The prognostic impact of KRAS, TP53, STK11 and KEAP1 mutations and their influence on the NLR in NSCLC patients treated with immunotherapy.

BACKGROUND: PD-L1 expression is used to predict NSCLC response to ICIs, but its performance is suboptimal. The impact of KRAS mutations in these patients is unclear. Studies evaluating co-mutations in TP53, STK11 and KEAP1 as well as the NLR showed that they may predict the benefit of ICIs. PATIENTS & METHODS: This is a retrospective study of patients with NSCLC treated with ICIs at the CHUM between July 2015 and June 2020. OS and PFS were compared using Kaplan-Meier and logrank methods. Co-mutations in TP53, STK11 and KEAP1 as well as the NLR were accounted for. ORR and safety were compared using Wald method. RESULTS: From 100 patients with known KRAS status, 50 were mutated (KRASMut). Mutation in TP53, STK11 and KEAP1 were present, and their status known in, respectively, 19/40 (47.5 %), 8/39 (20.5 %) and 4/38 (10.5 %) patients. STK11Mut and KEAP1Mut were associated with shorter overall survival when compared with wild type tumors (respectively median OS of 3.3 vs 20.4, p = 0.0001 and 10.1 vs 17.7, p = 0.24). When KRAS status was compounded with STK11/KEAP1, KRASMut trended to a better prognosis in STK11+KEAP1WT tumors (median OS 21.1 vs 15.8 for KRASWT, p = 0.15), but not for STK11+/-KEAP1Mut tumors. The NLR was strongly impacted by STK11 (6.0Mutvs 3.6WT, p = 0.014) and TP53 (3.2Mutvs 4.8WT, p = 0.048), but not by KEAP1 or KRAS mutations. CONCLUSION: STK11Mut and KEAP1Mut are adverse predictors of ICI therapy benefit. The NLR is strongly impacted by STK11Mut but not by KEAP1Mut, suggesting differences in their resistance mechanism. In STK11-KEAP1WT tumors, KRASMut seem associated with improved survival in NSCLC patients treated with ICIs. MICROABSTRACT: Response of NSCLC to immunotherapy is not easily predictable. We conducted a retrospective study in 100 patients with NSCLC and a known KRAS status. By accounting for different co-mutations, KRAS mutation was found to be associated with a better median overall survival in STK11 and KEAP1 wild-type tumors (21.1 vs 15.8, p = 0.15). NLR was impacted by STK11, but not KEAP1 mutation, suggesting a difference in their resistance mechanism.

Consensus Recommendations to Optimize the Detection and Reporting of NTRK Gene Fusions by RNA-Based Next-Generation Sequencing.

The detection of gene fusions by RNA-based next-generation sequencing (NGS) is an emerging method in clinical genetic laboratories for oncology biomarker testing to direct targeted therapy selections. A recent Canadian study (CANTRK study) comparing the detection of NTRK gene fusions on different NGS assays to determine subjects' eligibility for tyrosine kinase TRK inhibitor therapy identified the need for recommendations for best practices for laboratory testing to optimize RNA-based NGS gene fusion detection. To develop consensus recommendations, representatives from 17 Canadian genetic laboratories participated in working group discussions and the completion of survey questions about RNA-based NGS. Consensus recommendations are presented for pre-analytic, analytic and reporting aspects of gene fusion detection by RNA-based NGS.

Sequential targeting of PARP with carboplatin inhibits primary tumour growth and distant metastasis in triple-negative breast cancer.

BACKGROUND: Patients with triple-negative breast cancer (TNBC) develop early recurrence. While PARP inhibitors (PARPi) have demonstrated potential in BRCA1/2-mutant (BRCAMUT) TNBC, durable responses will likely be achieved if PARPi are used in combination. It is plausible that sequential administration of a potent PARPi like talazoparib in combination with carboplatin can enhance primary tumour and metastasis inhibition in BRCAMUT and BRCA1/2 wild-type (BRCAWT) TNBCs, and decrease toxicity. METHODS: We evaluated the impact of the concurrent combination of talazoparib and carboplatin on cell survival in 13 TNBC cell lines. We compared the concurrent and sequential combination upon fork replication, migration and invasion. We also used three orthotopic xenograft models to evaluate primary tumour growth, distant metastasis, and toxicity. RESULTS: Concurrent talazoparib and carboplatin was synergistic in 92.3% of TNBC cell lines, independent of BRCA1/2-mutation status. The sequential combination decreased fork speed in normal cells, but not in TNBC cells. The talazoparib-first sequential combination resulted in a strong reduction in migration (70.4%, P < 0.0001), invasion (56.9%, P < 0.0001), lung micrometastasis (56.4%, P < 0.0001), and less toxicity in a BRCAWT model. CONCLUSION: The sequential combination of talazoparib and carboplatin is an effective approach to inhibit micrometastatic disease, providing rationale for the use of this combination in early TNBC patients.

CANTRK: A Canadian Ring Study to Optimize Detection of NTRK Gene Fusions by Next-Generation RNA Sequencing.

The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

A Pan-Canadian Validation Study for the Detection of EGFR T790M Mutation Using Circulating Tumor DNA From Peripheral Blood.

INTRODUCTION: Genotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. METHODS: In phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. RESULTS: All laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. CONCLUSIONS: Discrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible.

Canadian ROS proto-oncogene 1 study (CROS) for multi-institutional implementation of ROS1 testing in non-small cell lung cancer.

Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories. Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor. Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases. This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.

DEVIL, VAAD and vLSC constitute a spectrum of HPV-independent, p53-independent intra-epithelial neoplasia of the vulva.

AIMS: We aimed to characterise a large cohort of non-invasive, human papillomavirus (HPV) and p53-independent verruciform lesions, such as 'vulvar acanthosis with altered differentiation' (VAAD), 'differentiated exophytic vulvar intra-epithelial lesion' (DEVIL) and 'verruciform lichen simplex chronicus' (vLSC). METHODS AND RESULTS: From January 2008 to December 2020 we retrospectively identified 36 eligible patients with verruciform non-invasive lesions (n = 36) and collected clinical, histological and follow-up parameters. Verruciform non-invasive lesions occurred at a median age of 71 years, with a median follow-up of 33.5 months. Clinically, pruritus was only reported in patients with VAAD (n = 3, 21%). Lesion colour was significantly different across categories (P = 0.028). Apart from the histopathological criteria already known to distinguish these entities (hypogranulosis, epithelial pallor and low-magnification architecture), no other significant criteria were discovered and significant overlap was observed, particularly between VAAD and DEVIL. Patients with vLSC trended towards longer survival without recurrence compared to VAAD and DEVIL (P = 0.082), but showed comparable invasion-free survival interval (P = 0.782). Squamous cell carcinomas (SCC) associated with either VAAD, DEVIL or vLSC displayed similar clinical, histopathological and biological parameters. In non-invasive precursor lesions, stromal oedema was associated with invasion (P = 0.015) and remained so upon Cox regression analysis (P = 0.009). CONCLUSION: Our study of HPV and p53 independent non-invasive verruciform lesions of the vulva highlights significant clinical, histopathological and biological overlap between VAAD, DEVIL and vLSC, suggesting that these pre-invasive lesions should be viewed as a spectrum. We also show that stromal features such as oedema might play an import role in progression to invasion.

Improvement of EGFR Testing over the Last Decade and Impact of Delaying TKI Initiation.

BACKGROUND: Epidermal growth factor receptor (EGFR) is the most common oncogenic mutation in lung adenocarcinoma and tyrosine kinase inhibitors (TKIs) have been considered standard treatment for more than a decade. However, time to initiation of TKIs (TTIT) from diagnosis is often delayed and represents a challenge for clinicians. We aimed to assess the impact of TTIT on clinical outcomes and complications. METHOD: TTIT was defined as the time between confirmed advanced diagnosis and the initiation of a TKI. Complications during this pre-TKI period were retrospectively collected from all patients with EGFR-mutant non small cell lung cancer (NSCLC) in our institution. RESULTS: 102 patients were diagnosed with EGFR mutated NSCLC between 2006 and 2019. The median PFS and OS were 12.9 and 22.5 months, respectively. TTIT was 5.7 months (95% CI 3.4-8) with a significant decrease in the latter years of this cohort. During the pre-TKI period, 23 patients received chemotherapy as first line treatment, of which 5 developed severe adverse events and 3 were not fit to receive TKI thereafter. Additionally, 29 patients had rapid clinical deterioration before initiation of first line TKI and 16 had to be hospitalized. Among the patients presenting a performance status deterioration, their prognosis was markedly affected compared to the remainder of the cohort (p = 0.01). CONCLUSION: Our real-world evidence study supports the concept that a delay to treat EGFR mutant NSCLC with TKIs is associated with adverse events, patient progression, hospitalization, and decreased overall survival. Rapid molecular diagnosis, including access to ctDNA technology may circumvent these deleterious delays.

Survival Impact of Aggressive Treatment and PD-L1 Expression in Oligometastatic NSCLC.

BACKGROUND: Studies have shown that aggressive treatment of non-small cell lung cancer (NSCLC) with oligometastatic disease improves the overall survival (OS) compared to a palliative approach and some immunotherapy checkpoint inhibitors, such as anti-programmed cell death ligand 1 (PD-L1), anti-programmed cell death protein 1 (PD-1), and T-Lymphocyte-associated antigen 4 (CTLA-4) inhibitors are now part of the standard of care for advanced NSCLC. However, the prognostic impact of PD-L1 expression in the oligometastatic setting remains unknown. METHODS: Patients with oligometastatic NSCLC were identified from the patient database of the Centre hospitalier de l'Université de Montréal (CHUM). "Oligometastatic disease" definition chosen is one synchronous metastasis based on the M1b staging of the eight IASLC (The International Association for the Study of Lung Cancer) Classification (within sixth months of diagnosis) or up to three cerebral metastasis based on the methodology of the previous major phase II randomized study of Gomez et al. We compared the OS between patients receiving aggressive treatment at both metastatic and primary sites (Group A) and patients receiving non-aggressive treatment (Group B). Subgroup analysis was performed using tumor PD-L1 expression. RESULTS: Among 643 metastatic NSCLC patients, we identified 67 patients with oligometastasis (10%). Median follow-up was 13.3 months. Twenty-nine patients (43%) received radical treatment at metastatic and primary sites (Group A), and 38 patients (57%) received non-aggressive treatment (Group B). The median OS (mOS) of Group A was significantly longer than for Group B (26 months vs. 5 months, p = 0.0001). Median progression-free survival (mPFS) of Group A was superior than Group B (17.5 months vs. 3.4 months, p = 0.0001). This difference was still significant when controlled for primary tumor staging: stage I (p = 0.316), stage II (p = 0.024), and stage III (p = 0.001). In the cohort of patients who were not treated with PD-L1 inhibitors, PD-L1 expression negatively correlated with mOS. CONCLUSIONS: Aggressive treatments of oligometastatic NSCLC significantly improve mOS and mPFS compared to a more palliative approach. PD-L1 expression is a negative prognostic factor which suggests a possible role for immunotherapy in this setting.

Tepotinib Efficacy in a Patient with Non-Small Cell Lung Cancer with Brain Metastasis Harboring an HLA-DRB1-MET Gene Fusion.

Alterations in c-MET, a tyrosine kinase receptor encoded by the MET gene, have been reported in approximately 3% of non-small cell lung cancer (NSCLC) cases and carry important treatment implications. The best studied genetic alterations are exon 14 skipping and gene amplification; however, gene rearrangement has also been described, and multiple fusion partners have been reported. Recently, in METex14-mutated NSCLC, multitarget tyrosine kinase inhibitors (TKIs), such as crizotinib and cabozantinib, as well as MET-selective TKIs, such as tepotinib and capmatinib, have demonstrated durable responses. In this study, we present the case of a 41-year-old woman with advanced NSCLC harboring an HLA-DRB1-MET gene fusion. The patient was offered successively two different MET multikinase inhibitors, crizotinib and cabozantinib, and the selective inhibitor tepotinib. Each time, including under tepotinib, the patient experienced rapid and complete responses associated with a tremendous improvement in her physical function. KEY POINTS: To our knowledge, this is the first report of a patient with non-small cell lung cancer harboring an HLA-DRB1-MET gene fusion demonstrating a clinical response to multiple MET inhibitors, including tepotinib. This finding illustrates the efficacy and rationale to targeting MET regardless of fusion partner and gives insight to pooling of patients with different MET fusion products in trials assessing safety and efficacy of novel molecules.

Amplification of a calcium channel subunit CACNG4 increases breast cancer metastasis.

BACKGROUND: Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling. METHODS: In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo. FINDINGS: CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ). INTERPRETATION: CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling. FUNDING: Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research.

Hematoxylin and Eosin Counterstaining Protocol for Immunohistochemistry Interpretation and Diagnosis.

Hematoxylin and eosin (H&E) staining is a well-established technique in histopathology. However, immunohistochemistry (IHC) interpretation is done exclusively with hematoxylin counterstaining. Our goal was to investigate the potential of H&E as counterstaining (H&E-IHC) to allow for visualization of a marker while confirming the diagnosis on the same slide. The quality of immunostaining and the fast-technical performance were the main criteria to select the final protocol. We stained multiple diagnostic tissues with class I IHC tests with different subcellular localization markers (anti-CK7, CK20, synaptophysin, CD20, HMB45, and Ki-67) and with double-staining on prostate tissues with anti-high molecular weight keratins/p63 (DAB detection) and p504s (alkaline phosphatase detection). To validate the efficacy of the counterstaining, we stained tissue microarrays from the Canadian Immunohistochemistry Quality Control (cIQc) with class II IHC tests (ER, PR, HER2, and p53 markers). Interobserver and intraobserver concordance was assessed by κ statistics. Excellent agreement of H&E-IHC interpretation was observed in comparison with standard IHC from our laboratory (κ, 0.87 to 1.00), and with the cIQc reference values (κ, 0.81 to 1.00). Interobserver and intraobserver agreement was excellent (κ, 0.89 to 1.00 and 0.87 to 1.00, respectively). We therefore show for the first time the potential of using H&E counterstaining for IHC interpretation. We recommend the H&E-IHC protocol to enhance diagnostic precision for the clinical workflow and research studies.

Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma.

Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development.

Molecular classification of infiltrating breast cancer: toward personalized therapy.

Breast cancer is a heterogeneous disease, which comprises several molecular and genetic subtypes, each with characteristic clinicobiologic behavior and imaging patterns. Traditional classification of breast cancer is based on the histopathologic features but offers limited prognostic value. Novel molecular characterization of breast cancer with cellular markers has allowed a new classification that offers prognostic value, with predictive categories of disease aggressiveness. These molecular signatures also open the door to personalized therapeutic options, with new receptor-targeted therapies. For example, invasive cancer subtypes such as the luminal A and B subtypes show better prognosis and response to hormone receptor-targeted therapies compared with the triple-negative subtypes; on the other hand, triple-negative tumors respond better than luminal tumors to chemotherapy. Tumors that display amplification of the oncogene ERBB2 (also known as the HER2/neu oncogene) respond to drugs directed against this oncogene, such as trastuzumab. The imaging aspects of tumors correlate with molecular subgroups, as well as other pathologic features such as nuclear grade. Smooth tumor margins at mammography may be suggestive of a triple-negative breast cancer, and a human epidermal growth factor receptor 2 (HER2)-positive tumor is characteristically a spiculated mass with calcifications. Low-grade ductal carcinoma in situ (DCIS) is better detected with mammography, although magnetic resonance (MR) imaging may allow better characterization of high-grade DCIS. MR imaging diffusion sequences show higher values for the apparent diffusion coefficient for triple-negative and HER2-positive subtypes, compared with luminal A and B tumors. MR imaging is also a useful tool in the prediction of tumor response after chemotherapy, especially for triple-negative and HER2-positive subtypes.

Canadian anaplastic lymphoma kinase study: a model for multicenter standardization and optimization of ALK testing in lung cancer.

INTRODUCTION: Fluorescence in situ hybridization (FISH) is currently the standard for diagnosing anaplastic lymphoma kinase (ALK)-rearranged (ALK+) lung cancers for ALK inhibitor therapies. ALK immunohistochemistry (IHC) may serve as a screening and alternative diagnostic method. The Canadian ALK (CALK) study was initiated to implement a multicenter optimization and standardization of laboratory developed ALK IHC and FISH tests across 14 hospitals. METHODS: Twenty-eight lung adenocarcinomas with known ALK status were used as blinded study samples. Thirteen laboratories performed IHC using locally developed staining protocols for 5A4, ALK1, or D5F3 antibodies; results were assessed by H-score. Twelve centers conducted FISH using protocols based on Vysis' ALK break-apart FISH kit. Initial IHC results were used to optimize local IHC protocols, followed by a repeat IHC study to assess the results of standardization. Three laboratories conducted a prospective parallel IHC and FISH analysis on 411 consecutive clinical samples using post-validation optimized assays. RESULTS: Among study samples, FISH demonstrated 22 consensus ALK+ and six ALK wild type tumors. Preoptimization IHC scores from 12 centers with 5A4 and the percent abnormal cells by FISH from 12 centers showed intraclass correlation coefficients of 0.83 and 0.68, respectively. IHC optimization improved the intraclass correlation coefficients to 0.94. Factors affecting FISH scoring and outliers were identified. Post-optimization concurrent IHC/FISH testing in 373 informative cases revealed 100% sensitivity and specificity for IHC versus FISH. CONCLUSIONS: Multicenter standardization study may accelerate the implementation of ALK testing protocols across a country/region. Our data support the use of an appropriately validated IHC assay to screen for ALK+ lung cancers.

Chronic granulomatous mastitis: Imaging, pathology and management.

PURPOSE: The aim of this study is to describe the clinical and radiological presentation of chronic granulomatous mastitis. MATERIAL AND METHODS: We retrospectively reviewed the clinical and radiological data of 11 women with histologically proven chronic granulomatous mastitis (CGM) diagnosed between March 2008 and September 2011. RESULTS: The diagnosis of CGM is often a challenging one that can mimic infectious and malignant breast conditions. Clinically, CGM most commonly presents as a mass, occasionally with associated erythema. The most frequent mammographic presentation is an asymmetric density, while ultrasound most commonly reveals a hypoechoic mass with tubular extensions and a striated echotexture. On MRI, the most specific finding is peripherally enhancing fluid or solid masses with fistulous tract to the skin, although the latter is not commonly encountered. Diagnosis can be reliably obtained by needle core or vacuum-assisted biopsy, and is established pathologically by the identification of granulomatous inflammation without caseous necrosis. CGM is a diagnosis of exclusion after infectious and foreign body causes are ruled out. Treatment options include oral steroids or surgery; both options are associated with similar recurrence rates. The disease tends to burn itself out and the option of conservative management with observation is a valid one. CONCLUSION: CGM is a rare benign disease with no specific features clinically or at imaging. There are no radiologic findings that are specific of CGM, but in the appropriate clinical setting, the diagnosis can be suggested by the radiologist.

Proteomic analyses reveal high expression of decorin and endoplasmin (HSP90B1) are associated with breast cancer metastasis and decreased survival.

BACKGROUND: Breast cancer is the most common malignancy among women worldwide in terms of incidence and mortality. About 10% of North American women will be diagnosed with breast cancer during their lifetime and 20% of those will die of the disease. Breast cancer is a heterogeneous disease and biomarkers able to correctly classify patients into prognostic groups are needed to better tailor treatment options and improve outcomes. One powerful method used for biomarker discovery is sample screening with mass spectrometry, as it allows direct comparison of protein expression between normal and pathological states. The purpose of this study was to use a systematic and objective method to identify biomarkers with possible prognostic value in breast cancer patients, particularly in identifying cases most likely to have lymph node metastasis and to validate their prognostic ability using breast cancer tissue microarrays. METHODS AND FINDINGS: Differential proteomic analyses were employed to identify candidate biomarkers in primary breast cancer patients. These analyses identified decorin (DCN) and endoplasmin (HSP90B1) which play important roles regulating the tumour microenvironment and in pathways related to tumorigenesis. This study indicates that high expression of Decorin is associated with lymph node metastasis (p<0.001), higher number of positive lymph nodes (p<0.0001) and worse overall survival (p = 0.01). High expression of HSP90B1 is associated with distant metastasis (p<0.0001) and decreased overall survival (p<0.0001) these patients also appear to benefit significantly from hormonal treatment. CONCLUSIONS: Using quantitative proteomic profiling of primary breast cancers, two new promising prognostic and predictive markers were found to identify patients with worse survival. In addition HSP90B1 appears to identify a group of patients with distant metastasis with otherwise good prognostic features.

CCN3 impairs osteoblast and stimulates osteoclast differentiation to favor breast cancer metastasis to bone.

Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.

Expression of Abl interactor 1 and its prognostic significance in breast cancer: a tissue-array-based investigation.

Abl interactor 1 (Abi1) is an adaptor protein involved in cell migration. Previous in vitro work suggested that Abi1 is a regulator of breast cancer proliferation, migration, and invasion. In the present study, we explore the expression of Abi1 and its downstream effector phospho-Akt (p-Akt) in a series of breast cancers and correlate their expression with clinicopathological and survival data. Using tissue microarrays, 988 patients with invasive breast carcinoma were evaluated by immunohistochemistry. Statistical correlation was performed to determine associations between Abi1 and p-Akt expression and standard breast clinicopathological factors. The prognostic value of Abi1 and p-Akt for disease-free (DFS) and overall survival (OS) was also evaluated. Abi1 expression was demonstrated in 33.7% (314/933) of invasive carcinomas, while p-Akt was expressed in 46.7% (441/944). There was a significant association between Abi1 and p-Akt expression (P=0.001). Abi1 expression showed significant positive correlation with older age at diagnosis and the Ki67 index. Most importantly, it was demonstrated to be an independent predictor of both DFS and OS (HR = 1.6 and 1.5, P<0.001, respectively). There was no association between p-Akt expression and survival. To the best of our knowledge, this is the first study evaluating Abi1 expression in a large group of breast cancers. Our analysis demonstrated that tumors expressing high levels of Abi1 are significantly associated with early recurrence and worse survival on multivariate analysis. This suggests that Abi1 expression has potential as a molecular marker to refine outcome prediction in breast cancer patients.

Amplification of the prolactin receptor gene in mammary lobular neoplasia.

The identification of lobular carcinoma in situ (LCIS) in a patient's specimen confers an appreciable increased risk of development of future invasive mammary carcinoma. However, the study of LCIS presents a challenge as it is usually only recognized in fixed specimens. Recent advances in high throughput genomics have made possible comprehensive copy number analysis of lesions such as this. Using array comparative genomic hybridization (aCGH), we characterized eight cases of lobular carcinoma (four invasive and four non-invasive) from microdissected samples of archival specimens and validated our results by quantitative real-time PCR (qRT-PCR). Immunohistochemistry (IHC) was performed on an independent set of 80 in situ ductal (DCIS) and lobular breast lesions to confirm our results. Amplification of the prolactin receptor gene (PRLr) was identified in 4/4 cases of LCIS by aCGH. We confirmed this amplification by qRT-PCR and demonstrated PRLr expression in 29/40 (73%) cases of lobular neoplasia by IHC. Amplification of PRLr was neither detected in 10 cases of DCIS nor in 5 areas of normal breast tissue by qRT-PCR and only 14/40 (35%) cases of DCIS showed PRLr expression by IHC (P = 0.0008). Our study suggests the prolactin receptor gene is a molecular target that may be important in the pathogenesis and progression of lobular neoplasia. Investigation of the status of this gene in cases of DCIS has indicated that it may not be as important in the progression of this type of breast cancer, supporting the view that lobular and ductal carcinomas may evolve along separate pathways.