Tumor metabolism assessed by FDG-PET/CT and tumor proliferation assessed by genomic grade index to predict response to neoadjuvant chemotherapy in triple negative breast cancer.

PURPOSE: Survival is increased when pathological complete response (pCR) is reached after neoadjuvant chemotherapy (NAC), especially in triple-negative breast cancer (TNBC) patients. Positron emission tomography/computed tomography (PET/CT) with 18F-fluorodeoxyglucose (FDG) and the genomic grade index (GGI), each separately, showed good potential to predict pCR. Our study was designed to evaluate the predictive value for the therapeutic response of a combination of parameters based on FDG-PET, histoclinical features and molecular markers of proliferation. METHODS: Molecular parameters were measured on pre-treatment biopsy. Tumor metabolic activity was measured using two PET/CT scans, one before and one after 2 cycles of NAC. The pCR was determined on specimen after NAC. Event-free survival (EFS) was estimated using the Kaplan Meier method. RESULTS: Of 55 TNBC patients, 19 (35%) reached pCR after NAC. Tumor grade and Ki67 were not associated with pCR whereas GGI (P = 0.04) and its component KPNA2 (P = 0.04) showed a predictive value. The change of FDG uptake between PET1 and PET2 (ΔSUVmax) was highly associated with pCR (P = 0.0001) but the absolute value of baseline SUVmax was not (P = 0.11). However, the AUC of pCR prediction increased from 0.63 to 0.76 when baseline SUVmax was combined with the GGI (P = 0.016). The only two parameters associated with EFS were ΔSUVmax (P = 0.048) and pathological response (P = 0.014). CONCLUSIONS: The early tumor metabolic change during NAC is a powerful parameter to predict pCR and outcome in TNBC patients. The GGI, determined on pretreatment biopsy, is also predictive of pCR and the combination GGI and baseline SUVmax improves the prediction.

Clinical and molecular characterisation of hereditary and sporadic metastatic colorectal cancers harbouring microsatellite instability/DNA mismatch repair deficiency.

BACKGROUND: Patients treated with chemotherapy for microsatellite unstable (MSI) and/or mismatch repair deficient (dMMR) cancer metastatic colorectal cancer (mCRC) exhibit poor prognosis. We aimed to evaluate the relevance of distinguishing sporadic from Lynch syndrome (LS)-like mCRCs. PATIENTS AND METHODS: MSI/dMMR mCRC patients were retrospectively identified in six French hospitals. Tumour samples were screened for MSI, dMMR, RAS/RAF mutations and MLH1 methylation. Sporadic cases were molecularly defined as those displaying MLH1/PMS2 loss of expression with BRAFV600E and/or MLH1 hypermethylation and no MMR germline mutation. RESULTS: Among 129 MSI/dMMR mCRC patients, 81 (63%) were LS-like and 48 (37%) had sporadic tumours; 22% of MLH1/PMS2-negative mCRCs would have been misclassified using an algorithm based on local medical records (age, Amsterdam II criteria, BRAF and MMR statuses when locally tested), compared to a systematical assessment of MMR, BRAF and MLH1 methylation statuses. In univariate analysis, parameters associated with better overall survival were age (P < 0.0001), metastatic resection (P = 0.001) and LS-like mCRC (P = 0.01), but not BRAFV600E. In multivariate analysis, age (hazard ratio (HR) = 3.19, P = 0.01) and metastatic resection (HR = 4.2, P = 0.001) were associated with overall survival, but not LS. LS-like patients were associated with more frequent liver involvement, metastatic resection and better disease-free survival after metastasectomy (HR = 0.28, P = 0.01). Median progression-free survival of first-line chemotherapy was similar between the two groups (4.2 and 4.2 months; P = 0.44). CONCLUSIONS: LS-like and sporadic MSI/dMMR mCRCs display distinct natural histories. MMR, BRAF mutation and MLH1 methylation testing should be mandatory to differentiate LS-like and sporadic MSI/dMMR mCRC, to determine in particular whether immune checkpoint inhibitors efficacy differs in these two populations.

Long-term survival of advanced triple-negative breast cancers with a dose-intense cyclophosphamide/anthracycline neoadjuvant regimen.

BACKGROUND: Triple-negative (TN) breast cancers exhibit major initial responses to neoadjuvant chemotherapy, but generally have a poor outcome. Because of the lack of validated drug targets, chemotherapy remains an important therapeutic tool in these cancers. METHODS: We report the survival of two consecutive series of 267 locally advanced breast cancers (LABC) treated with two different neoadjuvant regimens, either a dose-dense and dose-intense cyclophosphamide-anthracycline (AC) association (historically called SIM) or a conventional sequential association of cyclophosphamide and anthracycline, followed by taxanes (EC-T). We compared pathological responses and survival rates of these two groups and studied their association with tumours features. RESULTS: Although the two regimens showed equivalent pathological complete response (pCR) in the whole population (16 and 12%), the SIM regimen yielded a non-statistically higher pCR rate than EC-T (48% vs 24%, P=0.087) in TN tumours. In the SIM protocol, DFS was statistically higher for TN than for non-TN patients (P=0.019), although we showed that the TN status was associated with an increased initial risk of recurrence in both regimens. This effect gradually decreased and after 2 years, TN was associated with a significantly decreased likelihood of relapse in SIM-treated LABC (hazard ratio (HR)=0.25 (95% CI: 0.07-0.86), P=0.028). CONCLUSIONS: AC dose intensification treatment is associated with a very favourable long-term survival rate in TN breast cancers. These observations call for a prospective assessment of such dose-intense AC-based regimens in locally advanced TN tumours.

Challenging single- and multi-probesets gene expression signatures of pathological complete response to neoadjuvant chemotherapy in breast cancer: experience of the REMAGUS 02 phase II trial.

This study was designed to identify predictive signatures of pathological complete response (pCR) in breast cancer treated by taxane-based regimen, using clinicopathological variables and transcriptomic data (Affymetrix Hgu133 Plus 2.0 devices). The REMAGUS 02 trial (n = 153,training set) and the publicly available M.D. Anderson data set (n = 133, validation set) were used. A re-sampling method was applied. All predictive models were defined using logistic regression and their classification performances were tested through Area Under the Curve (AUC) estimation. A stable set of 42 probesets (31 genes) differentiate pCR or no pCR samples. Single-or 2-probesets signatures, mainly related to ER pathway, were equally predictive of pCR with AUC greater then 0.80. Models including probesets associated with ESR1, MAPT, CA12 or PIGH presented good classification performances. When clinical variables were entered into the model, only CA12 and PIGH, remained informative (p = 0.05 and p = 0.005) showing that a combination of a few genes provided robust and reliable prediction of pCR.

A refined molecular taxonomy of breast cancer.

The current histoclinical breast cancer classification is simple but imprecise. Several molecular classifications of breast cancers based on expression profiling have been proposed as alternatives. However, their reliability and clinical utility have been repeatedly questioned, notably because most of them were derived from relatively small initial patient populations. We analyzed the transcriptomes of 537 breast tumors using three unsupervised classification methods. A core subset of 355 tumors was assigned to six clusters by all three methods. These six subgroups overlapped with previously defined molecular classes of breast cancer, but also showed important differences, notably the absence of an ERBB2 subgroup and the division of the large luminal ER+ group into four subgroups, two of them being highly proliferative. Of the six subgroups, four were ER+/PR+/AR+, one was ER-/PR-/AR+ and one was triple negative (AR-/ER-/PR-). ERBB2-amplified tumors were split between the ER-/PR-/AR+ subgroup and the highly proliferative ER+ LumC subgroup. Importantly, each of these six molecular subgroups showed specific copy-number alterations. Gene expression changes were correlated to specific signaling pathways. Each of these six subgroups showed very significant differences in tumor grade, metastatic sites, relapse-free survival or response to chemotherapy. All these findings were validated on large external datasets including more than 3000 tumors. Our data thus indicate that these six molecular subgroups represent well-defined clinico-biological entities of breast cancer. Their identification should facilitate the detection of novel prognostic factors or therapeutical targets in breast cancer.

Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches.

BACKGROUND: Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT-PCR), but are not routinely used. We evaluated the relevance of Q-RT-PCR for HER2 status determination. METHODS: We compared IHC and Q-RT-PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection. RESULTS: We observed 97.3% concordance between Q-RT-PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT-PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones. CONCLUSION: Q-RT-PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT-PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

Multifocal Epstein-Barr virus-associated smooth muscle tumor in adults with AIDS: case report and review of the literature.

OBJECTIVES: The aim of this study was to provide a systematic review of Epstein-Barr virus-associated smooth muscle tumors (EBV-SMT) in human immunodeficiency virus (HIV)-infected adults, focusing on clinical and histopathologic features and outcome. METHODS: A literature search was performed using Medline, Embase and the Cochrane Library. RESULTS: We reviewed 35 cases including our case of a patient with a progressive multifocal EBV-SMT. Patients were mainly men (n = 24) with a mean age of 35.5 years. Median CD4 count was 21/mm(3). Main locations were brain (n = 12), liver (n = 8), spinal cord (n = 7) and adrenal gland (n = 6). The tumors were multifocal in 34% of cases, whereas analysis of clonality showed different clones in tumors from different sites. Treatment included removal surgery in 17 cases and/or radiotherapy in 9 and therapeutic abstention in 4. Mean follow-up after diagnosis was 12.3 months. Nine patients died during this period essentially from opportunistic infection and only 2 from the disease. CONCLUSION: EBV-SMT should be added to the list of virally induced tumors in severely immunocompromised HIV-infected adults. Multifocality of independent tumor clones, especially in liver, brain, spinal cord and adrenal gland, and a slow disease progression seem to be the key features of these tumors, the treatment of which remains poorly defined.

Changes in allelic imbalances in locally advanced breast cancers after chemotherapy.

In advanced breast cancers, TP53 mutation is highly predictive of complete response to high-dose epirubicin/cyclophosphamide chemotherapy. In these tumours with an altered control of genomic stability, accumulation of chemotherapy-induced genetic alterations may contribute to cell death and account for complete response. To explore the effects of chemotherapy on stability of the tumour genome, allelic profiles were obtained from microdissected tumour samples before and after chemotherapy in 29 unresponsive breast cancers (9 with TP53 mutation). Ninety-four per cent allelic profiles remained unchanged after treatment. Interestingly, 11 profiles (6%) showed important changes after treatment; allelic imbalances significantly increased (four cases) or decreased (seven cases) after chemotherapy in three distinct experiments, two of which using laser microdissected tumour cells. These genetic changes were not linked to the TP53 status, but one tumour showed complete disappearance of TP53-mutated cells in the residual tumour after treatment. Altogether, these observations carry important implications for the clonal evolution of breast cancers treated with DNA-damaging agents, as they point both to the importance of tumour heterogeneity and chemotherapy-driven selection of subclones.

Trastuzumab-based treatment of HER2-positive breast cancer: an antibody-dependent cellular cytotoxicity mechanism?

This study evaluated by immunohistochemistry (IHC) immune cell response during neoadjuvant primary systemic therapy (PST) with trastuzumab in patients with HER2-positive primary breast cancer. In all, 23 patients with IHC 3+ primary breast cancer were treated with trastuzumab plus docetaxel. Pathological complete and partial responses were documented for nine (39%) and 14 (61%) patients, respectively. Case-matched controls comprised patients treated with docetaxel-based PST without trastuzumab (D; n=23) or PST without docetaxel or trastuzumab (non-taxane, non-trastuzumab, NT-NT; n=23). All surgical specimens were blind-analysed by two independent pathologists, with immunohistochemical evaluation of B and T lymphocytes, macrophages, dendritic cells and natural killer (NK) cells. Potential cytolytic cells were stained for Granzyme B and TiA1. HER2 expression was also evaluated in residual tumour cells. Trastuzumab treatment was associated with significantly increased numbers of tumour-associated NK cells and increased lymphocyte expression of Granzyme B and TiA1 compared with controls. This study supports an in vivo role for immune (particularly NK cell) responses in the mechanism of trastuzumab action in breast cancer. These results suggest that trastuzumab plus taxanes lead to enhanced NK cell activity, which may partially account for the synergistic activity of trastuzumab and docetaxel in breast cancer.

Effect of mutated TP53 on response of advanced breast cancers to high-dose chemotherapy.

TP53 activation by genotoxic drugs can induce apoptosis or cell-cycle arrest. Thus, whether the gene is mutated or wild type could affect the response of a tumour to chemotherapy. Clinical data are unclear, possibly as a result of heterogeneity of tumours, drugs, methods of assessing response, or TP53 status. We studied 50 non-inflammatory, locally advanced breast cancers that had been treated with high doses of a combination of epirubicin and cyclophosphamide. We noted eight complete responses, which all occurred in the 14 patients with tumours containing mutated TP53 (p<0.0001). In high-grade, advanced breast cancers, inactivation of the TP53 pathway could greatly improve the response to this chemotherapy regimen.

Allelic loss detection in inflammatory breast cancer: improvement with laser microdissection.

Solid tumors are composed not only of tumor cells but also of stromal nonneoplastic cells. In whole tumor samples, stromal cells retaining their alleles may therefore obscure detection of loss of heterozygosity (LOH) in tumor cells. An increasing number of studies have used laser-assisted tissue microdissection to improve LOH detection, but the real gain in sensitivity has been poorly quantified. We studied a group of 16 inflammatory breast carcinomas that were submitted to both standard DNA extraction from frozen whole tumor samples and laser microdissection performed on paraffin-embedded tumor samples. Using PCR with fluorescence-labeled primers, we comparatively analyzed ten polymorphic markers with both sources of DNA. With the LOH detection threshold set at -25%, we showed that 25 LOHs could not be diagnosed with whole tumor samples out of 73 LOHs positively diagnosed in microdissected samples (34%). With the LOH detection threshold set at -50%, the respective figures were 39 LOHs not diagnosed out of 55 LOHs (71%). Measuring the intensity of the allelic decrease, we showed that the mean decrease of the lost allele is -34% with whole tumor samples and -67% with microdissected samples. The increase in sensitivity of LOH detection with microdissection is associated with the density of stromal cells. This strong improvement in LOH detection in this aggressive type of breast cancer indicates that many other molecular studies performed on heterogeneous solid tumors may benefit from a first step of laser microdissection.

Expression of amphiregulin and epidermal growth factor receptor in human breast cancer: analysis of autocriny and stromal-epithelial interactions.

Amphiregulin (AR) and its receptor, epidermal growth factor receptor (EGFR), were evaluated by dual immunostaining in a series of 84 invasive ductal breast carcinoma specimens, 33 of which were from locally advanced inflammatory (T4d) cancer. Co-expression of AR and EGFR was always found in non-malignant breast tissues adjacent to tumours (24/24). Alternatively, expression of AR and EGFR was found in invasive epithelial tumour cells in 50% and 17.8% of specimens, respectively. In tumour stroma, 59.5% and 30.9% of specimens, respectively, were positively stained. By univariate analysis, AR and EGFR expression in invasive carcinomas was correlated with large tumour size, inflammatory carcinoma, node involvement, Bloom-Richardson (SBR) grade III, and absence of oestrogen receptor. EGFR expression in stromal cells was correlated with non-inflammatory carcinoma. A putative autocrine loop with AR and EGFR expression in invasive carcinoma was detected in 14.3% of cases. Stromal expression of AR and EGFR expression in invasive tumour cells was detected in 11.9% of cases and related to poor prognostic parameters. By multivariate analysis, AR expression in invasive tumour was strongly related to inflammatory carcinoma (p=0.005) and marginally related to SBR grade III (p=0.07). EGFR expression in invasive tumour and stromal cells was correlated with absence of oestrogen receptor and non-inflammatory carcinoma (p=0.002 and p=0.015, respectively).

Characterization of the secreted form of endothelial-cell-specific molecule 1 by specific monoclonal antibodies.

Endothelial-cell-specific molecule 1 (ESM-1) is a recently identified endothelial cell molecule. As ESM-1 mRNA is preferentially expressed in human lung and kidney tissues, and as ESM-1 mRNA expression is regulated by inflammatory cytokines, ESM-1 is thought to play a role in the vascular contribution to organ-specific inflammation. In order to define its behavior, mouse anti-ESM-1 monoclonal antibodies were developed, and three distinct epitopes were mapped, which allowed development of a specific ELISA assay, immunohistological staining and immunoblot analysis. Here, we demonstrate that ESM-1 is present in cell lysates of human endothelial cells (human umbilical vein endothelial cells) with an apparent molecular weight of 20 kD. In contrast, the secreted form of ESM-1 is shifted to an apparent molecular weight of 50 kD, indicating that the secreted form of ESM-1 is posttranslationally modified. By ELISA, we show that the secretion of ESM-1 is significantly enhanced in the presence of TNFalpha. In contrast, the spontaneous as well as TNFalpha-induced secretion of ESM-1 is strongly inhibited by IFNgamma. Moreover, ESM-1 was detected in the serum of healthy subjects at an average concentration of 1.08 ng/ml, and we demonstrated that the serum level of ESM-1 is dramatically increased in patients presenting a septic shock. Analysis of ESM-1 expression in normal human tissues by immunohistochemistry showed that ESM-1 is localized in the vascular network, but also in the bronchial and renal epithelia. Our results demonstrate that ESM-1 is mainly expressed in the vascular endothelium both in vitro and in vivo, but also by different epithelia. ESM-1 may represent a new marker of endothelial cell activation, and may have a functional role in endothelium-dependent pathological disorders.

Recurrent cholestatic jaundice associated with generalized pustular psoriasis: evidence for a neutrophilic cholangitis.

Generalized pustular psoriasis can result in systemic complications. We report the case of a woman with relapsing generalized pustular psoriasis and recurring episodes of cholestatic jaundice. Liver biopsy performed during an attack showed a neutrophilic infiltrate surrounding and invading portal triad bile ducts. Ultrasonographic exams and retrograde cholangiography ruled out biliary tract disease. This observation suggests that recurring cholestatic jaundice in pustular psoriasis is related to a neutrophilic cholangitis.

The effects of transurethral resection and cystoprostatectomy on dissemination of epithelial cells in the circulation of patients with bladder cancer.

This study was undertaken to evaluate the risk of haematogenous dissemination of epithelial cells induced by endoscopic resection and/or cystoprostatectomy for transitional cell carcinoma of the bladder. Thirty-three patients were studied. Thirty-one had different stages and grades of bladder cancer and two patients had benign bladder conditions. Twenty-five cancer patients required transurethral resection of their bladder tumour. Of those, 20 had superficial disease (pTaG1-G2: n = 19; pT1G2: n = 1) and five had muscle invasive tumours (pT2G3: n = 2; pT3aG3: n = 1; pT4G3: n = 2). Five patients underwent radical cystoprostatectomy for muscle invasive cancers (pT2G3: n = 3; pT3bG3: n = 1; pT4G3: n = 1) and one man received chemotherapy for metastatic disease. Venous blood (10 ml) was obtained from the antecubital fossa in each patient, before and 1-2 h after completion of surgery, and prior to treatment in the metastatic patient. An indirect immunocytochemical technique was used to detect circulating epithelial cells after centrifugation on Ficoll gradient and fixation of mononuclear cells on slides, using a monoclonal antibody directed against three cytokeratins: CK8, CK18 and CK19. Circulating epithelial cells were detected only in the patient with metastatic disease. None of the other patients had evidence of epithelial circulating cells before or after surgery. The results suggest that irrespective of disease stage and grade, neither endoscopic nor open bladder surgery leads to detectable dissemination of urothelial cells in the peripheral circulation. These procedures are therefore unlikely to increase the risk of progression and metastasis in transitional cell carcinoma of the bladder.