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1.  HER2 and ESR1 mRNA expression levels and response to neoadjuvant trastuzumab plus chemotherapy in patients with primary breast cancer 
Recent data suggest that benefit from trastuzumab and chemotherapy might be related to expression of HER2 and estrogen receptor (ESR1). Therefore, we investigated HER2 and ESR1 mRNA levels in core biopsies of HER2-positive breast carcinomas from patients treated within the neoadjuvant GeparQuattro trial.
HER2 levels were centrally analyzed by immunohistochemistry (IHC), silver in situ hybridization (SISH) and qRT-PCR in 217 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) core biopsies. All tumors had been HER2-positive by local pathology and had been treated with neoadjuvant trastuzumab/ chemotherapy in GeparQuattro.
Only 73% of the tumors (158 of 217) were centrally HER2-positive (cHER2-positive) by IHC/SISH, with cHER2-positive tumors showing a significantly higher pCR rate (46.8% vs. 20.3%, P <0.0005). HER2 status by qRT-PCR showed a concordance of 88.5% with the central IHC/SISH status, with a low pCR rate in those tumors that were HER2-negative by mRNA analysis (21.1% vs. 49.6%, P <0.0005). The level of HER2 mRNA expression was linked to response rate in ESR1-positive tumors, but not in ESR1-negative tumors. HER2 mRNA expression was significantly associated with pCR in the HER2-positive/ESR1-positive tumors (P = 0.004), but not in HER2-positive/ESR1-negative tumors.
Only patients with cHER2-positive tumors - irrespective of the method used - have an increased pCR rate with trastuzumab plus chemotherapy. In patients with cHER2-negative tumors the pCR rate is comparable to the pCR rate in the non-trastuzumab treated HER-negative population. Response to trastuzumab is correlated to HER2 mRNA levels only in ESR1-positive tumors. This study adds further evidence to the different biology of both subsets within the HER2-positive group.
Introduction The human epidermal growth factor receptor 2 (HER2) is the prototype of a predictive biomarker for targeted treatment [1-8]. International initiatives have established the combination of immunohistochemistry (IHC) and in situ hybridization as the current gold standard [9,10]. As an additional approach determination of HER2 mRNA expression is technically feasible in formalin-fixed paraffin-embedded (FFPE) tissue [11-13]. Crosstalk between the estrogen receptor (ER) and the HER2 pathway has been suggested based on cell culture and animal models [14]. Consequently, the 2011 St Gallen panel has pointed out that HER2-positive tumors should be divided into two groups based on expression of the ER [15].
A retrospective analysis of the National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 study has suggested that mRNA levels of HER2 and ESR1 might be relevant for the degree of benefit from adjuvant trastuzumab. By subpopulation treatment effect pattern plot (STEPP) analysis in ER-positive tumors, benefit from trastuzumab was shown to be restricted to those with higher levels of HER2 mRNA (S Paik, personal communication, results summarized in [15]).
In our study we evaluated this hypothesis in the neoadjuvant setting in a cohort of 217 patients from the neoadjuvant GeparQuattro trial [5]. All patients had been HER2- positive by local pathology assessment and had received 24 to 36 weeks of neoadjuvant trastuzumab plus an anthracycline/taxane-based chemotherapy. For central evaluation we used three different methods, HER2 IHC, and HER2 silver in situ hybridization (SISH), as well as measurement of HER2 mRNA by quantitative real-time (qRT)-PCR [11].
The primary objective of this analysis was to investigate if pathological complete response (pCR) rate in HER2-positive breast cancer would depend on the level of HER2 mRNA expression, with a separate analysis for HR-positive and -negative tumors. Central evaluation of the HER2 status showed that 27% of the tumors with HER2 overexpression by local pathology were HER2-negative. This enabled us to compare response rates in patients with HER2-positive and -negative tumors as a secondary objective.
PMCID: PMC3672694  PMID: 23391338
2.  Exquisite Sensitivity of TP53 Mutant and Basal Breast Cancers to a Dose-Dense Epirubicin−Cyclophosphamide Regimen 
PLoS Medicine  2007;4(3):e90.
In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown.
Methods and Findings
In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m2 epirubicin and 1,200 mg/m2 cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status.
This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin–cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features.
Hugues de The and colleagues report thatTP53 status is a predictive factor for responsiveness in breast cancers to a dose-dense epirubicin-cyclophosphamide chemotherapy regimen, and suggests that this regimen might be well suited for patientsTP53 mutant tumors.
Editors' Summary
One woman in eight will develop breast cancer during her life. As with other cancers, breast cancer arises when cells accumulate genetic changes (mutations) that allow them to grow uncontrollably and to move around the body. These altered cells are called malignant cells. The normal human breast contains several types of cell, any of which can become malignant. In addition, there is more than one route to malignancy—different sets of genes can be mutated. As a result, breast cancer is a heterogeneous disease that cannot be cured with a single type of treatment. Ideally, oncologists would like to know before they start treating a patient which therapeutic approach is going to be successful for that individual. Recently, researchers have begun to identify molecular changes that might eventually allow oncologists to make such rational treatment decisions. For example, laboratory studies in cell lines or animals indicate that the status of a gene called TP53 determines the chemotherapy agents (drugs that preferentially kill rapidly dividing cancer cells) to which cells respond. p53, the protein encoded by TP53, is a tumor suppressor. That is, in normal cells it prevents unregulated growth by controlling the expression of proteins involved in cell division and cell death. Consequently, p53 is often inactivated during cancer development.
Why Was This Study Done?
Although laboratory studies have linked TP53 status to chemotherapy responses, little is known about this relationship in human breast cancers. The clinical studies that have investigated whether TP53 status affects chemotherapy responses have generally found that patients whose tumors contain mutant TP53 have a poorer response to therapy and/or a shorter survival time than those whose tumors contain normal TP53. In this study, the researchers have asked whether TP53 status affects tumor responses to a dose-intense chemotherapy regimen (frequent, high doses of drugs) given to women with advanced noninflammatory breast cancer before surgery. This type of treatment is called neoadjuvant chemotherapy and is used to shrink tumors before surgery.
What Did the Researchers Do and Find?
The researchers collected breast tumor samples from 80 women before starting six fortnightly cycles of chemotherapy with epirubicin and cyclophosphamide. After this, each woman had her affected breast removed and examined to see whether the chemotherapy had killed the tumor cells. The researchers determined which original tumor samples contained mutated TP53 and used a technique called microarray expression profiling to document gene expression patterns in them. Overall, 28 tumors contained mutated TP53. Strikingly, all 15 tumors that responded completely to neoadjuvant chemotherapy (no tumor cells detectable in the breast tissue after chemotherapy) contained mutated TP53. Nine of these responsive tumors were basal-cell–like breast tumors, a particularly aggressive type of breast cancer; only one basal-cell–like, TP53-mutated tumor did not respond to chemotherapy. Patients whose tumors were unresponsive to the neoadjuvant chemotherapy but contained mutated TP53 tended to die sooner than those whose tumors contained normal TP53 or those with chemotherapy-responsive TP53-mutated tumors. Finally, expression profiling identified changes in the expression of many p53-regulated genes, but did not identify an expression profile in the TP53-mutated tumors unique to those that responded to chemotherapy.
What Do These Findings Mean?
These findings indicate that noninflammatory breast tumors containing mutant TP53—in particular, basal-cell–like tumors—are very sensitive to dose-dense epirubicin and cyclophosphamide chemotherapy. Intensive regimens of this type have rarely been used in previous studies, which might explain the apparent contradiction between these results and the generally poor response to chemotherapy of TP53-mutated breast tumors. More tumors now need to be examined to confirm the association between complete response, TP53 status and basal-cell–like tumors. In addition, although complete tumor responses generally predict good overall survival, longer survival studies than those reported here are needed to show that the tumor response to this particular neoadjuvant chemotherapy regimen translates into improved overall survival. If the present results can be confirmed and extended, dose-dense neoadjuvant chemotherapy with epirubicin and cyclophosphamide could considerably improve the outlook for patients with aggressive TP53-mutant, basal-cell–like breast tumors.
Additional Information.
Please access these Web sites via the online version of this summary at
The US National Cancer Institute provides patient and physician information on breast cancer and general information on understanding cancer
Cancer Research UK offers patient information on cancer and breast cancer
The MedlinePlus encyclopedia has pages on breast cancer
Emory University's CancerQuest discusses the biology of cancer, including the role of tumor suppressor proteins
Wikipedia has pages on p53 (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1831731  PMID: 17388661
3.  Preoperative/Neoadjuvant Therapy in Pancreatic Cancer: A Systematic Review and Meta-analysis of Response and Resection Percentages 
PLoS Medicine  2010;7(4):e1000267.
Jörg Kleef and colleagues systematically reviewed studies on neoadjuvant therapy and tumor response, toxicity, resection, and survival percentages in pancreatic cancer and suggest that patients with locally nonresectable tumors should be included in neoadjuvant protocols.
Pancreatic cancer has an extremely poor prognosis and prolonged survival is achieved only by resection with macroscopic tumor clearance. There is a strong rationale for a neoadjuvant approach, since a relevant percentage of pancreatic cancer patients present with non-metastatic but locally advanced disease and microscopic incomplete resections are common. The objective of the present analysis was to systematically review studies concerning the effects of neoadjuvant therapy on tumor response, toxicity, resection, and survival percentages in pancreatic cancer.
Methods and Findings
Trials were identified by searching MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials from 1966 to December 2009 as well as through reference lists of articles and proceedings of major meetings. Retrospective and prospective studies analyzing neoadjuvant radiochemotherapy, radiotherapy, or chemotherapy of pancreatic cancer patients, followed by re-staging, and surgical exploration/resection were included. Two reviewers independently extracted data and assessed study quality. Pooled relative risks and 95% confidence intervals were calculated using random-effects models. Primary outcome measures were proportions of tumor response categories and percentages of exploration and resection. A total of 111 studies (n = 4,394) including 56 phase I–II trials were analyzed. A median of 31 (interquartile range [IQR] 19–46) patients per study were included. Studies were subdivided into surveys considering initially resectable tumors (group 1) and initially non-resectable (borderline resectable/unresectable) tumors (group 2). Neoadjuvant chemotherapy was given in 96.4% of the studies with the main agents gemcitabine, 5-FU (and oral analogues), mitomycin C, and platinum compounds. Neoadjuvant radiotherapy was applied in 93.7% of the studies with doses ranging from 24 to 63 Gy. Averaged complete/partial response probabilities were 3.6% (95% CI 2%–5.5%)/30.6% (95% CI 20.7%–41.4%) and 4.8% (95% CI 3.5%–6.4%)/30.2% (95% CI 24.5%–36.3%) for groups 1 and 2, respectively; whereas progressive disease fraction was estimated to 20.9% (95% CI 16.9%–25.3%) and 20.8% (95% CI 14.5%–27.8%). In group 1, resectability was estimated to 73.6% (95% CI 65.9%–80.6%) compared to 33.2% (95% CI 25.8%–41.1%) in group 2. Higher resection-associated morbidity and mortality rates were observed in group 2 versus group 1 (26.7%, 95% CI 20.7%–33.3% versus 39.1%, 95% CI 29.5%–49.1%; and 3.9%, 95% CI 2.2%–6% versus 7.1%, 95% CI 5.1%–9.5%). Combination chemotherapies resulted in higher estimated response and resection probabilities for patients with initially non-resectable tumors (“non-resectable tumor patients”) compared to monotherapy. Estimated median survival following resection was 23.3 (range 12–54) mo for group 1 and 20.5 (range 9–62) mo for group 2 patients.
In patients with initially resectable tumors (“resectable tumor patients”), resection frequencies and survival after neoadjuvant therapy are similar to those of patients with primarily resected tumors and adjuvant therapy. Approximately one-third of initially staged non-resectable tumor patients would be expected to have resectable tumors following neoadjuvant therapy, with comparable survival as initially resectable tumor patients. Thus, patients with locally non-resectable tumors should be included in neoadjuvant protocols and subsequently re-evaluated for resection.
Please see later in the article for the Editors' Summary
Editors' Summary
Pancreatic cancer is the fourth leading cause of cancer-related deaths worldwide. It begins when a cell in the pancreas (an organ lying behind the stomach that produces digestive enzymes and hormones such as insulin that controls blood sugar levels) acquires genetic changes that allow it to grow uncontrollably and, sometimes, to spread around the body (metastasize). Because pancreatic cancer rarely causes any symptoms early in its development, it is locally advanced in more than a third of patients and has already metastasized in another half of patients by the time it is diagnosed. Consequently, on average, people die within 5–8 months of a diagnosis of pancreatic cancer. At present, the only chance for cure is surgical removal (resection) of the tumor, part of the pancreas, and other nearby digestive organs. This procedure—the Whipple procedure—is only possible in the fifth of patients whose tumor is found when it is small enough to be resectable, and even in these patients, the cure rate associated with surgery is less than 25%, although radiotherapy or chemotherapy after surgery (adjuvant therapy) can be beneficial.
Why Was This Study Done?
For patients whose tumor has metastasized, palliative chemotherapy to slow down tumor growth and to minimize pain is the only treatment option. But, for the many patients whose disease is locally advanced and unresectable at diagnosis, experts think that “neoadjuvant” therapy might be helpful. Neoadjuvant therapy—chemotherapy and/or radiotherapy given before surgery—aims to convert unresectable tumors into resectable tumors by shrinking the visible tumor and removing cancer cells that cannot be seen with the naked eye. Randomized phase III trials—studies in which groups of patients are randomly assigned to different interventions and specific outcomes measured—are the best way to determine whether an intervention has any clinical benefits, but no randomized phase III trials of neoadjuvant therapy for unresectable pancreatic cancer have been undertaken. Therefore, in this systematic review (a study that uses predefined criteria to identify all the research on a given topic) and meta-analysis (a statistical method for combining the results of several studies), the researchers analyze data from other types of studies to investigate whether neoadjuvant therapy for pancreatic cancer provides any clinical benefits.
What Did the Researchers Do and Find?
In their systematic review, the researchers identified 111 studies involving 4,394 patients in which the effects of neoadjuvant chemotherapy and/or radiotherapy on tumor response, tumor resectability, and patient survival had been investigated. They subdivided the studies into two groups: group 1 studies included patients whose tumors were considered resectable on preoperative examination, and group 2 studies included patients whose tumors were borderline resectable or unresectable. In their meta-analysis, the researchers found that similar percentages of the tumors in both groups responded to neoadjuvant therapy by shrinking or regressing and that about a fifth of the tumors in each group grew larger or metastasized during neoadjuvant therapy. In the group 1 studies, three-quarters of the tumors were resectable after neoadjuvant therapy (a decrease in the proportion of tumors that could be treated surgically) whereas in the group 2 studies, a third of the tumors were resectable after neoadjuvant therapy (an increase in the proportion of tumors that could be treated surgically). After resection, the average survival time for group 1 patients was 23.3 months, a similar survival time to that seen in patients treated with surgery and adjuvant therapy. The average survival time for group 2 patients after resection was 20.5 months.
What Do These Findings Mean?
The finding that the average survival time after neoadjuvant therapy and surgery in patients whose tumor was judged resectable before neoadjuvant therapy was similar to that of patients treated with chemotherapy and/or radiotherapy after surgery suggests that for patients with resectable tumors, neoadjuvant therapy will not provide any clinical benefit. By contrast, the finding that a third of patients initially judged unresectable were able to undergo resection after neoadjuvant therapy and then had a similar survival rate to patients judged resectable before neoadjuvant treatment strongly suggests that patients presenting with locally advanced/unresectable tumors should be offered neoadjuvant therapy and then re-evaluated for resection. Randomized trials are now needed to confirm this finding and to determine the optimum neoadjuvant therapy for this group of patients.
Additional Information
Please access these Web sites via the online version of this summary at
The US National Cancer Institute provides information for patients and health professionals about all aspects of pancreatic cancer (in English and Spanish), including a booklet for patients
The American Cancer Society also provides detailed information about pancreatic cancer
The UK National Health Service and Cancer Research UK include information for patients on pancreatic cancer on their Web sites
MedlinePlus provides links to further resources on pancreatic cancer (in English and Spanish),, and the Pancreatic Cancer Action Network give more information to pancreatic cancer patients, their families, and caregivers
PMCID: PMC2857873  PMID: 20422030
4.  Thymosin beta 15A (TMSB15A) is a predictor of chemotherapy response in triple-negative breast cancer 
British Journal of Cancer  2012;107(11):1892-1900.
Biomarkers predictive of pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) of breast cancer are urgently needed.
Using a training/validation approach for detection of predictive biomarkers in HER2-negative breast cancer, pre-therapeutic core biopsies from four independent cohorts were investigated: Gene array data were analysed in fresh frozen samples of two cohorts (n=86 and n=55). Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was performed in formalin-fixed, paraffin-embedded (FFPE) samples from two neoadjuvant phase III trials (GeparTrio, n=212, and GeparQuattro, n=383).
A strong predictive capacity of thymosin beta 15 (TMSB15A) gene expression was evident in both fresh frozen cohorts (P<0.0001; P<0.0042). In the GeparTrio FFPE training cohort, a significant linear correlation between TMSB15A expression and pCR was apparent in triple-negative breast cancer (TNBC) (n=61, P=0.040). A cutoff point was then defined that divided TNBC into a low and a high expression group (pCR rate 16.0% vs 47.2%). Both linear correlation of TMSB15A mRNA levels (P=0.017) and the pre-defined cutoff point were validated in 134 TNBC from GeparQuattro (pCR rate 36.8% vs 17.0%, P=0.020). No significant predictive capacity was observed in luminal carcinomas from GeparTrio and GeparQuattro.
In TNBC, TMSB15A gene expression analysis might help to select patients with a high chance for pCR after NACT.
PMCID: PMC3504944  PMID: 23079573
predictive factor; neoadjuvant chemotherapy; triple negative; breast cancer; thymosin beta 15
5.  Neoadjuvant therapy of early stage human epidermal growth factor receptor 2 positive breast cancer: latest evidence and clinical implications 
Neoadjuvant therapy in human epidermal growth factor receptor 2 (HER2)-positive breast cancer is exactly the paradigm of targeted therapy and a suitable setting to develop and test rapidly novel therapies in early stages. Moreover, neoadjuvant approaches provide a significant source of tumour tissue to identify molecular heterogeneity and potential predictive biomarkers of response. The addition of trastuzumab to primary chemotherapy revolutionized the treatment of this tumour subtype, increasing pathological complete response rate (pCR) that, even with its limitations, has also been shown to be an early marker of survival in HER2-positive disease. HER2-positive breast cancer is a biological heterogeneous disease with different characteristics and clinical outcomes. Multiple promising anti-HER2 drugs with nonoverlapping mechanisms of action have recently been developed. Combined administration of two different HER2-targeted agents, that is, trastuzumab with lapatinib or pertuzumab, and primary chemotherapy shows enhanced antitumour activity, with an increase in pCR to values never reached in the past. Moreover, results of recent studies show that the combination of targeted therapy alone (dual HER2 blockade with or without endocrine therapy) also has activity in a substantial percentage of patients, eradicating HER2-positive tumours without chemotherapy and with a favourable toxicity profile. It is still necessary to be able to select the appropriate group of patients who can avoid chemotherapy (approximately 25%), and to establish robust predictive biomarkers of response or resistance to the anti-HER2 approach. Neoadjuvant therapy represents an enormous step forward in HER2-positive breast cancer. The results of the most relevant neoadjuvant studies and latest evidence are described in this review, though new questions have emerged.
PMCID: PMC4206611  PMID: 25342988
dual HER2 blockade; early breast cancer; HER2 positive; neoadjuvant therapy; pathological complete response; predictive biomarkers
6.  Predicting Response to Treatment in Gastroesophageal Junction Adenocarcinomas: Combining Clinical, Imaging, and Molecular Biomarkers 
The Oncologist  2010;15(3):270-284.
This review presents the evidence base and discusses novel experimental approaches for the combination of biomarker modalities to allow optimization of an individualized treatment approach in GEJ adenocarcinoma patients that may be relevant to other tumor types as well.
Learning Objectives
After completing this course, the reader will be able to: Contrast the subtypes of gastroesophageal adenocarcinoma in order to select optimal therapeutic approaches for given subtypes.Compare the various tools (CT, MRI, PET, PET-CT, etc.) for evaluating response to therapy in order to determine whether to initiate new therapy.Evaluate response to neoadjuvant therapy, utilizing imaging, histopathogy of resected specimens, and biomarkers, to plan postoperative treatment.
This article is available for continuing medical education credit at
The incidence of adenocarcinomas of the gastroesophageal junction (GEJ) is rapidly rising, and even in early-stage locoregional confined disease the 5-year survival rate rarely exceeds 25%–35%. Randomized trials and meta-analyses have demonstrated a benefit with neoadjuvant or perioperative chemotherapy and with neoadjuvant chemoradiotherapy. However, the optimal approach in individual patients is not clear and remains controversial. A consistent finding is that patients who have a histopathological response to neoadjuvant therapy are more likely to receive a survival benefit. These clinical data provide a strong argument for the urgent development of methods to predict histopathological response to neoadjuvant therapies for GEJ adenocarcinoma. Published data demonstrate that clinicopathological features (tumor location), imaging (fluorodeoxyglucose-positron emission tomography “metabolic response”), and tissue/molecular biomarkers may all have a predictive value for neoadjuvant therapies. However, it is uncertain from published data whether or not they will be useful for clinical decision making in individual patients. Existing candidate biomarkers need to be properly qualified and validated and novel biomarkers are required; and an optimal approach should involve the combination and integration of clinical, imaging, and molecular biomarkers. This review presents the evidence base and discusses novel experimental approaches for the combination of biomarker modalities to allow optimization of an individualized treatment approach in GEJ adenocarcinoma patients that may be relevant to other tumor types as well.
PMCID: PMC3227948  PMID: 20203174
Gastroesophageal junction; Adenocarcinoma; Response; Positron emission tomography; Biomarkers
7.  Extending neoadjuvant care through multi-disciplinary collaboration: proceedings from the fourth annual meeting of the Canadian Consortium for Locally Advanced Breast Cancer 
Current Oncology  2012;19(2):106-114.
The use of systemic therapy before surgery (“neoadjuvant therapy”) is the standard of care for the treatment of locally advanced and nonoperable breast cancer. The advantages of neoadjuvant therapy include improved rates of breast-conserving surgery, the possibility of early measurement of response, and potentially improved outcomes for certain subgroups of high-risk patients. The use of neoadjuvant therapy in operable breast cancer is increasing, although there are no clear guidelines in Canada to help guide patient selection and management.
Multidisciplinary experts in the diagnosis and treatment of locally advanced breast cancer (labc) converged at the fourth annual meeting of the Canadian Consortium for LABC (colab) to further their goals of improved standards for neoadjuvant care and clinical research through education and collaboration. Canadian clinical researchers were joined by Dr. Michael Untch of the Helios Hospital Berlin–Buch—representing the German neoadjuvant treatment groups German Gynecologic Oncology Working Group (Arbeitsgemeinschaft Gynakologische Onkologie) and German Breast Group—to discuss the advancement of research in the neoadjuvant setting and important issues of clinical care and investigator-led research. The group reached a consensus on the importance of multidisciplinary collaboration, the use of clips to mark tumour location, and core biopsy testing for the estrogen and progesterone receptors and the human epidermal growth factor receptor 2 at the time of diagnosis. Other initiatives—including creation of a prospective database, inception of the colab Neoadjuvant Network, and development of a clinical survey to evaluate current practice—continue to further the colab mandate of transforming the neoadjuvant treatment landscape in Canada.
PMCID: PMC3320223
Breast neoplasms; cancer treatment; clinical research; translational research; neoadjuvant therapy; surgery; radiation oncology; pathology
8.  Serum biomarker profiles and response to neoadjuvant chemotherapy for locally advanced breast cancer 
Neoadjuvant chemotherapy has become the standard of care for the diverse population of women diagnosed with locally advanced breast cancer. Serum biomarker levels are increasingly being investigated for their ability to predict therapy response and aid in the development of individualized treatment regimens. Multianalyte profiles may offer greater predictive power for neoadjuvant treatment response than the individual biomarkers currently in use.
Serum samples were collected from 44 patients enrolled in a phase I–II, open-label study of liposomal doxorubicin and paclitaxel in combination with whole breast hyperthermia for the neoadjuvant treatment of locally advanced breast cancer (stage IIB or stage III). Samples were collected prior to each of four rounds of treatment and prior to definitive surgery. Samples were assayed by Luminex assay for 55 serum biomarkers, including cancer antigens, growth/angiogenic factors, apoptosis-related molecules, metastasis-related molecules, adhesion molecules, adipokines, cytokines, chemokines, hormones, and other proteins.
Biomarker levels were compared retrospectively with clinical and pathologic treatment responses. Univariate analysis of the data identified several groups of biomarkers that differed significantly among treatment outcome groups early in the course of neoadjuvant chemotherapy. Multivariate statistical analysis revealed multibiomarker panels that could differentiate between treatment response groups with high sensitivity and specificity.
We demonstrate here that serum biomarker profiles may offer predictive power concerning treatment response and outcome in the neoadjuvant setting. The continued development of these findings will be of considerable clinical utility in the design of treatment regimens for individual breast cancer patients.
Trial registration
PMCID: PMC2481492  PMID: 18474099
9.  A 20 gene model for predicting nodal involvement in bladder cancer patients with muscle invasive tumors 
PLoS Currents  2011;3:RRN1248.
Bladder cancer is the fourth most common cancer in males worldwide and also the most expensive cancer to treat. Approximately 25% of patients with muscle invasive disease are found to harbor occult lymph node involvement at the time of cystectomy and this finding is associated with a 5-year survival rate of <30%. If these patients could be identified pre-operatively, use of neoadjuvant chemotherapy may be advantageous because this approach has been shown to confer a small survival advantage in patients with muscle invasive disease. However, because only a few patients benefit from this approach it has not been used extensively in the United States with fewer than 2% of patients undergoing this treatment. This is largely due to concerns that since neoadjuvant therapy is beneficial for only a few patients, it has the potential to delay surgery in the majority who do not benefit. However, since neoadjuvant therapy is most likely to benefit those patients at highest risk for progression of disease, it follows that patients with lymph node metastases would constitute an ideal group for such treatment. Hence, if patients with occult node involvement prior to cystectomy could be identified, they would constitute an ideal group for application of neoadjuvant therapy as they are most likely to benefit. In this summary, we describe the first multi-analyte gene expression model developed for predicting occult nodal involvement at cystectomy in bladder cancer patients, for the purpose of making better informed decisions regarding neoadjuvant therapy. The 20 gene model, which was developed on Affymetrix Human Genome U133A and U133 Plus 2.0 arrays, identified individuals with high relative risk (RR) of nodal involvement (RR = 1.74, 95% CI, 1.03 – 2.93) intermediate risk (RR = 1.05, 95% CI, .45 – 2.41), and low risk (RR = 0.74, 95% CI, 0.51 – 0.96), when evaluated in an independent test dataset. The 20 gene model can be applied to formalin-fixed paraffin embedded tissue with sufficient tumor content, making implementation in routine diagnostic tissue highly feasible. Although a clinical assay for the gene panel has not undergone analytic validation in a clinical laboratory setting, multiple platforms are available which could be utilized for routine testing, including real-time reverse transcriptase PCR directed against individual analytes as well as microarray approaches.
PMCID: PMC3157080  PMID: 21858252
10.  A Six-Gene Signature Predicts Survival of Patients with Localized Pancreatic Ductal Adenocarcinoma 
PLoS Medicine  2010;7(7):e1000307.
Jen Jen Yeh and colleagues developed and validated a six-gene signature in patients with pancreatic ductal adenocarcinoma that may be used to better stage the disease in these patients and assist in treatment decisions.
Pancreatic ductal adenocarcinoma (PDAC) remains a lethal disease. For patients with localized PDAC, surgery is the best option, but with a median survival of less than 2 years and a difficult and prolonged postoperative course for most, there is an urgent need to better identify patients who have the most aggressive disease.
Methods and Findings
We analyzed the gene expression profiles of primary tumors from patients with localized compared to metastatic disease and identified a six-gene signature associated with metastatic disease. We evaluated the prognostic potential of this signature in a training set of 34 patients with localized and resected PDAC and selected a cut-point associated with outcome using X-tile. We then applied this cut-point to an independent test set of 67 patients with localized and resected PDAC and found that our signature was independently predictive of survival and superior to established clinical prognostic factors such as grade, tumor size, and nodal status, with a hazard ratio of 4.1 (95% confidence interval [CI] 1.7–10.0). Patients defined to be high-risk patients by the six-gene signature had a 1-year survival rate of 55% compared to 91% in the low-risk group.
Our six-gene signature may be used to better stage PDAC patients and assist in the difficult treatment decisions of surgery and to select patients whose tumor biology may benefit most from neoadjuvant therapy. The use of this six-gene signature should be investigated in prospective patient cohorts, and if confirmed, in future PDAC clinical trials, its potential as a biomarker should be investigated. Genes in this signature, or the pathways that they fall into, may represent new therapeutic targets.
Please see later in the article for the Editors' Summary
Editors' Summary
Pancreatic cancer kills nearly a quarter of a million people every year. It begins when a cell in the pancreas (an organ lying behind the stomach that produces digestive enzymes and hormones such as insulin, which controls blood sugar levels) acquires genetic changes that allow it to grow uncontrollably and to spread around the body (metastasize). Nearly all pancreatic cancers are “pancreatic ductal adenocarcinomas” (PDACs)—tumors that start in the cells that line the tubes in the pancreas that take digestive juices to the gut. Because PDAC rarely causes any symptoms early in its development, it has already metastasized in about half of patients before it is diagnosed. Consequently, the average survival time after a diagnosis of PDAC is only 5–8 months. At present, the only chance for cure is surgical removal (resection) of the tumor, part of the pancreas, and other nearby digestive organs. The operation that is needed for the majority of patients—the Whipple procedure—is only possible in the fifth of patients whose tumor is found when it is small enough to be resectable but even with postoperative chemotherapy, these patients only live for 23 months after surgery on average, possibly because they have micrometastases at the time of their operation.
Why Was This Study Done?
Despite this poor overall outcome, about a quarter of patients with resectable PDAC survive for more than 5 years after surgery. Might some patients, therefore, have a less aggressive form of PDAC determined by the biology of the primary (original) tumor? If this is the case, it would be useful to be able to stratify patients according to the aggressiveness of their disease so that patients with very aggressive disease could be given chemotherapy before surgery (neoadjuvant therapy) to kill any micrometastases. At present neoadjuvant therapy is given to patients with locally advanced, unresectable tumors. In this study, the researchers compare gene expression patterns in primary tumor samples collected from patients with localized PDAC and from patients with metastatic PDAC between 1999 and 2007 to try to identify molecular markers that distinguish between more and less aggressive PDACs.
What Did the Researchers Do and Find?
The researchers identified a six-gene signature that was associated with metastatic disease using a molecular biology approach called microarray hybridization and a statistical method called significance analysis of microarrays to analyze gene expression patterns in primary tumor samples from 15 patients with localized PDAC and 15 patients with metastatic disease. Next, they used a training set of tumor samples from another 34 patients with localized and resected PDAC, microarray hybridization, and a graphical method called X-tile to select a combination of expression levels of the six genes that discriminated optimally between high-risk (aggressive) and low-risk (less aggressive) tumors on the basis of patient survival (a “cut-point”). When the researchers applied this cut-point to an independent set of 67 tumor samples from patients with localized and resected PDAC, they found that 42 patients had high-risk tumors. These patients had an average survival time of 15 months; 55% of them were alive a year after surgery. The remaining 25 patients, who had low-risk tumors, had an average survival time of 49 months and 91% of them were alive a year after resection.
What Do These Findings Mean?
These and other findings identify a six-gene signature that can predict outcomes in patients with localized, resectable PDAC better than, and independently of, established clinical markers of outcome. If the predictive ability of this signature can be confirmed in additional patients, it could be used to help patients make decisions about their treatment. For example, a patient wondering whether to risk the Whipple procedure (2%–6% of patients die during this operation and more than 50% have serious postoperative complications), the knowledge that their tumor was low risk might help them decide to have the operation. Conversely, a patient in poor health with a high-risk tumor might decide to spare themselves the trauma of major surgery. The six-gene signature might also help clinicians decide which patients would benefit most from neoadjuvant therapy. Finally, the genes in this signature, or the biological pathways in which they participate, might represent new therapeutic targets for the treatment of PDAC.
Additional Information
Please access these Web sites via the online version of this summary at
The US National Cancer Institute provides information for patients and health professionals about all aspects of pancreatic cancer (in English and Spanish), including a booklet for patients
The American Cancer Society also provides detailed information about pancreatic cancer
The UK National Health Service and Cancer Research UK include information for patients on pancreatic cancer on their Web sites
MedlinePlus provides links to further resources on pancreatic cancer (in English and Spanish)
Cure Pancreatic Cancer provides information about scientific and medical research related to the diagnosis, treatment, cure, and prevention of pancreatic cancer
Pancreatic Cancer Action Network is a US organization that supports research, patient support, community outreach, and advocacy for a cure for pancreatic cancer
PMCID: PMC2903589  PMID: 20644708
11.  Apoptosis – associated genes and their role in predicting responses to neoadjuvant breast cancer treatment 
Neoadjuvant chemotherapy is used in the treatment of breast carcinoma because it substantially reduces the size of the primary tumor and lymph node metastases. The present study investigated biomarkers that can predict a pathologic response to the therapy.
The role of apoptosis in regression of the tumors after neoadjuvant chemotherapy was determined by TUNEL and anti-active caspase 3 assay. The transcriptional profile of 84 key apoptosis genes was evaluated in both pre-therapeutically obtained tumor tissue by core needle biopsy and in specimens removed by final surgery, using a pathway-specific real-time PCR assay. Obtained data were analyzed by hierarchical cluster analysis and correlation analysis. The immunohistochemical profile of each tumor was determined using the standard ABC method.
On the basis of a hierarchical cluster analysis of 13 significantly changed genes, we divided patients into good and poor prognosis groups, which correlate well with progression-free survival. In the good prognosis group, we found a statistically significant down-regulation of the expression of MCL1 and IGF1R genes after neoadjuvant treatment. We also found a statistically significant overexpression of BCL2L10, BCL2AF1, CASP8, CASP10, CASP14, CIDEB, FADD, HRK, TNFRSF25, TNFSF8 and CD70 genes. In contrast, we found up-regulation of IGF1R after the treatment in the group with poor prognosis.
Gene expression profiling using real-time PCR assay is a valuable research tool for the investigation of molecular markers, which reflect tumor biology and treatment response.
PMCID: PMC3560664  PMID: 22207111
biomarkers; apoptosis; breast cancer; neoadjuvant chemotherapy
12.  High Expression of Class III β-Tubulin Predicts Good Response to Neoadjuvant Taxane and Doxorubicin/Cyclophosphamide-Based Chemotherapy in Estrogen Receptor–Negative Breast Cancer 
Clinical breast cancer  2012;13(2):103-108.
Class III β-tubulin (βIII-tubulin) has been associated with tumor response to taxane-based therapies in breast cancer. However its role in the neoadjuvant setting has not been explored. We evaluated βIII-tubulin expression by immunohistochemistry in 44 patients, and found high expression associated with good pathologic response in estrogen receptor–negative (ER−) breast cancers. Our results give strong reason to consider βIII-tubulin as a predictive biomarker for neoadjuvant chemotherapy response.
Expression of class III β–tubulin (βIII-tubulin) correlates with tumor progression and resistance to taxane-based therapies for several human malignancies including breast cancer. However its predictive value in a neoadjuvant setting in breast cancer remains unexplored. The objective of this explorative study was to determine whether βIII-tubulin expression in breast cancer correlated with pathologic characteristics and whether its expression was predictive of response to neoadjuvant chemotherapy.
Patients and Methods
We determined βIII-tubulin expression in 85 breast cancers, including 41 localized breast cancers treated with primary surgery and 44 treated with neoadjuvant chemotherapy before surgery. βIII-tubulin expression was evaluated by immunohistochemical methods and was correlated with pathologic characteristics and response to neoadjuvant chemotherapy using residual cancer burden (RCB) score.
High βIII-tubulin expression was significantly associated with poorly differentiated high-grade breast cancers (P = .003) but not with tumor size, estrogen receptor (ER) status, or human epidermal growth factor receptor 2 (HER2)/neu overexpression. In ER− tumors treated with neoadjuvant chemotherapy, high βIII-tubulin expression was associated with a significantly greater likelihood of achieving a good pathologic response to chemotherapy as reflected by lower RCB scores (P = .021).
This study reveals differential βIII-tubulin expression in breast cancers of different histologic grades, hormone receptors, and HER2/neu status. It also suggests a potential role for βIII-tubulin as a predictive biomarker for response in neoadjuvant chemotherapy for ER− breast cancer, which has not been previously reported. These data provide a strong rationale for considering βIII-tubulin status and further validation of this marker in a large study.
PMCID: PMC4039021  PMID: 23218766
Breast cancer; Chemotherapy; Class III β-tubulin; Neoadjuvant; Predictive biomarker
13.  Identification and Validation of an Anthracycline/Cyclophosphamide–Based Chemotherapy Response Assay in Breast Cancer 
There is no method routinely used to predict response to anthracycline and cyclophosphamide–based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection.
DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided.
In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population.
A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide–based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.
PMCID: PMC3906990  PMID: 24402422
14.  Role of neoadjuvant therapy in the management of pancreatic cancer: is the era of biomarker-directed therapy here? 
Current Oncology  2014;21(4):e650-e657.
Pancreatic cancer is the 4th leading cause of cancer-related death. Complete surgical resection (CR0) is considered the only curative treatment. Most patients present with unresectable or borderline resectable disease. Many small phase i/ii trials have tried to address the role of neoadjuvant treatment using chemotherapy with or without chemoradiation in the management of locally advanced disease. However, many of them looked at the rate of CR0 resection and the feasibility of such treatment. A trend for improved overall survival has been observed in the group of patients with borderline resectable disease who completed neoadjuvant treatment. A large proportion of patients progress while on treatment, sparing them from unnecessary surgery.
We searched the PubMed database (using the key words “pancreatic cancer,” or “pancreatic neoplasm,” or “pancreatic adenocarcinoma,” and “neoadjuvant treatment,” or “neoadjuvant chemotherapy,” or “neoadjuvant radiation therapy,” or “neoadjuvant chemo-radiation,” or “adjuvant therapy” [all fields] and “clinical trial” or “study”) and abstracts presented at the American Society of Clinical Oncology meetings on gastrointestinal cancers. Here, we review the most recent papers that present results on neoadjuvant therapy in pancreatic cancer. All but one report used overall survival as an endpoint. Unfortunately, there are no valid biomarkers predicting tumour progression or recurrence, and response to treatment than can help to guide therapeutic choices.
Our recommendation is to consider neoadjuvant treatment in cases of borderline resectable disease. In patients with primary resectable tumours, surgery followed by adjuvant treatment and enrollment on adjuvant treatment studies would be appropriate.
PMCID: PMC4117630  PMID: 25089113
Pancreatic cancer; neoadjuvant therapy; chemoradiation; borderline resectability; biomarkers
15.  Neoadjuvant therapy for localized prostate cancer: Examining mechanism of action and efficacy within the tumor 
Urologic oncology  2014;S1078-1439(13)00500-0 10.1016/j.urolonc.2013.12.001.
Efforts to improve the clinical outcome for patients with localized high-risk prostate cancer have led to the development of neoadjuvant systemic therapies. We review the different modalities of neoadjuvant therapies for localized prostate cancer and highlight emerging treatment approaches including immunotherapy and targeted therapy.
We performed a PubMed search of clinical trials evaluating preoperative systemic therapies for treating high-risk prostate cancer published after 2000, and those studies with the highest clinical relevance to current treatment approaches were selected for review. The database at was queried for neoadjuvant studies in high-risk prostate cancer, and those evaluating novel targeted therapies and immunotherapies are spotlighted here.
Neoadjuvant chemotherapy has become standard of care for treating some malignancies, including breast and bladder cancers. In prostate cancer, preoperative hormonal therapy or chemotherapy has failed to demonstrate improvements in overall survival. Nevertheless, the emergence of novel treatment modalities such as targeted small molecules and immunotherapy has spawned neoadjuvant clinical trials that provide a unique vantage from which to study mechanism of action and biological potency. Tissue-based biomarkers are being developed to elucidate the biological efficacy of these treatments. With targeted therapy, these can include phospho-proteomic signatures of target pathway activation and deactivation. With immunotherapies, including sipuleucel-T and ipilimumab, recruitment of immune cells to the tumor microenvironment can also be used as robust markers of a biological effect. Such studies can provide insight not only into mechanism of action for these therapies but can also provide paths forward to improving clinical efficacy like with rationally designed combinations and dose selection.
The use of neoadjuvant androgen-deprivation therapy and chemotherapy either singly or in combination before radical prostatectomy is generally safe and feasible while reducing prostate volume and tumor burden. However, pathologic complete response rates are low and no long-term survival benefit has been observed with the addition of neoadjuvant therapies over surgery alone at present, and therefore preoperative therapy is not the current standard of care in prostate cancer treatment.
PMCID: PMC4499005  PMID: 24495446
Prostate cancer; Neoadjuvant; Preoperative; Targeted therapy; Immunotherapy
16.  Association between Pathological Complete Response and Outcome Following Neoadjuvant Chemotherapy in Locally Advanced Breast Cancer Patients 
Journal of Breast Cancer  2014;17(4):376-385.
We aimed to determine the rate of pathological complete response (pCR), clinicopathological factors associated with pCR, and clinical outcomes following neoadjuvant chemotherapy in locally advanced breast cancer.
Medical records of patients who had undergone neoadjuvant chemotherapy for breast cancer between January 2007 and September 2011 were retrospectively reviewed, and the pCR rates were calculated according to three sets of criteria: the National Surgical Adjuvant Breast and Bowel Project (NSABP), the MD Anderson Cancer Center (MDACC), and the German Breast Group (GBG). Tumors were classified as luminal A like, luminal B like, human epidermal growth factor receptor 2 (HER2), or triple-negative. pCR and clinical outcome, including overall survival (OS) and disease-free survival (DFS) rates were analyzed at the median follow-up of 54.2 months.
Of a total of 179 patients who had received neoadjuvant chemotherapy, 167 patients (93.3%) had locally advanced breast cancer and 12 patients (6.7%) had early-stage breast cancer. The majority of patients (152 patients, 89.4%) received anthracycline-based neoadjuvant chemotherapy. The objective clinical response rate was 61.5%, comprising clinical partial response in 5.5% and clinical complete response in 3.9% of patients. Twenty-one (11.7%), 20 (11.2%), and 17 patients (9.5%) achieved pCR according to NSABP, MDACC, and GBG definitions, respectively. pCR rates, as defined by NSABP, according to breast cancer subtype were 4.4%, 9.7%, 24.2%, and 19.2% in luminal A like, luminal B like, HER2, and triple-negative subtypes, respectively. Patients who achieved pCR had significantly better DFS (5-year DFS rates, 80% vs. 53%, p=0.030) and OS (5-year OS rates, 86% vs. 54%, p=0.042) than those who did not.
The pCR rate following neoadjuvant chemotherapy for breast cancer in Thai women attending our institution was 11.7%; pCR was more frequently observed in HER2 and triple-negative breast tumor subtypes. Patients who achieved pCR had significantly improved survival.
PMCID: PMC4278058  PMID: 25548587
Antineoplastic combined chemotherapy protocols; Breast neoplasms; Neoadjuvant therapy; Surgery; Treatment outcome
17.  Safety of Minimally Invasive Esophagectomy (MIE) After Neoadjuvant Chemoradiotherapy and as Primary Resection at a Non-University Tertiary Care Center (NUTCC) 
Minimally invasive techniques for esophagectomy have improved patient outcomes while maintaining oncologic principles. Neoadjuvant therapy can provide sufficient tumor downstaging such that more patients are able to undergo R0 resection. The aim of this study was to assess the role of MIE in patients who undergo neoadjuvant therapy at a non-university tertiary care center (NUTCC).
MIE by combined thoracoscopic and laparoscopic approaches performed cooperatively by two surgeons between September 2005 and August 2008 were reviewed. The patients were studied as two groups, one group that received neoadjuvant chemoradiotherapy (group A) and the other receiving no neoadjuvant therapy (group B). Preoperative, intraoperative, postoperative, and histopathologic data were evaluated.
Thirty one (31) patients underwent minimally invasive esophagectomy for esophageal malignancies. Of these, 58% (18 patients) received neoadjuvant therapy; in the neoadjuvant therapy group, 61% of patients were preoperative stage IIA, 11% were stage IIB, and 28% were stage III. The median operating time was 286 minutes in the neoadjuvant group and 266 minutes in the non-neoadjuvant group. Median estimated blood loss (EBL) in the two groups was 300 mL (range, 100–700 mL) and 263 mL (range, 150–600 mL), respectively. Six (6) patients from the neoadjuvant group and only 2 from the non-neoadjuvant group received intraoperative transfusions. Extension to a mini-celiotomy was required in 2 of the neoadjuvant patients and none of the non-neoadjuvant patients. The median length of hospital stay (LOS) was 12 days (range, 8–22 d) and 11 days (range, 8–54 d) in the two groups, respectively. There was no mortality reported in the series and no evidence of anastomotic leak in either group. The rate of major morbidity was 67% vs. 54% in the neoadjuvant vs. non-neoadjuvant groups. Complete pathologic response (CR) on final histopathology was achieved in 6/18 patients (33%) receiving neoadjuvant therapy. Three patients had positive margins, all from the neoadjuvant group.
MIE can be safely performed after neoadjuvant chemoradiotherapy. Pathologic CR can be achieved with a preoperative approach in 33% of patients with esophageal cancer. MIE should be considered as safe as open resection after preoperative treatment.
PMCID: PMC3047022
18.  Topoisomerase II alpha and TLE3 as predictive markers of response to anthracycline and taxane-containing regimens for neoadjuvant chemotherapy in breast cancer 
OncoTargets and therapy  2014;7:2111-2120.
Anthracyclines and taxanes are considered the standard for neoadjuvant chemotherapy of breast cancer, although they are often associated with serious side effects and wide variability of individual response. In this study, we analyzed the value of topoisomerase II alpha (TOP2A) and transducin-like enhancer of split 3 (TLE3) as predictive markers of response to therapy with anthracyclines and taxanes.
Materials and methods
TOP2A and TLE3 protein expressions were evaluated using immunohistochemistry on 28 samples, obtained by core needle biopsy in patients with locally advanced breast carcinoma, subsequently subjected to epirubicin- and paclitaxel-based neoadjuvant chemotherapy. The immunohistochemical staining was correlated with the clinical response measured by the tumor size reduction evaluated by breast magnetic resonance imaging, prior and after chemotherapy, and by pathologic evaluation of the surgical specimen.
Neoadjuvant chemotherapy achieved a size reduction in 26/28 tumors (92.9%), with an average percentage decrease of 45.6%. A downstaging was achieved in 71.4% of the cases of locally advanced carcinoma. TOP2A positivity was correlated with a greater reduction in tumor diameter (P=0.06); negative staining for TLE3 was predictive of a better response to neoadjuvant chemotherapy (P=0.07). A higher reduction in tumor diameter (P=0.03) was also found for tumors that were concurrently TLE3-negative and TOP2A-positive.
TOP2A and TLE3 showed a correlation with response to neoadjuvant chemotherapy. While TOP2A is a well-known marker of response to anthracyclines-based chemotherapy, TLE3 is a new putative predictor of response to taxanes. Data from the current study suggest that TOP2A and TLE3 warrant further investigation in a larger series as predictors of response to neoadjuvant chemotherapy for locally advanced breast carcinoma.
PMCID: PMC4240193  PMID: 25484594
breast carcinoma; advanced stage disease; immunohistochemical markers
19.  Assessment of Ki67 in Breast Cancer: Recommendations from the International Ki67 in Breast Cancer Working Group 
Uncontrolled proliferation is a hallmark of cancer. In breast cancer, immunohistochemical assessment of the proportion of cells staining for the nuclear antigen Ki67 has become the most widely used method for comparing proliferation between tumor samples. Potential uses include prognosis, prediction of relative responsiveness or resistance to chemotherapy or endocrine therapy, estimation of residual risk in patients on standard therapy and as a dynamic biomarker of treatment efficacy in samples taken before, during, and after neoadjuvant therapy, particularly neoadjuvant endocrine therapy. Increasingly, Ki67 is measured in these scenarios for clinical research, including as a primary efficacy endpoint for clinical trials, and sometimes for clinical management. At present, the enormous variation in analytical practice markedly limits the value of Ki67 in each of these contexts. On March 12, 2010, an international panel of investigators with substantial expertise in the assessment of Ki67 and in the development of biomarker guidelines was convened in London by the cochairs of the Breast International Group and North American Breast Cancer Group Biomarker Working Party to consider evidence for potential applications. Comprehensive recommendations on preanalytical and analytical assessment, and interpretation and scoring of Ki67 were formulated based on current evidence. These recommendations are geared toward achieving a harmonized methodology, create greater between-laboratory and between-study comparability, and allow earlier valid applications of this marker in clinical practice.
PMCID: PMC3216967  PMID: 21960707
20.  The potential prognostic value of connexin 26 and 46 expression in neoadjuvant-treated breast cancer 
BMC Cancer  2013;13:50.
Several classification systems are available to assess pathological response to neoadjuvant chemotherapy in breast cancer, but reliable biomarkers to predict the efficiency of primary systemic therapy (PST) are still missing. Deregulation of gap junction channel forming connexins (Cx) has been implicated in carcinogenesis and tumour progression through loss of cell cycle control. In this study we correlated Cx expression and cell proliferation with disease survival and pathological response to neoadjuvant chemotherapy in breast cancers using existing classification systems.
The expression of Cx26, Cx32, Cx43, Cx46 and Ki67 was evaluated in 96 breast cancer patients prior to and after neoadjuvant chemotherapy using duplicate cores in tissue microarrays (TMA). Cx plaques of <1μm were detected with multilayer, multichannel fluorescence digital microscopy. Current classifications to assess residual tumour burden after primary systemic therapy included the EWGBSP, CPS-EG, Miller-Payne, Sataloff and NSABP systems.
In our cohort dominated by hormone receptor (ER/PR) positive and HER2 negative cases, only the CPS-EG classification showed prognostic relevance: cases with scores 1–2 had significantly better overall survival (p=0.015) than cases with scores 3–5. Pre-chemotherapy Cx43 expression correlated positively with hormone receptor status both before and after chemotherapy and had a negative correlation with HER2 expression pre-chemotherapy. There was a positive correlation between Cx32 and HER2 expression pre-chemotherapy and between Cx32 and Ki67 expression post-chemotherapy. A negative correlation was found between post-chemotherapy Cx46 and Ki67 expression. Decreased post-chemotherapy Cx26 expression (<5%) statistically correlated with better overall survival (p=0.011). Moderate or higher Cx46 expression (>20%) pre- and post-chemotherapy correlated with significantly better survival in the intermediate prognostic subgroups of EWGBSP TR2b (ppre-chemo=0.006; Sataloff TB (ppre-chemo=0.005; ppost-chemo=0.029) and in Miller-Payne G3 (ppre-chemo=0.002; ppost-chemo=0.012) classifications. Pre-chemotherapy, Cx46 expression was the only marker that correlated with overall survival within these subgroups.
Our results suggest that Cx46 and Cx26 expression in breast cancer may improve the assessment of pathological response and refine intermediate prognostic subgroups of residual tumour classifications used after neoadjuvant chemotherapy.
PMCID: PMC3583680  PMID: 23374644
Breast cancer; Connexin; Gap junction; Preoperative chemotherapy; Prognosis
21.  Biomarker Profiling by Nuclear Magnetic Resonance Spectroscopy for the Prediction of All-Cause Mortality: An Observational Study of 17,345 Persons 
PLoS Medicine  2014;11(2):e1001606.
In this study, Würtz and colleagues conducted high-throughput profiling of blood specimens in two large population-based cohorts in order to identify biomarkers for all-cause mortality and enhance risk prediction. The authors found that biomarker profiling improved prediction of the short-term risk of death from all causes above established risk factors. However, further investigations are needed to clarify the biological mechanisms and the utility of these biomarkers to guide screening and prevention.
Please see later in the article for the Editors' Summary
Early identification of ambulatory persons at high short-term risk of death could benefit targeted prevention. To identify biomarkers for all-cause mortality and enhance risk prediction, we conducted high-throughput profiling of blood specimens in two large population-based cohorts.
Methods and Findings
106 candidate biomarkers were quantified by nuclear magnetic resonance spectroscopy of non-fasting plasma samples from a random subset of the Estonian Biobank (n = 9,842; age range 18–103 y; 508 deaths during a median of 5.4 y of follow-up). Biomarkers for all-cause mortality were examined using stepwise proportional hazards models. Significant biomarkers were validated and incremental predictive utility assessed in a population-based cohort from Finland (n = 7,503; 176 deaths during 5 y of follow-up). Four circulating biomarkers predicted the risk of all-cause mortality among participants from the Estonian Biobank after adjusting for conventional risk factors: alpha-1-acid glycoprotein (hazard ratio [HR] 1.67 per 1–standard deviation increment, 95% CI 1.53–1.82, p = 5×10−31), albumin (HR 0.70, 95% CI 0.65–0.76, p = 2×10−18), very-low-density lipoprotein particle size (HR 0.69, 95% CI 0.62–0.77, p = 3×10−12), and citrate (HR 1.33, 95% CI 1.21–1.45, p = 5×10−10). All four biomarkers were predictive of cardiovascular mortality, as well as death from cancer and other nonvascular diseases. One in five participants in the Estonian Biobank cohort with a biomarker summary score within the highest percentile died during the first year of follow-up, indicating prominent systemic reflections of frailty. The biomarker associations all replicated in the Finnish validation cohort. Including the four biomarkers in a risk prediction score improved risk assessment for 5-y mortality (increase in C-statistics 0.031, p = 0.01; continuous reclassification improvement 26.3%, p = 0.001).
Biomarker associations with cardiovascular, nonvascular, and cancer mortality suggest novel systemic connectivities across seemingly disparate morbidities. The biomarker profiling improved prediction of the short-term risk of death from all causes above established risk factors. Further investigations are needed to clarify the biological mechanisms and the utility of these biomarkers for guiding screening and prevention.
Please see later in the article for the Editors' Summary
Editors' Summary
A biomarker is a biological molecule found in blood, body fluids, or tissues that may signal an abnormal process, a condition, or a disease. The level of a particular biomarker may indicate a patient's risk of disease, or likely response to a treatment. For example, cholesterol levels are measured to assess the risk of heart disease. Most current biomarkers are used to test an individual's risk of developing a specific condition. There are none that accurately assess whether a person is at risk of ill health generally, or likely to die soon from a disease. Early and accurate identification of people who appear healthy but in fact have an underlying serious illness would provide valuable opportunities for preventative treatment.
While most tests measure the levels of a specific biomarker, there are some technologies that allow blood samples to be screened for a wide range of biomarkers. These include nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. These tools have the potential to be used to screen the general population for a range of different biomarkers.
Why Was This Study Done?
Identifying new biomarkers that provide insight into the risk of death from all causes could be an important step in linking different diseases and assessing patient risk. The authors in this study screened patient samples using NMR spectroscopy for biomarkers that accurately predict the risk of death particularly amongst the general population, rather than amongst people already known to be ill.
What Did the Researchers Do and Find?
The researchers studied two large groups of people, one in Estonia and one in Finland. Both countries have set up health registries that collect and store blood samples and health records over many years. The registries include large numbers of people who are representative of the wider population.
The researchers first tested blood samples from a representative subset of the Estonian group, testing 9,842 samples in total. They looked at 106 different biomarkers in each sample using NMR spectroscopy. They also looked at the health records of this group and found that 508 people died during the follow-up period after the blood sample was taken, the majority from heart disease, cancer, and other diseases. Using statistical analysis, they looked for any links between the levels of different biomarkers in the blood and people's short-term risk of dying. They found that the levels of four biomarkers—plasma albumin, alpha-1-acid glycoprotein, very-low-density lipoprotein (VLDL) particle size, and citrate—appeared to accurately predict short-term risk of death. They repeated this study with the Finnish group, this time with 7,503 individuals (176 of whom died during the five-year follow-up period after giving a blood sample) and found similar results.
The researchers carried out further statistical analyses to take into account other known factors that might have contributed to the risk of life-threatening illness. These included factors such as age, weight, tobacco and alcohol use, cholesterol levels, and pre-existing illness, such as diabetes and cancer. The association between the four biomarkers and short-term risk of death remained the same even when controlling for these other factors.
The analysis also showed that combining the test results for all four biomarkers, to produce a biomarker score, provided a more accurate measure of risk than any of the biomarkers individually. This biomarker score also proved to be the strongest predictor of short-term risk of dying in the Estonian group. Individuals with a biomarker score in the top 20% had a risk of dying within five years that was 19 times greater than that of individuals with a score in the bottom 20% (288 versus 15 deaths).
What Do These Findings Mean?
This study suggests that there are four biomarkers in the blood—alpha-1-acid glycoprotein, albumin, VLDL particle size, and citrate—that can be measured by NMR spectroscopy to assess whether otherwise healthy people are at short-term risk of dying from heart disease, cancer, and other illnesses. However, further validation of these findings is still required, and additional studies should examine the biomarker specificity and associations in settings closer to clinical practice. The combined biomarker score appears to be a more accurate predictor of risk than tests for more commonly known risk factors. Identifying individuals who are at high risk using these biomarkers might help to target preventative medical treatments to those with the greatest need.
However, there are several limitations to this study. As an observational study, it provides evidence of only a correlation between a biomarker score and ill health. It does not identify any underlying causes. Other factors, not detectable by NMR spectroscopy, might be the true cause of serious health problems and would provide a more accurate assessment of risk. Nor does this study identify what kinds of treatment might prove successful in reducing the risks. Therefore, more research is needed to determine whether testing for these biomarkers would provide any clinical benefit.
There were also some technical limitations to the study. NMR spectroscopy does not detect as many biomarkers as mass spectrometry, which might therefore identify further biomarkers for a more accurate risk assessment. In addition, because both study groups were northern European, it is not yet known whether the results would be the same in other ethnic groups or populations with different lifestyles.
In spite of these limitations, the fact that the same four biomarkers are associated with a short-term risk of death from a variety of diseases does suggest that similar underlying mechanisms are taking place. This observation points to some potentially valuable areas of research to understand precisely what's contributing to the increased risk.
Additional Information
Please access these websites via the online version of this summary at
The US National Institute of Environmental Health Sciences has information on biomarkers
The US Food and Drug Administration has a Biomarker Qualification Program to help researchers in identifying and evaluating new biomarkers
Further information on the Estonian Biobank is available
The Computational Medicine Research Team of the University of Oulu and the University of Bristol have a webpage that provides further information on high-throughput biomarker profiling by NMR spectroscopy
PMCID: PMC3934819  PMID: 24586121
22.  Quantitative measurements of HER2 and phospho-HER2 expression: correlation with pathologic response to neoadjuvant chemotherapy and trastuzumab 
BMC Cancer  2014;14:326.
Preoperative therapy with chemotherapy and the HER2-targeted monoclonal antibody trastuzumab is valuable for patients with large or locally advanced HER2-positive (HER2+) breast cancers but traditional methods of measuring HER2 expression do not accurately stratify patients for likelihood of response. Quantitative immunofluorescent approaches have the potential to provide a mathematically continuous measure of HER2. Here we seek to determine whether quantitative measurement of HER2 or phospho-HER2 correlates with likelihood of response to trastuzumab- containing neoadjuvant therapy.
We evaluated core biopsy samples from 27 HER2+ breast cancer patients enrolled in a preoperative clinical trial using trastuzumab, nab-paclitaxel and carboplatin combination therapy (BrUOG BR-211B (NCT00617942)). Tumor core biopsies were taken before initiation of treatment and 9–13 days after patients received "run-in" doses of either single agent trastuzumab or nab-paclitaxel. The AQUA method of quantitative immunofluorescence was used for analysis of in situ protein expression. Patients then received 18 weeks of treatment, followed by surgery to assess pathologic response to the neoadjuvant regimen.
A HER2 score of 2111 by AQUA analysis has been shown to be equivalent to HER2 3+ by immunohistochemical staining in previous studies. Of 20 evaluable patients, 10 cases who achieved a pathologic complete response (pathCR) with neoadjuvant treatment had a mean HER2 level of 10251 compared with 4766 in the patients without pathCR (p = 0.0021). Measurement of phospho-HER2 showed no difference in pathCR vs non-pathCR groups. In 9 patients who had HER2 levels repeated after a single treatment with trastuzumab there was no evidence of a reduction in the HER2 or phospho-HER2 levels following that exposure.
High levels of HER2 are associated with achievement of a pathCR in the preoperative setting, while levels of Phospho-HER2 were not predictive of response. This data suggests that accurate measurement of HER2 may help determine the likelihood of response in the pre-surgical setting. Further validation in larger cohorts is required, but this pilot data shows the feasibility of this approach.
PMCID: PMC4037428  PMID: 24885187
Immunohistochemistry; Immunofluorescence
23.  Modulation of plasma complement by the initial dose of epirubicin/docetaxel therapy in breast cancer and its predictive value 
British Journal of Cancer  2010;103(8):1201-1208.
Despite the widespread use of neoadjuvant chemotherapy in breast cancer patients, prediction of individual response to treatment remains an unsolved clinical problem. Particularly, administration of an inefficient chemotherapeutic regimen should be avoided. Therefore, a better understanding of the molecular mechanisms underlying response to neoadjuvant chemotherapy is of particular clinical interest. Aim of the present study was to test whether neoadjuvant chemotherapy with epirubicin/docetaxel induces early changes in the plasma proteome of breast cancer patients and whether such changes correlate with response to therapy.
Plasma samples of 25 breast cancer patients obtained before and 24 h after initiation of epirubicin/docetaxel-based neoadjuvant chemotherapy were analysed using two-dimensional differential gel electrophoresis (2D-DIGE). Protein spots found to be differentially expressed were identified using mass spectrometry and then correlated with the pathological response after six cycles of therapy. Markers identified in a discovery set of patients (n=12) were confirmed in an independent validation set (n=13).
2D-DIGE revealed 33 protein spots to be differentially expressed in response to chemotherapy, including the complement factors C1, C3 and C4, inter-α-trypsin inhibitor, α-1-antichymotrypsin and α-2-Heremans-Schmid glycoprotein (AHSG). With respect to cytokines, only interleukin (IL)-6, IL-10 and soluble intracellular adgesion molecule 3 (sICAM3) were minimally modulated. Moreover, two protein spots within the complement component C3 significantly correlated with response to therapy.
We have identified acute phase proteins and the complement system as part of the early host response to epirubicin/docetaxel chemotherapy. As complement C3 cleavage correlates with the efficacy of docetaxel/epirubicin-based chemotherapy, it has the potential as an easily accessible predictive biomarker.
PMCID: PMC2967072  PMID: 20877360
breast cancer; response to therapy; epirubicin; docetaxel; complement system
24.  CCND1 as a Predictive Biomarker of Neoadjuvant Chemotherapy in Patients with Locally Advanced Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2011;6(10):e26399.
Cyclin D1 (CCND1) has been associated with chemotherapy resistance and poor prognosis. In this study, we tested the hypothesis that CCND1 expression determines response and clinical outcomes in locally advanced head and neck squamous cell carcinoma (HNSCC) patients treated with neoadjuvant chemotherapy followed by surgery and radiotherapy.
Methodology and Findings
224 patients with HNSCC were treated with either cisplatin-based chemotherapy followed by surgery and radiotherapy (neoadjuvant group, n = 100) or surgery and radiotherapy (non-neoadjuvant group, n = 124). CCND1 expression was assessed by immunohistochemistry. CCND1 levels were analyzed with chemotherapy response, disease-free survival (DFS) and overall survival (OS). There was no significant difference between the neoadjuvant group and non-neoadjuvant group in DFS and OS (p = 0.929 and p = 0.760) when patients treated with the indiscriminate administration of cisplatin-based chemotherapy. However, in the neoadjuvant group, patients whose tumors showed a low CCND1 expression more likely respond to chemotherapy (p<0.001) and had a significantly better OS and DFS than those whose tumors showed a high CCND1 expression (73% vs 8%, p<0.001; 63% vs 6%, p<0.001). Importantly, patients with a low CCND1 expression in neoadjuvant group received more survival benefits than those in non-neoadjuvant group (p = 0.016), however patients with a high CCND1 expression and treated with neoadjuvant chemotherapy had a significantly poor OS compared to those treated with surgery and radiotherapy (p = 0.032). A multivariate survival analysis also showed CCND1 expression was an independent predictive factor (p<0.001).
This study suggests that some but not all patients with HNSCC may benefit from neoadjuvant chemotherapy with cisplatin-based regimen and CCND1 expression may serve as a predictive biomarker in selecting patients undergo less than two cycles of neoadjuvant chemotherapy.
PMCID: PMC3204964  PMID: 22065993
25.  Current status of predictive biomarkers for neoadjuvant therapy in esophageal cancer 
Neoadjuvant therapy has been proven to be extremely valuable and is widely used for advanced esophageal cancer. However, a significant proportion of treated patients (60%-70%) does not respond well to neoadjuvant treatments and develop severe adverse effects. Therefore, predictive markers for individualization of multimodality treatments are urgently needed in esophageal cancer. Recently, molecular biomarkers that predict the response to neoadjuvant therapy have been explored in multimodal approaches in esophageal cancer and successful examples of biomarker identification have been reported. In this review, promising candidates for predictive molecular biomarkers developed by using multiple molecular approaches are reviewed. Moreover, treatment strategies based on the status of predicted biomarkers are discussed, while considering the international differences in the clinical background. However, in the absence of adequate treatment options related to the results of the biomarker test, the usefulness of these diagnostic tools is limited and new effective therapies for biomarker-identified nonresponders to cancer treatment should be concurrent with the progress of predictive technologies. Further improvement in the prognosis of esophageal cancer patients can be achieved through the introduction of novel therapeutic approaches in clinical practice.
PMCID: PMC4133529  PMID: 25133032
Esophageal cancer; Neoadjuvant therapy; Response prediction; Molecular biomarker; Chemoradiation

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