Susan Fineberg

The mammary gland iodide transporter is expressed during lactation and in breast cancer

The sodium/iodide symporter mediates active iodide transport in both healthy and cancerous thyroid tissue. By exploiting this activity, radioiodide has been used for decades with considerable success in the detection and treatment of thyroid cancer. Here we show that a specialized form of the sodium/iodide symporter in the mammary gland mediates active iodide transport in healthy lactating (but not in nonlactating) mammary gland and in mammary tumors. In addition to characterizing the hormonal regulation of the mammary gland sodium/iodide symporter, we demonstrate by scintigraphy that mammary adenocarcinomas in transgenic mice bearing Ras or Neu oncogenes actively accumulate iodide by this symporter in vivo. Moreover, more than 80% of the human breast cancer samples we analyzed by immunohistochemistry expressed the symporter, compared with none of the normal (nonlactating) samples from reductive mammoplasties. These results indicate that the mammary gland sodium/iodide symporter may be an essential breast cancer marker and that radioiodide should be studied as a possible option in the diagnosis and treatment of breast cancer.

An international study to increase concordance in Ki67 scoring

Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 ‘training’ and ‘test’ web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90–0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes.

Hsa‐miR‐375 is differentially expressed during breast lobular neoplasia and promotes loss of mammary acinar polarity

Invasive lobular carcinoma (ILC) of the breast, characterized by loss of E‐cadherin expression, accounts for 5–15% of invasive breast cancers and it is believed to arise via a linear histological progression. Genomic studies have identified a clonal relationship between ILC and concurrent lobular carcinoma in situ (LCIS) lesions, suggesting that LCIS may be a precursor lesion. It has been shown that an LCIS diagnosis confers a 15–20% risk of progression to ILC over a lifetime. Currently no molecular test or markers can identify LCIS lesions likely to progress to ILC. Since microRNA (miRNA) expression changes have been detected in a number of other cancer types, we explored whether their dysregulation might be detected during progression from LCIS to ILC. Using the Illumina miRNA profiling platform, designed for simultaneous analysis of 470 mature miRNAs, we analysed the profiles of archived normal breast epithelium, LCIS lesions found alone, LCIS lesions concurrent with ILC, and the concurrent ILCs as a model of linear histological progression towards ILC. We identified two sets of differentially expressed miRNAs, the first set highly expressed in normal epithelium, including hsa‐miR‐224, ‐139, ‐10b, ‐450, 140, and ‐365, and the second set up‐regulated during lobular neoplasia progression, including hsa‐miR‐375, ‐203, ‐425‐5p, ‐183, ‐565, and ‐182. Using quantitative RT‐PCR, we validated a trend of increasing expression for hsa‐miR‐375, hsa‐miR‐182, and hsa‐miR‐183 correlating with ILC progression. As we detected increased expression of hsa‐miR‐375 in LCIS lesions synchronous with ILC, we sought to determine whether hsa‐miR‐375 might induce phenotypes reminiscent of lobular neoplasia by expressing it in the MCF‐10A 3D culture model of mammary acinar morphogenesis. Increased expression of hsa‐miR‐375 resulted in loss of cellular organization and acquisition of a hyperplastic phenotype. These data suggest that dysregulated miRNA expression contributes to lobular neoplastic progression. Copyright

Pseudoangiomatous stromal hyperplasia of the breast: sonographic features with histopathologic correlation.

Abstract:  The objective of this study was to evaluate the spectrum of sonographic findings in pseudoangiomatous stromal hyperplasia (PASH) of the breast when it presents as a tumoral mass with pathologic correlation. Breast sonogram studies of 13 patients with 13 pathologically proven PASH lesions were retrospectively reviewed. The morphologic characteristics of the lesions as seen on ultrasound were evaluated and correlated with histopathologic findings. Sonography demonstrated most lesions, 11 of 13, to be hypoechoic in echotexture. One lesion was isoechoic in echotexture, also demonstrating small internal cysts, and one was predominantly hyperechoic. Two of the 11 hypoechoic lesions also demonstrated a complex heterogeneous pattern with a central hypoechoic area and a peripheral echogenic rim. All lesions were oval in shape with the long axis of the lesion parallel to the chest wall. None of the lesions demonstrated posterior acoustic shadowing. PASH lesions of the breast have a varied sonographic appearance. Knowledge of the spectrum of morphologic features shown on sonography can be helpful in the diagnosis of this entity. 

Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.

Can Features Evaluated in the Routine Pathologic Assessment of Lymph Node–Negative Estrogen Receptor–Positive Stage I or II Invasive Breast Cancer Be Used to Predict the Oncotype DX Recurrence Score?

CONTEXT Oncotype DX is a multigene reverse transcription-polymerase chain reaction assay used to quantify recurrence risk in patients with stage I or II estrogen receptor-positive, lymph node-negative invasive breast cancer. The results are reported as a Recurrence Score (RS). The 16 cancer genes evaluated include a proliferation set, hormone receptor set, and HER2 set. The activity of these genes is addressed by pathologic assessment of breast cancers. OBJECTIVE To determine if factors evaluated in pathologic evaluation of breast cancer could be used to predict Oncotype DX results. DESIGN We studied 138 cases of invasive breast cancer for which Oncotype DX results and pathology data were available. Grading was performed by using Nottingham grading system. For hormone receptor immunostaining, 10% nuclear staining was considered a positive result. RESULTS Oncotype DX RS was low in 81 cases, intermediate in 44 cases, and high in 13 cases. All 6 cases with both a negative progesterone receptor (PR) and a mitotic count score of 3 had a high RS. All 12 cases with both a negative PR and a mitotic count score greater than 1 had either an intermediate or high RS. Although Nottingham grade, PR status, mitotic count score, tumor size, and nuclear grade were each significantly associated with RS, in bivariate analyses the only variables that remained independently predictive of an intermediate or high RS score in a multivariate logistic regression model were negative PR and mitotic count score greater than 1. CONCLUSIONS Our study suggests that a mitotic count score greater than 1 combined with a negative PR result, as determined by pathologic assessment, could serve as a marker for an intermediate or high Oncotype DX RS.

Phase II Trial of Tipifarnib plus Neoadjuvant Doxorubicin-Cyclophosphamide in Patients with Clinical Stage IIB-IIIC Breast Cancer

Purpose: Tipifarnib is a farnesyl transferase (FTase) inhibitor that has activity in metastatic breast cancer and enhances the efficacy of cytotoxic agents in preclinical models. We evaluated the biological effects of tipifarnib in primary breast cancers in vivo, whether adding tipifarnib to preoperative chemotherapy increased the pathologic complete response rate (pCR) at surgery, and determined whether biomarkers predictive of pCR could be identified. Experimental Design: Forty-four patients with stage IIB-IIIC breast cancer received up to four cycles of neoadjuvant doxorubicin-cyclophosphamide (AC) every 2 weeks plus tipifarnib and filgrastim followed by surgery. Enzymatic assays measuring FTase activity and Western blotting for phospho (p)-signal transducer and activator of transcription 3 (STAT3), phospho-extracellular signal-regulated kinase, p-AKT, and p27 were done in 11 patients who agreed to optional tissue biopsies before therapy and 2 hours after the final dose of tipifarnib during the first cycle, and predictive biomarkers were evaluated by immunohistochemistry in 33 patients. The trial was powered to detect an improvement in breast pCR rate of 10% or less expected for AC alone to 25% for AC-tipifarnib (α = 0.05, β = 0.10). Results: Eleven patients had a breast pCR (25%; 95% confidence interval, 13-40%). FTase enzyme activity decreased in all patients (median, 91%; range, 24-100%) and p-STAT3 expression decreased in 7 of 9 (77%) patients. Low tumor Ki-67 expression (below the median of 60%) at baseline was significantly associated with resistance to therapy (P = 0.01). Conclusion: Tipifarnib inhibits FTase activity in human breast tumors in vivo, is associated with down-regulation of p-STAT3, and enhances the breast pCR rate, thus meriting further evaluation.

Assessing tumor-infiltrating lymphocytes in solid tumors: a practical review for pathologists and proposal for a standardized method from the International Immuno-Oncology Biomarkers Working Group: part 2: TILs in melanoma, gastrointestinal tract carcinomas, non-small cell lung carcinoma and mesothelioma, endometrial and ovarian carcinomas, squamous cell carcinoma of the head and neck, genitourinary carcinomas, and primary brain Tumors

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Analytical validation of a standardized scoring protocol for Ki67: phase 3 of an international multicenter collaboration

Pathological analysis of the nuclear proliferation biomarker Ki67 has multiple potential roles in breast and other cancers. However, clinical utility of the immunohistochemical (IHC) assay for Ki67 immunohistochemistry has been hampered by unacceptable between-laboratory analytical variability. The International Ki67 Working Group has conducted a series of studies aiming to decrease this variability and improve the evaluation of Ki67. This study tries to assess whether acceptable performance can be achieved on prestained core-cut biopsies using a standardized scoring method. Sections from 30 primary ER+ breast cancer core biopsies were centrally stained for Ki67 and circulated among 22 laboratories in 11 countries. Each laboratory scored Ki67 using three methods: (1) global (4 fields of 100 cells each); (2) weighted global (same as global but weighted by estimated percentages of total area); and (3) hot-spot (single field of 500 cells). The intraclass correlation coefficient (ICC), a measure of interlaboratory agreement, for the unweighted global method (0.87; 95% credible interval (CI): 0.81–0.93) met the prespecified success criterion for scoring reproducibility, whereas that for the weighted global (0.87; 95% CI: 0.7999–0.93) and hot-spot methods (0.84; 95% CI: 0.77–0.92) marginally failed to do so. The unweighted global assessment of Ki67 IHC analysis on core biopsies met the prespecified criterion of success for scoring reproducibility. A few cases still showed large scoring discrepancies. Establishment of external quality assessment schemes is likely to improve the agreement between laboratories further. Additional evaluations are needed to assess staining variability and clinical validity in appropriate cohorts of samples.

Expression, Clinical Significance, and Receptor Identification of the Newest B7 Family Member HHLA2 Protein

Purpose: HHLA2 (B7H7/B7-H5/B7y) is a newly identified B7 family member that regulates human T-cell functions. However, its protein expression in human organs and significance in human diseases are unknown. The objective of this study was to analyze HHLA2 protein expression in normal human tissues and cancers, as well as its prognostic significance, to explore mechanisms regulating HHLA2 expression, and to identify candidate HHLA2 receptors. Experimental Design: An immunohistochemistry protocol and a flow cytometry assay with newly generated monoclonal antibodies were developed to examine HHLA2 protein. HHLA2 gene copy-number variation was analyzed from cancer genomic data. The combination of bioinformatics analysis and immunologic approaches was established to explore HHLA2 receptors. Results: HHLA2 protein was detected in trophoblastic cells of the placenta and the epithelium of gut, kidney, gallbladder, and breast, but not in most other organs. In contrast, HHLA2 protein was widely expressed in human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, and esophagus. In a cohort of 50 patients with stage I–III triple-negative breast cancer, 56% of patients had aberrant expression of HHLA2 on their tumors, and high HHLA2 expression was significantly associated with regional lymph node metastasis and stage. The Cancer Genome Atlas revealed that HHLA2 copy-number gains were present in 29% of basal breast cancers, providing a potential mechanism for increased HHLA2 protein expression in breast cancer. Finally, Transmembrane and Immunoglobulin Domain Containing 2 (TMIGD2) was identified as one of the receptors for HHLA2. Conclusions: Wide expression of HHLA2 in human malignancies, together with its association with poor prognostic factors and its T-cell coinhibitory capability, suggests that the HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and an attractive target for human cancer therapy. Clin Cancer Res; 21(10); 2359–66. ©2014 AACR. See related commentary by Xiao and Freeman, p. 2201