: NCT04022616.

PD-L1 expression and CD8-positive T cells are associated with favorable survival in HER2-positive invasive breast cancer.

Programmed cell death 1 (PD-1) and its ligand (PD-L1) are key physiologic suppressors of the cytotoxic immune reaction. However, to date, the combination of PD1/PD-L1 expression and tumor-infiltrating lymphocytes (TILs) and antigen-presenting cells has been only minimally reported in breast carcinoma, in particular in relation to HER2-positive cases. The goal of this study was to evaluate both cellular tumoral immune reaction and PD-L1/PD1 distribution in HER2-positive cases, as well as any associations with clinical outcome using conventional chemotherapy combined with HER2 blocking. Multicolor immunohistochemical multiplex assays simultaneously demonstrating PD1, PD-L1, and CD8 or PD-L1, CD3, and CD163 were performed on tissue microarrays (TMA) representing 216 pretreatment cases of HER2-positive invasive breast carcinoma. PD-L1 expression was identified in 38 cases (18%), including 12 cases (6%) with PD-L1 labeling of tumor cells and 26 cases (12%) with PD-L1 labeling of immune cells only. Ten of 12 cases with PD-L1 staining of tumor cells showed staining of associated immune cells as well. With this assay method, PD1 was detectable in many fewer cases (6 cases or 3%). PD-L1 expression was positively associated with high Nottingham grade, negative ER and PR, the absence of lymph node metastasis, and high levels of CD8+ cells. The overall survival by univariate analysis was positively associated with lower tumor stage, the absence of lymph node metastasis, PD-L1 expression, and high levels of CD8+ cells. Therefore, our data suggest cytotoxic immune reaction mediated by CD8-positive T cells and PD-L1 expression may predict a better outcome in patients with HER2-positive breast carcinoma managed with conventional chemotherapy and HER2-blocking therapy. These findings recommend clinical trials utilizing checkpoint blocking immunotherapy in some form for HER2-positive breast cancer.

HER2 intratumoral heterogeneity is independently associated with incomplete response to anti-HER2 neoadjuvant chemotherapy in HER2-positive breast carcinoma.

PURPOSE: Anti-HER2 neoadjuvant chemotherapy has been widely used in HER2-positive breast cancer patients; however, pathologic complete response (pCR) is achieved in only 40-50% of patients. The aim of this study was to investigate the association of HER2 intratumoral heterogeneity (ITH) with response to anti-HER2 neoadjuvant chemotherapy. METHODS: Assessment of HER2 ITH was performed on whole tissue sections of pre-treatment samples from a cohort of 64 invasive breast carcinoma cases originally considered positive for HER2 and treated with anti-HER2 neoadjuvant chemotherapy. Both HER2 gene signal and protein expression were simultaneously evaluated by means of a single-slide dual assay, designated as a HER2 gene-protein assay (GPA). HER2 GPA was carried out as well on surgical resection tissues from 25 cases with incomplete therapeutic response. RESULTS: Nineteen of 64 cases (30%) showed HER2 ITH. Significantly more cases with HER2 ITH were found in the incomplete response group (56%, 14/25) than in the pCR group (13%, 5/39). Patients without ITH detectable by GPA had a 76% pCR outcome (34/45), as compared to 26% (5/19) for those with detectable ITH. Multivariate analysis demonstrated HER2 ITH, progesterone receptor positivity, and relatively low HER2/chromosome 17 centromere ratio to be significantly associated with incomplete response. CONCLUSIONS: HER2 ITH analyses conducted with GPA method revealed that HER2 ITH is an independent factor predicting incomplete response to anti-HER2 neoadjuvant chemotherapy.

HER2 intratumoral heterogeneity analyses by concurrent HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer patients.

HER2 gene-protein assay (GPA) is a new method for the simultaneous evaluation of HER2 immunohistochemistry (IHC) and HER2 dual in situ hybridization (DISH) on single tissue sections of breast cancer. We investigated the presence of HER2 gene and protein discrepancy and HER2-heterogeneity using HER2-GPA. HER2 status was analyzed for the correlation between the presence of HER2-heterogeneity and patient prognosis. Consecutive 280 invasive breast cancer were examined. Statuses of HER2 protein and gene were evaluated in whole tumor sections of HER2 GPA slides. HER2 protein and gene combination patterns were classified to six phenotypic and genotypic types for each case, as well as at individual cell levels: (A) IHC and DISH positive; (B) IHC positive and DISH negative; (C) IHC equivocal and DISH positive; (D) IHC equivocal and DISH negative; (E) IHC negative and DISH positive; and (F) IHC and DISH negative. The presence of HER2-heterogeneity was determined by the existence of at least two of six types within one tumor. HER2-IHC positive patients had significantly worse survival than IHC negative patients and HER2-DISH positive patients had significantly worse survival than DISH negative patients. HER2 IHC negative and DISH positive patients had significantly worse recurrence-free survival than IHC and DISH negative patients. In the HER2 IHC and DISH negative group, the HER2 heterogeneous group had significantly worse survival than the nonheterogeneous group. Notably, among triple negative breast cancer (TNBC), the HER2 heterogeneous group had significantly worse survival than the nonheterogeneous group. Our study suggests that the presence of HER2-heterogeneity might be a prognostic factor in HER2 negative breast cancer patients, especially in TNBC.

Gene copy number gain of EGFR is a poor prognostic biomarker in gastric cancer: evaluation of 855 patients with bright-field dual in situ hybridization (DISH) method.

BACKGROUND: EGFR overexpression is a prognostic biomarker and is expected to be a predictive biomarker for anti-EGFR therapies in gastric cancer. However, few studies have reported the clinical impact of EGFR gene copy number (GCN) and its correlation with EGFR overexpression. METHODS: We used dual in situ hybridization (DISH) to detect EGFR GCN and chromosome 7 centromere (CEN7) in a set of tissue microarrays representing 855 patients with gastric cancer. These data were compared with those of immunohistochemical (IHC) analysis of EGFR expression to evaluate prognostic value. RESULTS: EGFR GCN gain (≥ 2.5 EGFR signals per cell) was detected in 194 patients (22.7%) and indicated poor prognosis. Among 194 patients, EGFR amplification (EGFR/CEN7 ≥ 2.0) was observed in 29 patients (14.9%), which was almost identical to the IHC 3+ subgroup and worst prognostic subgroup. Patients with EGFR GCN gain but not amplification, including those exhibiting polysomy, also exhibited poorer prognosis than GCN non-gain patients and were distributed between IHC 0/1+ and 2+ subgroups. GCN gain was frequently observed in patients with more advanced disease, but served as an independent prognostic factor regardless of the pathological stage. CONCLUSIONS: EGFR GCN gain is a more accurate prognostic biomarker than EGFR overexpression in patients with gastric cancer.

The assessment of HER2 status in breast cancer: the past, the present, and the future.

Humanized monoclonal anti-human growth factor receptor 2 (HER2) antibody trastuzumab was approved for HER2 positive breast cancer patient treatment 11 years after the demonstration of HER2 gene amplification associated with the HER2 protein overexpression in breast cancer in 1987. HER2 positive status of breast cancer patients is assessed by HER2 gene amplification with in situ hybridization (ISH) and/or HER2 protein overexpression with immunohistochemistry (IHC). Because the discordance between quantitative HER2 ISH and subjective, semi-quantitative HER2 IHC assay results is a well-recognized issue of HER2 testing, we developed an assay combining HER2 ISH and HER2 IHC assays (HER2 gene-protein assay; HER2 GPA) as one test on the same tissue section. HER2 GPA allows pathologists to score the HER2 gene and HER2 protein status simultaneously at the individual cell level. The possibility that HER2 GPA may become the next generation of HER2 testing is discussed, particularly for cases in which it is difficult to assess the HER2 status of breast cancer patients due to the HER2 heterogeneity.

A novel gene-protein assay for evaluating HER2 status in gastric cancer: simultaneous analyses of HER2 protein overexpression and gene amplification reveal intratumoral heterogeneity.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) protein overexpression and gene amplification are important biomarkers for trastuzumab treatment in breast and gastric cancer patients. Gastric cancer presents high rates of tumor heterogeneity, which may influence the results of HER2 testing. A novel gene-protein assay (GPA) can allow the simultaneous analysis of HER2 protein and gene status on a single slide. METHODS: Using the tissue microarray technique, the HER2 status of each of 875 gastric cancer cases was evaluated by immunohistochemistry (IHC), brightfield dual-color in situ hybridization (DISH), and GPA. Intratumoral phenotypic and genotypic heterogeneity were evaluated by comparing the HER2 statuses of two tissue cores from each case. RESULTS: There was excellent concordance between GPA and IHC (99.2 %), as well as between GPA and DISH results (99.3 %). HER2 positivity obtained by GPA was almost identical (99.8 %) to the results obtained by IHC and DISH assays. Intratumoral phenotypic heterogeneity was more frequently observed in IHC 2+ cases (63.5 %) compared with IHC 3+ cases (28.3 %). Phenotypic heterogeneity (48.8 %) was more frequently observed than genotypic heterogeneity (26.8 %). Tumor heterogeneity was consistently observed from early to advanced stages. CONCLUSIONS: HER2-positive gastric cancers presented different levels of HER2 protein expression and gene amplification statuses within the same lesion in almost half the cases examined. Evaluating both phenotypic and genotypic heterogeneity may contribute to a deeper understanding and improved prediction of clinical outcome in gastric cancer patients treated with trastuzumab. This newly established GPA technology may also be useful for developing biomarkers for other molecularly targeted therapies.

Epidermal growth factor receptor mutation-specific immunohistochemical antibodies in lung adenocarcinoma.

AIMS: We investigated the sensitivity and specificity of two novel Epidermal growth factor receptor (EGFR) mutation-specific antibodies in the detection of the most common EGFR mutations in lung adenocarcinoma. METHODS AND RESULTS: A total of 241 resected lung adenocarcinoma specimens and six resected post-neoadjuvant gefitinib adenocarcinomas were analysed for EGFR mutation using mass spectrometry, fragment analysis and direct PCR sequencing platforms. Tissue arrays and/or full sections of these cases were evaluated using immunohistochemistry with two novel antibodies (clones SP125 and SP111) and two previously reported antibodies (clones 43B2 and 6B6), specific for L858R or 15-nucleotide exon-19 deletion EGFR mutations. SP125 antibody detected EGFR L858R mutation with a sensitivity of 76% and positive predictive value of 73%. SP111 antibody stained the 15-nucleotide EGFR exon-19 deletions with a sensitivity of 83% and a positive predictive value of 94%. Pretreatment with gefitinib did not affect antibody performance. Full-section immunohistochemical staining detected heterogeneous mutant EGFR proteins expression in tumours, and revealed L858R mutation in the non-neoplastic bronchial epithelium adjacent to EGFR L858R-carrying carcinomas in three of 16 (19%) cases. CONCLUSIONS: Immunohistochemistry using EGFR mutant-specific antibodies may be useful in shortening the diagnostic time of lung adenocarcinoma with most common EGFR mutations, especially in samples with low tumour cellularity.

HER2 Intratumoral Heterogeneity in Breast Cancer, an Evolving Concept.

Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is associated with an adverse prognosis. The introduction of anti-HER2 targeted therapy has dramatically improved the clinical outcomes of patients with HER2-positive breast cancer. Unfortunately, a significant number of patients eventually relapse and develop distant metastasis. HER2 intratumoral heterogeneity (ITH) has been reported to be associated with poor prognosis in patients with anti-HER2 targeted therapies and was proposed to be a potential mechanism for anti-HER2 resistance. In this review, we described the current definition, common types of HER2 ITH in breast cancer, the challenge in interpretation of HER2 status in cases showing ITH and the clinical applications of anti-HER2 agents in breast cancer showing heterogeneous HER2 expression. Digital image analysis has emerged as an objective and reproducible scoring method and its role in the assessment of HER2 status with ITH remains to be demonstrated.

HER2 Gene Protein Assay: A Robust Tool for Evaluating HER2 Status and Intratumoral Heterogeneity in Endometrial Cancers.

OBJECTIVES: Human epidermal growth factor receptor 2 (HER2) status in endometrial cancer is usually determined by immunohistochemistry and/or in situ hybridization. We employed a novel HER2 gene protein assay (GPA) to simultaneously assesses HER2 gene amplification and protein expression in high-grade endometrial cancers. METHODS: We performed GPA in 180 endometrial cancers, including 106 serous carcinomas, 34 carcinosarcomas, and 40 mixed epithelial carcinomas. HER2 status was determined using the 2018 HER2 guidelines for breast carcinoma, and HER2 intratumoral heterogeneity (ITH) was examined. Clinicopathologic characteristics were collected and correlated with HER2 status. RESULTS: HER2 positivity was noted in 32% of serous carcinomas, significantly higher than in carcinosarcomas (5.9%) and mixed carcinomas (12.5%). HER2 ITH was detected in 32% of serous carcinomas, significantly greater than in carcinosarcomas (8.8%) and mixed carcinomas (10%). Patients with carcinosarcoma had a significantly lower overall survival than patients with serous or mixed epithelial carcinoma, but HER2 status caused no difference in survival in patients with serous carcinoma. CONCLUSIONS: HER2 GPA can be used to accurately determine HER2 status in endometrial cancers and is a highly valuable tool for identifying HER2 heterogeneity.

Artificial intelligence reveals features associated with breast cancer neoadjuvant chemotherapy responses from multi-stain histopathologic images.

Advances in computational algorithms and tools have made the prediction of cancer patient outcomes using computational pathology feasible. However, predicting clinical outcomes from pre-treatment histopathologic images remains a challenging task, limited by the poor understanding of tumor immune micro-environments. In this study, an automatic, accurate, comprehensive, interpretable, and reproducible whole slide image (WSI) feature extraction pipeline known as, IMage-based Pathological REgistration and Segmentation Statistics (IMPRESS), is described. We used both H&E and multiplex IHC (PD-L1, CD8+, and CD163+) images, investigated whether artificial intelligence (AI)-based algorithms using automatic feature extraction methods can predict neoadjuvant chemotherapy (NAC) outcomes in HER2-positive (HER2+) and triple-negative breast cancer (TNBC) patients. Features are derived from tumor immune micro-environment and clinical data and used to train machine learning models to accurately predict the response to NAC in breast cancer patients (HER2+ AUC = 0.8975; TNBC AUC = 0.7674). The results demonstrate that this method outperforms the results trained from features that were manually generated by pathologists. The developed image features and algorithms were further externally validated by independent cohorts, yielding encouraging results, especially for the HER2+ subtype.

A Multiparameter Molecular Classifier to Predict Response to Neoadjuvant Lapatinib plus Trastuzumab without Chemotherapy in HER2+ Breast Cancer.

PURPOSE: Clinical trials reported 25% to 30% pathologic complete response (pCR) rates in HER2+ patients with breast cancer treated with anti-HER2 therapies without chemotherapy. We hypothesize that a multiparameter classifier can identify patients with HER2-"addicted" tumors who may benefit from a chemotherapy-sparing strategy. EXPERIMENTAL DESIGN: Baseline HER2+ breast cancer specimens from the TBCRC023 and PAMELA trials, which included neoadjuvant treatment with lapatinib and trastuzumab, were used. In the case of estrogen receptor-positive (ER+) tumors, endocrine therapy was also administered. HER2 protein and gene amplification (ratio), HER2-enriched (HER2-E), and PIK3CA mutation status were assessed by dual gene protein assay (GPA), research-based PAM50, and targeted DNA-sequencing. GPA cutoffs and classifier of response were constructed in TBCRC023 using a decision tree algorithm, then validated in PAMELA. RESULTS: In TBCRC023, 72 breast cancer specimens had GPA, PAM50, and sequencing data, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio ≥ 4.6 and %3+ IHC staining ≥ 97.5%. With PAM50 and sequencing data, the model added HER2-E and PIK3CA wild-type (WT). For clinical implementation, the classifier was locked as HER2 ratio ≥ 4.5, %3+ IHC staining ≥ 90%, and PIK3CA-WT and HER2-E, yielding 55% and 94% positive (PPV) and negative (NPV) predictive values, respectively. Independent validation using 44 PAMELA cases with all three biomarkers yielded 47% PPV and 82% NPV. Importantly, our classifier's high NPV signifies its strength in accurately identifying patients who may not be good candidates for treatment deescalation. CONCLUSIONS: Our multiparameter classifier differentially identifies patients who may benefit from HER2-targeted therapy alone from those who need chemotherapy and predicts pCR to anti-HER2 therapy alone comparable with chemotherapy plus dual anti-HER2 therapy in unselected patients.

Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models.

Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5' and 3' probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved.

Predictive significance of HER2 intratumoral heterogeneity, determined by simultaneous gene and protein analysis, for resistance to trastuzumab-based treatments for HER2-positive breast cancer.

Gene-protein assay (GPA), a combination of immunohistochemistry and dual in situ hybridization, allows simultaneous visualization of HER2 protein and gene on a single slide. We aimed to clarify the clinical significance of HER2 intratumoral heterogeneity (ITH) using GPA. We investigated the relationships between various HER2 ITH indicators and clinical course in 102 patients with HER2-positive breast cancer, treated with neoadjuvant trastuzumab and chemotherapy. Five representative microscopic images were captured from each GPA slide of pre-therapeutic biopsy materials. All evaluable cancer cells in the images were individually assessed for HER2 gene copy number and protein expression. Mean and coefficient of variation (CV) of both gene copy number and protein category were calculated, and each was divided into negative, equivocal, and positive. Based on their combined status, cancer cells were classified into nine types. Pathological complete response (pCR) to neoadjuvant treatments showed positive relationships to mean gene copy number (P < 0.001), mean protein category (P < 0.001), and proportion of gene- and protein-positive tumor cells (P < 0.001) and showed negative relationships to the CV of protein category (P < 0.001) and the proportion of gene-amplified but protein-negative tumor cells (P = 0.002). Two diagnostic models, created by combining clinicopathological factors and ITH indicators, showed excellent potential diagnostic ability for pCR (mean gene copy number and protein category CV; AUC = 0.837, proportion of gene- and protein-positive tumor cells; AUC = 0.831). HER2 ITH quantified by GPA is a potential predictive indicator for efficacy of HER2-targeted treatment.

Increased MYC gene copy number correlates with increased mRNA levels in diffuse large B-cell lymphoma.

BACKGROUND: Translocations involving the MYC gene and increased MYC mRNA levels are associated with poor outcome in diffuse large B-cell lymphoma. However, the presence of increased MYC gene copy number and/or polysomy of chromosome 8 have not been previously described. DESIGN AND METHODS: Utilizing dual color chromogenic in situ hybridization, we investigated MYC gene copy and chromosome 8 centromere numbers in 52 cases of diffuse large B-cell lymphoma. Cases were divided into those with "increased" or "not increased" MYC gene copy number for comparison with MYC mRNA levels, Ki-67 values, and survival. RESULTS: Increased MYC gene copy number was present in 38% of cases. Overall, the average MYC mRNA level was 2398 (range, 342 - 9783) and the percentage of nuclei positive for Ki-67 was 57.5% (range, 20-87%). Within the group with increased MYC copy number, the MYC mRNA values ranged from 816 to 5912 (average, 2843) and the Ki-67 values ranged from 23% to 83% (average, 57%). Within the group with not increased MYC copy number, MYC mRNA values ranged from 342 to 9783 (average, 2118) and the Ki-67 values ranged from 20% to 87% (average, 58%). There was a statistically significant relationship between increased MYC gene copy number and increased MYC mRNA (P=0.034) and a trend toward a relationship between increased mRNA and higher Ki-67 values. CONCLUSIONS: This is the first report that low level copy number increases are common in diffuse large B-cell lymphoma and that these changes correlate with MYC mRNA in a statistically significant manner. MYC copy number changes are an additional possible molecular mechanism that may result in increased mRNA and, likely, high proliferation and poor outcome.

The pre-B-cell receptor associated protein VpreB3 is a useful diagnostic marker for identifying c-MYC translocated lymphomas.

BACKGROUND: During B-cell development, precursor B cells transiently express the pre-B-cell receptor composed of µ heavy chain complexed with VpreB and λ5 surrogate light chain polypeptides. Recent profiling studies unexpectedly revealed abundant transcripts of one member of the VpreB family, VpreB3, in a subset of mature B cells and Burkitt lymphoma. DESIGN AND METHODS: Here we used a novel antibody to investigate the normal expression pattern of VpreB3 protein in human hematopoietic and lymphoid tissues, and to determine whether VpreB3 could serve as a useful diagnostic biomarker for select B-cell lymphomas. RESULTS: We found that VpreB3 protein is normally expressed by precursor B cells in bone marrow and by a subset of normal germinal center B cells in secondary lymphoid organs. Among lymphoid malignancies, we found an association between VpreB3 expression and B-cell tumors with c-MYC abnormalities. VpreB3 was highly expressed in all cases of Burkitt lymphoma, whether of endemic or sporadic origin (44/44 cases, 100%), all cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (5/5 cases, 100%), and the majority of diffuse large B-cell lymphomas harboring a c-MYC translocation (15/18 cases, 83%). The expression of VpreB3 in diffuse large B-cell lymphomas without a c-MYC translocation was associated with c-MYC polysomy in 25/75 cases (33%) but only rarely observed in diffuse large B-cell lymphomas lacking a c-MYC abnormality (9/98 cases, 9%). CONCLUSIONS: We conclude that for B-cell tumors with features suggesting a possible c-MYC translocation, such as intermediate to large cell size and high proliferation rate, the presence of VpreB3 should prompt subsequent confirmatory genetic testing, whereas the absence of VpreB3 is virtually always associated with wild-type c-MYC alleles.

Breast HER2 Intratumoral Heterogeneity as a Biomarker for Improving HER2-Targeted Therapy.

Development of HER2-targeted therapy drugs, particularly trastuzumab, demonstrated significant improvement of clinical outcomes among HER2 positive breast cancer patients during the last two decades. The exact biological mechanism of HER2 gene amplification occurrence remains unsolved. HER2 gene amplification and/or HER2 protein overexpression are the primary predictors for selecting invasive breast cancer patients as candidates for anti-HER2 agent-based chemotherapy protocol. However, HER2-targeted therapy is not completely successful: as it is well-documented, only one half of HER2 positive breast cancer patients achieve a pathological complete response after such a precision therapy. In the past, various HER2 drug resistance mechanisms were proposed for explaining incomplete the efficacy with anti-HER2 drugs. Recent studies suggested that HER2 intratumoral heterogeneity (ITH) determined by a concomitant HER2 gene and protein analyses are a significant primary resistance mechanism to HER2-targeted therapy. Recent discovery of undocumented "nonclassic" HER2-positive tumor cells with the amplified HER2 gene but no HER2 protein overexpression redefined HER2 ITH. The HER2 ITH consists of two groups of tumor heterogeneity subtypes: (1) genetic ITH (a mixture of HER2 negative tumor cells and classic HER2 positive tumor cells) and (2) nongenetic ITH (a mixture of classic HER2 positive tumor cells and nonclassic HER2 positive tumor cells). The mechanism underlining these nonclassic HER2 positive tumor cells with the amplified HER2 gene, but no HER2 protein overexpression, is unknown. Investigation of impaired HER2 and/or protein translation in these tumor cells could lead to a further improvement of cancer therapy by identifying new therapeutic targets for patients with HER2 ITH.

Details of human epidermal growth factor receptor 2 status in 454 cases of biliary tract cancer.

Human epidermal growth factor receptor 2 (HER2)-targeted therapy has improved clinical outcomes in patients with HER2-positive breast and gastric cancers, although ineffective or recurrent cases are present. One reason for this is the heterogeneity of HER2 expression in cancer cells. The aim of this study was to investigate the clinicopathological characteristics and HER2 status of patients with biliary tract cancers (BTCs). We examined HER2 protein expression by immunohistochemistry, HER2 gene amplification by fluorescence in situ hybridization, and both HER2 protein and gene levels simultaneously by gene-protein assay. Samples were collected from 454 patients who underwent surgical resection for BTCs (110 intrahepatic cholangiocarcinomas [ICC], 67 perihilar extrahepatic cholangiocarcinomas [ECC-Bp], 119 distal extrahepatic cholangiocarcinomas [ECC-Bd], 80 gallbladder carcinomas [GBC], and 79 ampullary carcinomas [AVC]). HER2 status was assessed according to the guidelines for HER2 testing in gastroesophageal adenocarcinoma. HER2-positive status was detected in 14.5% of BTCs (3.7% of ICC, 3.0% of ECC-Bp, 18.5% of ECC-Bd, 31.3% of GBC, and 16.4% of AVC). Furthermore, HER2-positivity tended to correlate with low histological grade, tumor histology, and macroscopic features in certain tumors. HER2 heterogeneity was common and highly frequent (83%) in BTC cases. Reduced HER2 protein expression in the deeper invasive areas with simultaneous dedifferentiation was frequently observed in HER2-positive cancer cells. The findings of this study suggest that a large subgroup of HER2-positive BTC cases can be considered for HER2-targeted therapy. Moreover, the HER2 status in BTCs should be determined carefully using a sensitive approach toward larger cancer tissues.