The landscape of genetic aberrations in myxofibrosarcoma.

Myxofibrosarcoma (MFS) is a rare subtype of sarcoma, whose genetic basis is poorly understood. We analyzed 69 MFS cases using whole-genome (WGS), whole-exome (WES) and/or targeted-sequencing (TS). Newly sequenced genomic data were combined with additional deposited 116 MFS samples. WGS identified a high number of structural variations (SVs) per tumor most frequently affecting the TP53 and RB1 loci, 40% of tumors showed a BRCAness-associated mutation signature, and evidence of chromothripsis was found in all cases. Most frequently mutated/copy number altered genes affected known disease drivers such as TP53 (56.2%), CDKN2A/B (29.7%), RB1 (27.0%), ATRX (19.5%) and HDLBP (18.9%). Several previously unappreciated genetic aberrations including MUC17, FLG and ZNF780A were identified in more than 20% of patients. Longitudinal analysis of paired diagnosis and relapse time points revealed a 1.2-fold mutation number increase accompanied with substantial changes in clonal composition over time. Our study highlights the genetic complexity underlying sarcomagenesis of MFS.

Comparison of risk assessment in 1652 early ER positive, HER2 negative breast cancer in a real-world data set: classical pathological parameters vs. 12-gene molecular assay (EndoPredict).

BACKGROUND: Risk assessment on the molecular level is important in predictive pathology to determine the risk of metastatic disease for ERpos, HER2neg breast cancer. The gene expression test EndoPredict (EP) was trained and validated for prediction of a 10-year risk of distant recurrence to support therapy decisions regarding endocrine therapy alone or in combination with chemotherapy. The EP test provides the 12-gene Molecular Score (MS) and the EPclin-Score (EPclin), which combines the molecular score with tumor size and nodal status. In this project we investigated the correlation of 12-gene MS and EPclin scores with classical pathological markers. METHODS: EndoPredict-based gene expression profiling was performed prospectively in a total of 1652 patients between 2017 and 2020. We investigated tumor grading and Ki67 cut-offs of 20% for binary classification as well as 10% and 30% for three classes (low, intermediate, high), based on national and international guidelines. RESULTS: 410 (24.8%) of 1652 patients were classified as 12-gene MS low risk and 626 (37.9%) as EPclin low risk. We found significant positive associations between 12-gene MS and grading (p < 0.001), EPclin and grading (p = 0.001), 12-gene MS and Ki67 (p < 0.001), and EPclin and Ki67 (p < 0.001). However, clinically relevant differences between EP test results, Ki67 and tumor grading were observed. For example, 118 (26.3%) of 449 patients with Ki67 > 20% were classified as low risk by EPclin. Same differences were seen comparing EP test results and tumor grading. CONCLUSION: In this study we could show that EP risk scores are distributed differentially among Ki67 expression groups, especially in Ki67 low and high tumors with a substantial proportion of patients with EPclin high risk results in Ki67 low tumors and vice versa. This suggests that classical pathological parameters and gene expression parameters are not interchangeable, but should be used in combination for risk assessment.

Two case reports: Breast schwannoma and a rare case of an axillary schwannoma imitating an axillary lymph node metastasis.

A 79-year-old woman with a newly detected oval circumscribed lump in subcutaneous location on mammography and ultrasound turned out to be a Schwannoma after ultrasound-guided core needle biopsy. A 72-year-old woman with breast cancer in medical history demonstrated a new axillary mass in follow up, initially regarded as a lymph node metastasis. Core needle biopsy did not lead to a sufficient diagnosis. Pathologic examination after intraoperative sampling revealed a Schwannoma. These 2 case reports illustrate the importance of diagnostic imaging and remind to include Schwannomas in the differential diagnosis of breast and axillary masses.

TFE3 activation in a TSC1-altered malignant PEComa: challenging the dichotomy of the underlying pathogenic mechanisms.

Perivascular epithelioid cell tumors (PEComas) form a family of rare mesenchymal neoplasms that typically display myomelanocytic differentiation. Upregulation of mTOR signaling due the inactivation of TSC1/2 (Tuberous Sclerosis 1 and 2) is believed to be a key oncogenic driver in this disease. Recently, a subgroup of PEComas harboring TFE3 (Transcription Factor E3) rearrangements and presenting with a distinctive morphology has been identified. TSC1/2 and TFE3 aberrations are deemed to be mutually exclusive in PEComa, with two different pathogenic mechanisms assumed to lead to tumorigenesis. Here, we challenge this dichotomy by presenting a case of a clinically aggressive TCS1-mutated PEComa displaying a TFE3-altered phenotype. FISH analysis was suggestive of a TFE3 inversion; however, RNA and whole genome sequencing was ultimately unable to identify a fusion involving the gene. However, a copy number increase of the chromosomal region encompassing TFE3 was detected and transcriptome analysis confirmed upregulation of TFE3, which was also seen at the protein level. Therefore, we believe that the TSC1/2-mTOR pathway and TFE3 overexpression can simultaneously contribute to tumorigenesis in PEComa. Our comprehensive genetic analyses add to the understanding of the complex pathogenic mechanisms underlying PEComa and harbor insights for clinical treatment options.

Combination therapy with Olaratumab/doxorubicin in advanced or metastatic soft tissue sarcoma -a single-Centre experience.

BACKGROUND: The antibody targeting platelet-derived growth factor receptor alpha (PDGFRA), olaratumab, was approved in 2016 for metastatic soft tissue sarcoma (STS) in combination with doxorubicin based on promising results of a phase Ib/II trial by the Food and Drug Administration (FDA). However, recently the phase III ANNOUNCE trial could not confirm the additional value of olaratumab in this context. METHODS: Here, in a retrospective analysis we share our single-centre experience with olaratumab/doxorubicin in STS by including n = 32 patients treated with olaratumab/doxorubicin between 2016 and 2019. RESULTS: Median progression-free survival (PFS) in the overall cohort was 3.1 months (range 0.6-16.2). A response [complete remission (CR), partial remission (PR) or stable disease (SD)] was seen in n = 11 (34%) cases, whereas n = 21 (66%) patients showed progressive disease (PD). In n = 9 patients surgery was performed subsequently in an individual therapeutic approach. Out of n = 5 patients receiving additional regional hyperthermia, n = 3 achieved PR or SD. CONCLUSIONS: This single-centre experience does also not support the promising phase Ib/II results for olaratumab/doxorubicin in STS. However, our findings do not preclude that olaratumab combination therapy could be valuable in a neoadjuvant setting. This warrants further exploration also taking into account the heterogeneous nature of STS.

PARP-1 expression as a prognostic factor in Desmoid-type fibromatosis.

Desmoid-type fibromatoses (or desmoid tumors) are entities of intermediate biological potential and are locally invasive. Radical surgery, as state of the art therapy, is frequently limited by incomplete resections. Hormone modifying therapies are promising but further research is required. Poly Adenosine Diphosphate Ribose Polymerase-1 (PARP-1), a DNA repairing enzyme, might be a pathogenetic factor and could become a potential target for therapy as shown by the successful treatment of selected carcinomas and sarcomas by PARP-inhibition. In this study, we investigated the expression of estrogen receptors (ER) α (1) and ß (2), progesterone receptor (PR), androgen receptor (AR), as well as PARP-1 via immunohistochemistry and quantitative RT-PCR in 69 tissue samples of desmoid tumors. Immunohistochemistry was quantified using the Immunoreactivity Score (IRS). Overall expression patterns were correlated with clinical-pathologic parameters to determine their value as a prognostic factor. Among the investigated hormone receptors only ERß showed partial cytoplasmic reactivity. PARP-1 revealed variable nuclear positivity with IRS ranging from 0 to 6. Univariate survival analysis showed that higher expression of estrogen receptor 1 was associated with shorter disease-free survival (p = 0.005). Uni- (p = 0.03) and multivariate (p = 0.003) analyses of mRNA data revealed that higher PARP-1 expression correlated with earlier recurrence. According to this study PARP-1 expression is associated with poorer prognosis, that is faster recurrence, highlighting the possibility of PARP-1-targeting agents as a therapeutic option. Hormone receptors were of minor prognostic relevance in this study.

Bioactive coating of zirconia toughened alumina ceramic implants improves cancellous osseointegration.

Bioactive coatings have the potential to improve the bony integration of mechanically loaded orthopedic ceramic implants. Using the concept of mimicking the natural bone surface, four different coatings of varying thickness on a zirconia toughened alumina (ZTA) ceramic implant were investigated regarding their osseointegration in a drill-hole model in sheep. The hypothesis that a bioactive coating of ZTA ceramics would facilitate cancellous bone integration was investigated. The bioactive coatings consisted of either a layer of covalently bound multi phosphonate molecules (chemical modification = CM), a nano hydoxyapatite coating (HA), or two different bioactive glass (BG) coatings in micrometer thickness, forming a hydroxyl-carbonate apatite layer on the implant surface in vivo (dip-coated 45S5 = DipBG; sol-gel 70S30C = SGBG). Coated surfaces were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. After 12 weeks, osseointegration was evaluated via mechanical push-out testing and histology. HA enhanced the maximum push-out force (HA: mean 3573.85 ± 1119.91 N; SGBG: mean 1691.57 ± 986.76 N; p = 0.046), adhesive shear strength (HA: mean 9.82 ± 2.89 MPA; SGBG: mean 4.57 ± 2.65 MPA; p = 0.025), and energy release rate (HA: mean 3821.95 ± 1474.13 J/mm2; SGBG: mean 1558.47 ± 923.47 J/mm2; p = 0.032) compared to SGBG. The implant-bone interfacial stiffness increased by CM compared to SGBG coating (CM: mean 6258.06 ± 603.80 N/mm; SGBG: mean 3565.57 ± 1705.31 n/mm; p = 0.038). Reduced mechanical osseointegration of SGBG coated implants could be explained histologically by a foreign body reaction surrounding the implants.

Clinical and analytical validation of Ki-67 in 9069 patients from IBCSG VIII + IX, BIG1-98 and GeparTrio trial: systematic modulation of interobserver variance in a comprehensive in silico ring trial.

PURPOSE: Ki-67 has been clinically validated for risk assessment in breast cancer, but the analytical validation and cutpoint-definition remain a challenge. Intraclass correlation coefficients (ICCs) are a statistical parameter for Ki-67 interobserver performance. However, the maximum degree of variance among pathologists allowed for meaningful biomarker results has not been defined. METHODS: Different amounts of variance were added to central pathology Ki-67 data (n = 9069) from three cohorts (IBCSGVIII + IX, BIG1-98, GeparTrio) by simulation of 4500 evaluations for each cohort, which were grouped by ICCs, ranging from excellent (ICC = 0.9) to poor concordance (ICC = 0.1). Endpoints were disease-free survival (DFS) and pathological complete response (pCR, GeparTrio). RESULTS: Ki-67 was a significant continuous prognostic marker for DFS over a wide range of cutpoints between 8% and 30% in all three cohorts. In our modelling approach, Ki-67 was a stable prognostic marker despite increased interpathologist variance. Even for a poor ICC of 0.5, one or more significant Ki-67 cutoffs were detected in 86.8% (GeparTrio), 92.4% (IBCSGVIII + IX) and 100% of analyses (BIG1-98). Similarly, in GeparTrio, even with an extremely low ICC of 0.2, 99.6% of analyses were significant for pCR. CONCLUSIONS: Our study shows that Ki-67 is a continuous marker which is extremely robust to pathologist variation. Even if only 50% of variance is attributable to true Ki-67-based proliferation (ICC = 0.5), this information is sufficient to obtain statistically significant differences in clinical cohorts. This stable performance explains the observation that many Ki-67 studies achieve significant results despite relevant interobserver variance and points to a high clinical validity of this biomarker. For clinical decisions based on analysis of individual patient data, ongoing efforts to further reduce interobserver variability, including ring trials and standardized guidelines as well as image analysis approaches, should be continued.

DNA methylation profiling reliably distinguishes pulmonary enteric adenocarcinoma from metastatic colorectal cancer.

Pulmonary enteric adenocarcinoma is a rare non-small cell lung cancer subtype. It is poorly characterized and cannot be distinguished from metastatic colorectal or upper gastrointestinal adenocarcinomas by means of routine pathological methods. As DNA methylation patterns are known to be highly tissue specific, we aimed to develop a methylation-based algorithm to differentiate these entities. To this end, genome-wide methylation profiles of 600 primary pulmonary, colorectal, and upper gastrointestinal adenocarcinomas obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database were used as a reference cohort to train a machine learning algorithm. The resulting classifier correctly classified all samples from a validation cohort consisting of 680 primary pulmonary, colorectal and upper gastrointestinal adenocarcinomas, demonstrating the ability of the algorithm to reliably distinguish these three entities. We then analyzed methylation data of 15 pulmonary enteric adenocarcinomas as well as four pulmonary metastases and four primary colorectal adenocarcinomas with the algorithm. All 15 pulmonary enteric adenocarcinomas were reliably classified as primary pulmonary tumors and all four metastases as well as all four primary colorectal cancer samples were identified as colorectal adenocarcinomas. In a t-distributed stochastic neighbor embedding analysis, the pulmonary enteric adenocarcinoma samples did not form a separate methylation subclass but rather diffusely intermixed with other pulmonary cancers. Additional characterization of the pulmonary enteric adenocarcinoma series using fluorescence in situ hybridization, next-generation sequencing and copy number analysis revealed KRAS mutations in nine of 15 samples (60%) and a high number of structural chromosomal changes. Except for an unusually high rate of chromosome 20 gain (67%), the molecular data was mostly reminiscent of standard pulmonary adenocarcinomas. In conclusion, we provide sound evidence of the pulmonary origin of pulmonary enteric adenocarcinomas and in addition provide a publicly available machine learning-based algorithm to reliably distinguish these tumors from metastatic colorectal cancer.

Risk Assessment after Neoadjuvant Chemotherapy in Luminal Breast Cancer Using a Clinicomolecular Predictor.

Purpose: This study aimed to evaluate a modified EPclin test (mEPclin), a combination of EndoPredict (EP) score, post-neoadjuvant pathologic tumor size and nodal status, for predicting the risk of distance recurrence after neoadjuvant chemotherapy (NACT) in patients with residual estrogen receptor (ER)-positive/HER2-negative breast cancer. We also compared the prognostic power of the mEPclin with that of the CPS-EG score.Experimental Design: A total of 428 formalin-fixed, paraffin-embedded tumor samples from GeparTrio and GeparQuattro studies were evaluated for mRNA expression of eight cancer-related and three reference genes. The mEPclin score was computed using a modified algorithm and predefined cut-off values were used to classify each patient at low or high risk. Primary endpoint was disease-free survival (DFS).Results: A higher continuous mEPclin score was significantly associated with increased risk of relapse [HR, 2.16; 95% confidence interval (CI), 1.86-2.51; P < 0.001] and death (HR, 2.28; 95% CI, 1.90-2.75; P < 0.001). Similarly, patients classified at high risk by dichotomous mEPclin showed significantly poorer DFS and overall survival compared with those at low risk. In contrast with CPS-EG, the mEPclin remained significantly prognostic for DFS in multivariate analysis (HR, 2.13; 95% CI, 1.73-2.63; P < 0.001). Combining CPS-EG and other clinicopathological variables with mEPclin yielded a significant improvement of the prognostic power for DFS versus without mEPclin (c-indices: 0.748 vs. 0.660; P < 0.001).Conclusions: The mEPclin score independently predicted the risk of distance recurrence and provided additional prognostic information to the CPS-EG score to assess more accurately the prognosis after NACT in the luminal non-pCR patient population. Therefore, this approach can be used to select patients for additional post-neoadjuvant therapies. Clin Cancer Res; 24(14); 3358-65. ©2018 AACR.

Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are predictive for response to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) and HER2-positive breast cancer, but their role in luminal breast cancer and the effect of TILs on prognosis in all subtypes is less clear. Here, we assessed the relevance of TILs for chemotherapy response and prognosis in patients with TNBC, HER2-positive breast cancer, and luminal-HER2-negative breast cancer. METHODS: Patients with primary breast cancer who were treated with neoadjuvant combination chemotherapy were included from six randomised trials done by the German Breast Cancer Group. Pretherapeutic core biopsies from 3771 patients included in these studies were assessed for the number of stromal TILs by standardised methods according to the guidelines of the International TIL working group. TILs were analysed both as a continuous parameter and in three predefined groups of low (0-10% immune cells in stromal tissue within the tumour), intermediate (11-59%), and high TILs (≥60%). We used these data in univariable and multivariable statistical models to assess the association between TIL concentration and pathological complete response in all patients, and between the amount of TILs and disease-free survival and overall survival in 2560 patients from five of the six clinical trial cohorts. FINDINGS: In the luminal-HER2-negative breast cancer subtype, a pathological complete response (pCR) was achieved in 45 (6%) of 759 patients with low TILs, 48 (11%) of 435 with intermediate TILs, and 49 (28%) of 172 with high TILs. In the HER2-positive subtype, pCR was observed in 194 (32%) of 605 patients with low TILs, 198 (39%) of 512 with intermediate TILs, and 127 (48%) of 262 with high TILs. Finally, in the TNBC subtype, pCR was achieved in 80 (31%) of 260 patients with low TILs, 117 (31%) of 373 with intermediate TILs, and 136 (50%) of 273 with high TILs (p<0·0001 for each subtype, χ2 test for trend). In the univariable analysis, a 10% increase in TILs was associated with longer disease-free survival in TNBC (hazard ratio [HR] 0·93 [95% CI 0·87-0·98], p=0·011) and HER2-positive breast cancer (0·94 [0·89-0·99], p=0·017), but not in luminal-HER2-negative tumours (1·02 [0·96-1·09], p=0·46). The increase in TILs was also associated with longer overall survival in TNBC (0·92 [0·86-0·99], p=0·032), but had no association in HER2-positive breast cancer (0·94 [0·86-1·02], p=0·11), and was associated with shorter overall survival in luminal-HER2-negative tumours (1·10 [1·02-1·19], p=0·011). INTERPRETATION: Increased TIL concentration predicted response to neoadjuvant chemotherapy in all molecular subtypes assessed, and was also associated with a survival benefit in HER2-positive breast cancer and TNBC. By contrast, increased TILs were an adverse prognostic factor for survival in luminal-HER2-negative breast cancer, suggesting a different biology of the immunological infiltrate in this subtype. Our data support the hypothesis that breast cancer is immunogenic and might be targetable by immune-modulating therapies. In light of the results in luminal breast cancer, further research investigating the interaction of the immune system with different types of endocrine therapy is warranted. FUNDING: Deutsche Krebshilfe and European Commission.

Clinical relevance and concordance of HER2 status in local and central testing-an analysis of 1581 HER2-positive breast carcinomas over 12 years.

Human epidermal growth factor receptor 2 (HER2) is a central predictive biomarker in breast cancer. Inaccurate HER2 results in different laboratories could be as high as 20%. However, this statement is based on data generated more than 13 years ago and may not reflect the standards of modern diagnostic pathology. We compared central and local HER2 testing in a total of 1581 HER2-positive tumors from five clinical trials. We evaluated the clinical relevance for pathological complete response (pCR) and disease-free survival in a subgroup of 677 tumors, which received an anti-HER2 therapy. Over the period of 12 years, the discordance rate for HER2 decreased from 52.4 (GeparTrio) to 8.4% (GeparSepto). Discordance rates were significantly higher in hormone receptor (HR)-positive tumors (26.6%), compared to HR-negative tumors (16.3%, P<0.0001), which could be explained by a different distribution of HER2 mRNA levels in HR-positive and HR-negative tumors. pCR rates were significantly lower in discordant tumors (13.7%) compared to concordant tumors (32.2%, GeparQuattro and GeparQuinto, P<0.001). In survival analysis, tumors with discordant HER2 testing had a reduced overall survival (OS) in the HR-negative group (P=0.019) and a trend for improved OS in the HR-positive group (P=0.125). The performance of local HER2 testing was considerably improved over time and has reached a 92% concordance, which shows that quality initiatives in diagnostic pathology are working. Tumors with discordant HER2 testing had a reduced therapy response and different survival rates.

In vivo effects of bupivacaine and gadobutrol on the intervertebral disc following discoblock and discography: a histological analysis.

OBJECTIVES: The aim of the present study was to histologically compare chondrotoxicity in surgically harvested intervertebral discs (IVDs) of patients following discoblock, discography, or no preoperative intervention. METHODS: Thirty patients (IVD degeneration Modic ≥ 2°, Pfirrmann 3° or 4°) at L4/5 or L5/S1 who were planned for anterior lumbar interbody fusion were randomly assigned to three groups (open MRI: group DG - discography with gadobutrol; group DB - discoblock with bupivacaine at 4 weeks prior to surgery; group C - no intervention). The intervertebral discs were histologically evaluated and compared using ANOVA and Bonferroni tests for cell count, apoptosis, and proliferation. RESULTS: A reduced cell count (groups DG vs. DB vs. C: 14.9 ± 7.1, 9.2 ± 3.8, and 16.6 ± 5.2 cells/mm2, respectively; p ANOVA = 0.016), increased apoptosis (groups DG vs. DB vs. C: 34.9 ± 10.2, 47.4 ± 16.3, 32.6 ± 12.2 %, respectively; p ANOVA = 0.039) and increased cell proliferation (post hoc pDB vs. DG or C p < 0.001; for 3-7 cell monoclonal cell nests: groups DG vs. DB vs. C: 2.4 ± 1, 3.9 ± 1, 2.2 ± 1.1, respectively; p interventionx nest size = 0.006) were found in the IVDs of patients in group DB. CONCLUSIONS: This in vivo study suggests that chondrotoxic effects occur in IVD cells after the intradiscal injection of bupivacaine but not after gadobutrol administration. KEY POINTS: • Local bupivacaine administration to intervertebral discs leads to cell toxicity and proliferation. • Gadobutrol demonstrated no significant effect on cell count, apoptosis, or cell proliferation. • In vivo cytotoxicity was demonstrated histologically in humans for the first time. • Addition/administration of bupivacaine during discographies must be judged critically.

Tumor-Infiltrating Lymphocytes: A Predictive and Prognostic Biomarker in Neoadjuvant-Treated HER2-Positive Breast Cancer.

PURPOSE: We elucidated the value of tumor-infiltrating lymphocytes (TIL) as an independent predictor for pathologic complete response (pCR) rate and as a prognostic marker for disease-free survival (DFS) in patients with HER2-positive breast cancer in the neoadjuvant setting. EXPERIMENTAL DESIGN: We evaluated stromal TILs in 498 HER2-positive breast cancer samples of the neoadjuvant GeparQuattro (G4) and GeparQuinto (G5) trials. Levels of TILs were determined as a continuous parameter per 10% increase and as lymphocyte-predominant breast cancer (LPBC; ≥ 60% TILs), and correlated with pCR rate and DFS. RESULTS: In the complete cohort, HER2-positive LPBC cases had a significantly increased pCR rates compared with non-LPBC types. They were significant predictors for pCR in univariate (10% TILs: OR 1.12, P = 0.002; LPBC: OR 2.02, P = 0.002) and multivariate analyses (10% TILs: OR 1.1, P = 0.014; LPBC: OR 1.87, P = 0.009). This effect was also detectable in the trastuzumab-treated (10% TILs: OR 1.12, P = 0.018; LPBC: OR 2.08, P = 0.013) but not in the lapatinib-treated subgroup. We identified a low-risk (pCR/LPBC) and a high-risk group (no pCR/no LPBC) regarding DFS. In triple-positive breast cancer, TILs are of more prognostic relevance than pCR. CONCLUSIONS: We could demonstrate the predictive and prognostic impact of TILs in HER2-positive breast cancer in the neoadjuvant setting. In combination with pCR rate, TILs may help to stratify prognostic subgroups, thereby guiding future therapy decisions. Clin Cancer Res; 22(23); 5747-54. ©2016 AACR.

Integrated Analysis of PTEN and p4EBP1 Protein Expression as Predictors for pCR in HER2-Positive Breast Cancer.

BACKGROUND: The PI3K/AKT pathway and phosphatase and tensin homolog (PTEN) aberrations are common in breast cancer. We investigated the correlation between phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), PTEN, p4EBP1 (phosphorylated E4 binding protein 1), and pathologic complete response (pCR) in patients receiving neoadjuvant therapy. EXPERIMENTAL DESIGN: We retrospectively evaluated PIK3CA, PTEN, and p4EBP1 protein expression in centrally HER2-positive patients (n = 181) who received epirubicin cyclophosphamide/trastuzumab followed by docetaxel/trastuzumab alone or concomitant/followed by capecitabine within the GeparQuattro study. PTEN was assessed using the automated quantitative immunofluorescence analysis and was analyzed as a dichotomic variable. p4EBP1 was assessed by immunohistochemistry and used as a continuous and dichotomic variable. RESULTS: p4EBP1 was available from 137, PTEN from 108, and PIK3CA genotype from 83 patients. Overall, the pCR rate in PTEN-low tumors was 27.6%, and in PTEN-high tumors, it was 57.1% (P = 0.010). pCR rates were not statistically different between PIK3CA wild-type and mutant (35% vs. 22%) or p4EBP1 IRS ≤ 4 and IRS > 4 (39% vs. 33%). pCR rate was 57.1% (8/14) in PTEN-high/PIK3CA wild-type and decreased to 15.4% in PTEN-low/PIK3CA-mutant tumors (P = 0.023). In multivariable analysis adjusted for baseline parameters, PTEN independently predicted pCR in the following cohorts: overall [OR, 7.54; 95% confidence interval (CI), 2.03-28.06; P = 0.003], PIK3CA wild-type (OR, 23.81; 95% CI, 1.75-324.05; P = 0.017), p4EBP1 IRS > 4 (OR, 11.53; 95% CI, 1.84-72.24; P = 0.009), and hormone receptor-positive (OR, 40.91; 95% CI, 2.93-570.44; P = 0.006). p4EBP1 was independently predictive for pCR in PIK3CA wild-type tumors (OR, 0.14; 95% CI, 0.03-0.78; P = 0.025). CONCLUSIONS: The study showed the potential role of PIK3CA genotype, PTEN, and p4EBP in predicting pCR after anthracycline-taxane-based chemotherapy and anti-HER2 treatment. Clin Cancer Res; 22(11); 2675-83. ©2016 AACR.

Tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy with or without carboplatin in human epidermal growth factor receptor 2-positive and triple-negative primary breast cancers.

PURPOSE: Modulation of immunologic interactions in cancer tissue is a promising therapeutic strategy. To investigate the immunogenicity of human epidermal growth factor receptor 2 (HER2) -positive and triple-negative (TN) breast cancers (BCs), we evaluated tumor-infiltrating lymphocytes (TILs) and immunologically relevant genes in the neoadjuvant GeparSixto trial. PATIENTS AND METHODS: GeparSixto investigated the effect of adding carboplatin (Cb) to an anthracycline-plus-taxane combination (PM) on pathologic complete response (pCR). A total of 580 tumors were evaluated before random assignment for stromal TILs and lymphocyte-predominant BC (LPBC). mRNA expression of immune-activating (CXCL9, CCL5, CD8A, CD80, CXCL13, IGKC, CD21) as well as immunosuppressive factors (IDO1, PD-1, PD-L1, CTLA4, FOXP3) was measured in 481 tumors. RESULTS: Increased levels of stromal TILs predicted pCR in univariable (P < .001) and multivariable analyses (P < .001). pCR rate was 59.9% in LPBC and 33.8% for non-LPBC (P < .001). pCR rates ≥ 75% were observed in patients with LPBC tumors treated with PMCb, with a significant test for interaction with therapy in the complete (P = .002) and HER2-positive (P = .006), but not the TNBC, cohorts. Hierarchic clustering of mRNA markers revealed three immune subtypes with different pCR rates (P < .001). All 12 immune mRNA markers were predictive for increased pCR. The highest odds ratios (ORs) were observed for PD-L1 (OR, 1.57; 95% CI, 1.34 to 1.86; P < .001) and CCL5 (OR, 1.41; 95% CI, 1.23 to 1.62; P < .001). CONCLUSION: Immunologic factors were highly significant predictors of therapy response in the GeparSixto trial, particularly in patients treated with Cb. After further standardization, they could be included in histopathologic assessment of BC.

Glutamate enrichment as new diagnostic opportunity in breast cancer.

Exogenous glutamine is an important source of energy and molecular building blocks for many tumors. There is a renewed interest in therapeutically targeting glutamine metabolism due to the recent discovery of two novel glutaminase inhibitors. To quantify the dysregulation of the glutamate-glutamine equilibrium in breast cancer, metabolomics analysis of 270 clinical breast cancer samples and 97 normal breast samples was carried out using gas chromatography combined with time-of-flight mass spectrometry. Positive correlation between glutamate and glutamine in normal breast tissues switched to negative correlation between glutamate and glutamine in breast cancer tissues. Compared with the ratio of glutamate to glutamine in normal tissues, we found 56% of the ER+ tumor tissues and 88% of the ER- tumor tissues glutamate-enriched. The glutamate-to-glutamine ratio (GGR) significantly correlated with ER status (p = 8.0E-09) and with tumor grade (p = 3.3E-05). Higher levels of GGR were associated with prolonged overall survival in univariate analysis (HR = 0.77, p = 0.027) and in multivariate analysis (HR = 0.73, p = 0.038). GGR levels were reflected in an unsupervised clustering of metabolomics profiles. In a supervised analysis of metabolomics data and of genome-wide expression data, replacement of GGR by metabolite surrogate markers was feasible, while replacement of GGR by RNA markers had a limited accuracy. Functional analysis of the gene expression data showed negative correlation between glutamate enrichment and activation of peroxisome proliferator-activated receptor (PPAR) pathway. Our findings may have important implications for patient stratification related to utilization of glutaminase inhibitors.

Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer.

Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology.

PIK3CA mutations are associated with lower rates of pathologic complete response to anti-human epidermal growth factor receptor 2 (her2) therapy in primary HER2-overexpressing breast cancer.

PURPOSE: Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in breast cancer, with mutations in PIK3CA being the most common. This study investigated the association between PIK3CA genotype and pathologic complete response (pCR) rates in human epidermal growth factor receptor 2 (HER2)-positive breast cancer treated with either dual or single anti-HER2 treatment in addition to neoadjuvant chemotherapy. PATIENTS AND METHODS: PIK3CA mutations in 504 tumor samples from participants in the neoadjuvant GeparQuattro, GeparQuinto, and GeparSixto studies were evaluated. All HER2-positive patients received either trastuzumab or lapatinib or the combination plus anthracycline-taxane chemotherapy. PIK3CA mutations were evaluated in formalin-fixed, paraffin-embedded tissues from core biopsies with a tumor cell content of ≥ 20% by using classical Sanger sequencing of exon 9 and exon 20. RESULTS: Overall, 21.4% of the patients harbored a PIK3CA mutation. Detection of a PIK3CA mutation was significantly associated with a lower pCR rate (19.4% with PIK3CA mutation v 32.8% with PIK3CA wild-type; odds ratio [OR], 0.49; 95% CI, 0.29 to 0.83; P = .008). In the 291 hormone receptor (HR) -positive tumors, pCR rate was 11.3% with a PIK3CA mutation compared with 27.5% with PIK3CA wild-type (OR, 0.34; 95% CI, 0.15 to 0.78; P = .011). In 213 patients with HR-negative tumors, pCR rate was 30.4% with PIK3CA mutation and 40.1% without (OR, 0.65; 95% CI, 0.32 to 1.32; P = .233; interaction test P = .292). In multivariable analysis, HR status and PIK3CA status provided independent predictive information. In patients with PIK3CA mutation, the pCR rates were 16%, 24.3%, and 17.4% with lapatinib, trastuzumab, and the combination, respectively (P = .654) and in the wild-type group, they were 18.2%, 33.%, and 37.1%, respectively (P = .017). Disease-free survival and overall survival were not statistically significantly different between patients with mutant and wild-type PIK3CA. CONCLUSION: HER2-positive breast carcinomas with a PIK3CA mutation are less likely to achieve a pCR after neoadjuvant anthracycline-taxane-based chemotherapy plus anti-HER2 treatment, even if a dual anti-HER2 treatment is given.

Co-expression of MET and CD47 is a novel prognosticator for survival of luminal breast cancer patients.

Although luminal-type primary breast cancer can be efficiently treated, development of metastatic disease remains a significant clinical problem. We have previously shown that luminal-type circulating tumor cells (CTCs) co-expressing the tyrosine-kinase MET and CD47, a ligand involved in cancer cell evasion from macrophage scavenging, are able to initiate metastasis in xenografts. Here, we investigated the clinical relevance of MET-CD47 co-expression in 255 hormone receptor positive breast tumors by immunohistochemistry and found a 10.3- year mean overall-survival difference between MET-CD47 double-positive and double-negative patients (p<0.001) MET-CD47 co-expression defined a novel independent prognosticator for overall-survival by multivariate analysis (Cox proportional hazards model: HR: 4.1, p<0.002) and CD47 expression alone or in combination with MET was strongly associated with lymph node metastasis. Furthermore, flow cytometric analysis of metastatic patient blood revealed consistent presence of MET+CD47+ CTCs (range 0.8 - 33.3% of CTCs) and their frequency was associated with increased metastatic spread. Finally, primary uncultured CTCs with high MET+CD47+ content showed an enhanced capacity to initiate metastasis in mice. Detection and targeting of MET and CD47 may thus provide a rational basis for risk stratification and treatment of patients with luminal-type breast cancer.