Journal of Chemistry-JuniperPublishers

                                Abstract

Quality by Design is the new-age path chosen towards achieving the demanding quality standards in pharmaceutical industry. The present paper aims to throw light on Pharmaceutical Quality byDesign (QbD) and how its implementation will help manufacture better quality of Pharmaceuticals. Quality by Design is introduced along with its key elements to help make the understanding process easier. To attain built-in quality is the primary objective of Quality by Design. Finally, it can be said that the quality that is achieved by end product testing is not something that can be guaranteed unlike the quality assurance that can be provided by Quality by Design.

Keywords: Quality by Design (QbD); Quality Target Product Profile; Design Space; Critical Quality Attributes

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Introduction

“Quality Can Be Planned.”-Joseph Juran

The above quote is self-explanatory when it comes to product quality in the pharmaceutical manufacturing industry. Quality by design (QbD) is not very old but a recent inclusion in the pharmaceutical industry. It`s sole objective is to achieve better quality standards that is especially important in the pharmaceutical industry. The QbD approach consists of various components, important ones being risk assessment, assessment and management of the identified risks, design of experiments (DoE), quality target product profile (QTPP), and establishing a control strategy to keep the product within the design space that was created with the QbD study [1]. Out of all the components, a lot of pharmaceutical development studies have incorporated DoE for a more rational approach [2].

The target of analytical QbD approach is to establish a design space (DS) of critical process parameters (CPPs) where the critical quality attributes (CQAs) of the method have been assured to fulfil the desired requirements with a selected probability [3-4].

The principles that are involved in the pharmaceutical development and are relevant to QbD are all described in the ICH guidelines (ICHQ8-11) [5].

Any Pharmaceutical Development Process Typically Covers the Following Sections:

a) Complete portfolio including all the details as well as analysis of the Reference Listed Drug Product

b) Quality Target Product Profile (QTTP) compilation.

c) Figuring out the Critical Quality Attributes (CQA)

d) Complete characterization of API &CMA (Components of Drug product) identification of the API

e) Excipient selection& excipients CMA identification

f) Formulation Development

g) Manufacturing Process Development [6]

Quality Target Product Profile (QTPP) describes the design criteria for the product, and should therefore form the basis for development of the CQAs, CPPs, and control strategy.

Critical Quality Attributes (CQA) – A physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality (ICH Q8) Critical Process Parameter (CPP) – A process parameter whose variability has an impact on a CQA and therefore should be monitored or controlled to ensure the process produces the desired quality. (ICH Q8) Critical Process Parameters (CPP) identification and their impact analysis is done by conducting a preliminary risk analysis for every process parameter (PP) that is involved in the individual unit operations.

Need for QbD in Pharmaceutical Industry [7,8]:

a) To integratepatient needs, quality requirements and scientific knowledge all in one design while the pharmaceutical product is still under developmentand further extending to the manufacturing process.

b) To have a better understanding about the impact of raw materials and process parameters on the quality of the final product. This is especially important for biopharmaceutical products since raw materials like cell culture media can be the risk for variability, effecting important factors likecellular viability, cell growth and specific productivity.

c) To collaborate closely with rest of the industries and the regulators and successfully keep up with the regulatory reviews

d) To maintain harmonization in all the regions so that a single CMC submission worldwide is all that is needed.

e) To encourage continuous quality improvement for the benefit of patients.

f) To enable better product design that will have less problems while manufacturing, thus facilitating more efficiency in the manufacturing process.

g) To make post-approval changes easier since it will be contained within a pre-defined design space, thus resulting in regulatory flexibility.

Every production process in a pharmaceutical industry to implement certain control strategies with the ultimate goal of a robust process. A robust process is the gateway to high product quality at the end of the day [9]. Process variability stands as a hurdle to process robustness, and this originates from lack of control on the process parameters. Thus, QbD steps-in to avoidbatch to batch variability in pharmaceutical products [10].

The net outcome of the detailed QbD study (applied in any product) is the segregation of process parameters with respect to their criticality and the finalization of a proven acceptable range (PAR) for every operation. The knowledge that is gained post the QbD evaluation encompasses every minute detail of the operational process as well as the product in general, and lead to the defining of a Design Space. This way, the impact that the manufacturing process might have with regard to the variability of the CQAs becomes apparent, which helps in strategizing testing, quality and monitoring of batches [11].

Process Evaluation: Linking Process Parameters to Quality Attributes

It is important to carefully evaluate the process completely before applying QbD to it. The better knowledge you have of the process, the more effective your QbD will be. Moreover, process characterization is required to specify the proven acceptable ranges (PAR) for critical process parameters (CPPs). In the traditional approach that is implemented in biopharmaceutical production, existing empirical process knowledge is used on a daily basis. However, this approach leads tolaborious and time consuming post approval changes during process adaptation and any new technology implementation that may have become necessary for raising the efficiency of the process. Also, the effects aprocess scale-up can have on the quality of the final product cannot be predicted when using the empirical process development.

This can increase costs and also can cause difficulty in implementing any changes in the set manufacturing process. Thus was born a way to achieve deeper understanding of processes which would lead to greater flexibility and freedom to effect changes. The concept of operation under a pre-defined design space gave this flexibility. Design space is nothing but a concept that is a part of the “Quality by Design” (QbD) paradigm. Now, manufacturers are to follow a science-based process development than their empirical counterpart.

The QbD Concept is Best Explained in this Flowchart Below

Define a Quality Target Product Profile (QTPP) for product performance

⇩

Identify its Critical Quality Attributes (CQAs)

⇩

Create experimental design (DoE)

⇩

Analysis done to understand the impact of Critical Process Parameters (CPP) on CQAs

⇩

Identify and control the sources of variability.

Process characterization sets the ball rolling in any process development, which employs a sound risk assessment rating the various critical process parameters according to their importance [12-14].

Downstream Processing in Biotransformation

Downstream processes of biopharmaceutical industry essentially include the following steps:

a) Harvesting

b) Isolation

c) Purification

Various unit operations that constitute any biopharmaceutical process follow a designed sequence to form an integrated process [15]. Thus, any change in any one of the one-unit operation can affect the functioning of the subsequent unit operations. This is the reason why interaction effects between participating parameters across unit operations should also be taken into account during the process development. Interactions are said to happen when setting of a parameter will show effect on the response of another parameter. Due to this dependence between the parameters, the combined effects of any two parameters hailing from different unit operations cannot be predicted from their individual effects. Regulatory authorities demand inclusion of interactions of parameters within the QbD approach during any process optimization [16]

Example: Downstream processing of 1, 3-propanediol

Process: Fermentation

Fermentation broth that uses flocculation, reactive extraction, and distillation was studied. Flocculation of soluble protein as well as cellular debris that were present in the broth was carried out by using optimal concentrations of chitosan (150 ppm) and polyacrylamide (70 ppm). It was seen that the soluble protein that was present in the broth decreased to 0.06 g L-1. Recovery ratio (supernatant liquor: broth was found to be greater than 99% (Figure 1) [17,18].

The above flowchart shows a typical fermentation process broken down in steps. Glycerol fermentation process is taken as example for the illustration [19].

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Case Study for API

API product development from the very nascent stages require a lot of planning when implementing QbD at every stage. Whether it is two-step process or a multi-step process, each and every operation and parameter needs to be scrutinized before creating a relevant design space. Brainstorming every possible roadblock that might threaten the quality of the final pharma product is what will help design a top-quality process. A futuristic vision is important in the initial steps of QbD planning. The most important part is to pay sufficient attention to detail lest critical aspects might be missed. This is best done by sitting with the entire development team and taking every minor detail into account. Given below is a case study for a API intermediate development process with the help of QbD that highlights the important steps as to how to go about implementing it from the very beginning of your research. QbD is done best, when it is implemented from the very nascent stage of product development.

Quality Target Product Profile

When making your QTPP, make sure you list down everything from your vendor details to target costing. This step basically asks you to think of every aspect of your product and make a comprehensive profile of it. The specification of quality must be highlighted here with all the challenging impurities that might threaten your quality. Everything from stability testing requirements to raw material quality [20] is encompassed in this stage of QbD.

CQA Determination

Given below are some typical CQA parameters that are considered in most of the enzymatic methods of API intermediate preparations.

a) Purity

b) Chiral purity

c) Enzyme residue

d) Assay

e) Appearance

f) Residual Solvent

g) Yield

h) Polymorphic forms

i) Moisture content

j) Melting point (Table 1)

Initial Risk Assessment

The risk assessment can be done in various ways and is the customizable step in QbD. This part calls for a group-discussion or a team meeting where everyone can list down all possible risks related to the project in discussion and grade each one in the list with the amount of risk that it poses. The simplest module suggests you number them 1, 2, 3 with the increasing or decreasing order of the risk threat. A more complicated and detailed risk assessment requires linking of CQAs and CPAs to highlight the risk of their interdependence (Figure 2) [21].

Post risk assessment, comes the control strategies to be followed to tackle the possible risks that are probable. The control strategies are for you to think and execute to achieve your target quality specifications.

Design of Experiment

This is a valuable tool for channelizing your experimental work, to move ahead in a systematic manner. Design of experiments can be of several types: comparative, screening, response surface modeling, and regression modeling [1].

Comparative Experiments: The aim of this study is simple, i.e., picking best out of two options. The selection can be done by the comparison data generated, which is the average of the sample of data.

Screening Experiments: If you want to zero-in on key factors affecting a response, screening experiments would be the best bet. For this, list down concise list of factors that might have critical effects on response that you desire. This model serves as preliminary analysis during development studies.

Response Surface Modeling: Once you have identified the critical factors that affect your desired response, response surface modeling comes handy to identify a target and/or minimize or maximize a response.

Regression Modeling: This is used to estimate the dependence of a response variable on the process inputs.

A step by step guide is given for the DoE step of the QbD process (Figure 3).

Response columns were filled post experimentation as per the design creation (Figure 4).

Factorial Design Analysis Done as Given Under

Analysis Done First for One of the Responses, “Yield”: (Table 2)

P-Values Were Checked for Significance and Higher P-Value Term Eliminated First to Create a Reduced Model:

(Table 3) (Figures 5 & 6)

Observation

From the above graph, significant interaction between the two terms can be inferred.

Analysis Done for the Response “Diacid”: (Table 4)

P-values Checked for Significance and Higher P-Value Term Eliminated First to Create a Reduced Model: (Table 5) (Figure 7)

Observation

From the above graph, significant interaction between the two terms can be inferred.

Response Optimizer Was Used to Optimize Both The Terms With Respective to The Given Responses- Yield and Diacid: (Table 6) (Figure 8)

The optimized parameters predicted for maximum yield and minimum impurity (of di-acid) was found to be 8pH and 37C.

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Case Study 2

As mentioned before, regression analysis is another important tool that can be used to study existing data. This means that if you have done some experiments (without designing them beforehand), you can quickly run a regression analysis of the collected data to derive a relationship between CPPs and the reaction results.

A lot of times, when one follows the one-factor-at-a-time optimization process, by the time any CPP is optimized, a lot of data stands generated. Instead of just tabulating the data and wasting time manually making sense out of them, regression analysis can come to your rescue. As always, graphical data representation seems much easier to understand and also saves your valuable time.

The effect of pH was studied [22] separately in the preparation of deoxynojirimycin base (stage III). The reaction involved N-formyl amino sorbitol, water, oxygen and whole cells of Gluconobacter oxydans DSM2003. Later involvement of sodium hydroxide and sodium borohydride gave rise to deoxynojirimycin. Further work-up and 2-methoxy ethanol facilitated crystallization yielded Deoxynojirimycin base. In this experiment, pH of the reaction was changed to find out its role during the reaction and a regression analysis was run using Minitab to study this affect.

Observations recorded showed that reaction did not occur at pH2 and at pH8, the reaction did not reach completion. The optimum pH range between 4 to 6 showed certain effect on yield and purity. The significance of pH variation during the reaction was thus established as described below (Graphs 1-3):

When null hypothesis p-test was carried out, no significant effect of pH was to be found on product purity, impurity1 and impurity2, but its significant influence was seen in minimizing impurity3.

Furthermore, large-scale batches conducted were statistically analyzed as well to achieve better understanding of the influence of list of parameters on the output obtained. The following parameters were studied during the stage III reaction described above:

a) pH, RPM and Oxygen cylinders consumed during the course of the reaction.

Their effect on the output and reaction completion time was studied. It was seen that only RPM showed statistically significant effect on the reaction completion time and rest of the factors did not contribute to any significant effect on the output or reaction completion time.

During biotransformation process, i.e. during oxidation of N-formyl using Gluconobacter oxydans DSM2003 whole cell, three main unknown impurities peaks were observed in HPLC chromatogram while reaction monitoring. This process is capable of removing these three impurities during down streaming, work up & isolation to the levels mentioned below:

a) Impurity 1 (has defined RRT on HPLC chromatogram) not more than 3%

b) Impurity 2 (any other unknown impurity) not more than 1%

c) Impurity 3 (has defined RRT on HPLC chromatogram) not more than 10 %

Since higher level of impurities affect the yield of the process, efforts were carried out to study the factors which can reduce the formation of process impurities.

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Conclusion

The concept of Quality by Design (QbD) is highly reliable when it comes to achieving foolproof quality of your product. This is a modern tool that is going viral in Pharmaceutical industry especially because this industry demands high quality standards and tolerates no compromise when it comes to the quality. Breaking down QbD, it essentially comes down to identifying the critical parameters of the process and assigning a particular design space for every single critical attribute. Thus, QbD can be considered as an intelligent approach to quality that yields robust processes. QbD also ensures that there is continuous improvement in the process during the entire lifecycle of a Pharmaceutical product [23].

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Acknowledgement

Our group would like to thank the Department of Scientific and Industrial Research India, Dr. Hari Babu (COO Mylan), Sanjeev Sethi (Chief Scientific Office Mylan Inc); Dr. Abhijit Deshmukh (Head of Global OSD Scientific Affairs); Dr. Yasir Rawjee {Head - Global API}, Dr. Sureshbabu Jayachandra (Head of Chemical Research); Dr. Suryanarayana Mulukutla (Head Analytical Dept MLL API R & D) as well as analytical development team of Mylan Laboratories Limited for their encouragement and support. We would also like to thank Dr. Narahari Ambati (AGC- India IP) & his Intellectual property team for their support.

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Dear Editor,

Communication, especially delivery of difficult news (DDN), remains a key part of clinical practice. Despite its importance, many medical providers lack the skill and ability to effectively DDN to their patients. Due to lack of data specific to sub-Saharan Africa and to help us develop an appropriate training tool for this geographical area, we sought to explore what challenges and barriers residents at our institution faced when they deliver difficult news to their patients.

Methodology

A qualitative approach using thematic analysis was adopted and a self-administered open ended survey was administered amongst residents currently doing their Masters in Medicine at the Aga Khan University Hospital in Nairobi and Dar es Salaam. The survey was emailed to all the residents within the two campuses using the REDCap software. Two independent researchers identified recurring themes and categorized barriers into either: physician, cultural or institutional. All residents provided written informed consent prior to participation in this study.

Results

A total of 105 residents participated in the survey (response rate 70%). The most frequent physician barriers identified were lack of training opportunities and time to DDN. More than fifty percent of the residents stated that cultural diversity, gender role-play and discussing dying as a taboo subject were the most common cultural barriers. Approximately 4 out of 10 residents faced institutional barriers such as lack of time due to busy schedules especially when on call, lack of private spaces to hold these conversations and limited mentorship/guidance by the faculty when having difficult conversations with patients.

Discussion

Physician barriers: Only 42.1% of the residents in our study had received any formal training in DDN. A study conducted in Nigeria looking at breaking bad news among nurses and physicians showed that only 21% of the respondents had formal training to do so [1]. Similarly, minimal formal training in conducting complex discussions were reported by health care providers in a Tanzanian health care facility [2]. In addition, our resident expressed lack of mentorship and support from faculty members in delivering difficult news to their patients.

Cultural barriers: Our residents felt that the diversity of cultures at our institutions greatly influenced the DDN to their patients, especially if different than their own. Similarly, Periyakoil and colleagues also found that many physicians struggle with end of life conversations especially in patients with a different ethnicity than their own [3]. Female residents at our institution were not taken seriously and struggled with delivery of difficult news compared to their male counterparts. Residents expressed that a majority of patients preferred a male relative be present during DDN. In addition, extended family members, rather than the female spouse, were involved in the decision making process. Our residents also expressed that talking about dying was often seen as taboo for many patients and their families. Further studies are needed to better understand how specific cultures affects effective communication especially in sub-Saharan Africa.

Institutional barriers: Similar to other studies, our residents reported a lack of adequate space and privacy when DDN to patients [2]. Opportunities supported by the institution to train and enhance communication skills were scarce. Similar to other studies, time constraint due to other clinical duties was also reported as a significant barrier [4].

Conclusion

Our study helps shed light on the barriers and challenges our residents face within our institution in delivering difficult news to their patients. This study will help us design and implement effective strategies, unique to sub-Saharan Africa, to better train our resident in DDN to their patients.

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In breast cancer great endeavors are going on to find new markers in determining the prognosis. In addition to well-known prognostic factors such as tumour size, grade, nodal status, molecular subtype, proliferation index, gene-expression based recurrence score, recently there is increasing evidence that tumor-infiltrating lymphocytes (TILs) are of important role as an immune biomarker in breast carcinoma [1-3]. TILs might be a reflection of host immune response which plays an important role in cancer control and patient’s clinical outcome. TILs are mononuclear immune cells that infiltrate tumour tissue and mainly comprised of cytotoxic (CD8+) T cells as well as macrophages, helper (CD4+) T cells, B cells, and NK cells [4,5]. There are two types of TILs: Stromal TILs (sTILs) are defined as the percentage of tumour stromal field in which lymphocytes are located without direct connection to tumour cells. In contrast, intratumoural TILs reflect immune cells within the tumour cells. Stromal TILs are commonly detected in H&E stained sections by light microscopy, and consist the majority of TILs in breast cancer [1]. Its assessment is the most reproducible parameter by pathologists. Moreover, lymphocytes-predominant breast cancer (LPBC) is defined as those cancer with TILs occupies at least 50 % to 60 % of the stroma [4].

The clinical importance and amount of TILs appear to vary significantly among different subtypes of breast cancer [2,6]. TILs are more common in triple-negative breast cancer (TNBC) and HER2-overexpressing breast cancer, with LPBC frequency of 15-25 % in these groups. Contrary, luminal tumour is the least immune infiltrated breast cancer subtype [3,6,7]. Large series suggest that tumour with higher TILs levels are associated with improvement of prognosis by longer survival rates and with reduction of locoregional recurrences [8,9]. This positive prognostic effect is especially striking on TNBC and HER2 positive patient populations [6,10]. In addition to prognostic value of TILs, trials also indicate the predictive role of TILs in response to neoadjuvant chemotherapy [11,12]. The aim of this study was to determine the association between TILs level and other clinicopathological parameters and to explore the prognostic and predictive roles of TILs in our breast cancer patients treated with neoadjuvant therapy.

Materials and Methods

The study was conducted to determine the association of TILs value with the clinical outcomes at neoadjuvant settings. Breast cancer patients treated after neoadjuvant chemotherapy at Bakırköy Sadi Konuk Training and Research Hospital from July 2011 to January 2016 were included in this study. All were followed up in our hospital. Patient’s demographic information, characteristics, outcomes and follow-up were obtained from hospital records. The study protocol was approved by the ethics committee of our institute (No:2017/09-20). Neoadjuvant therapy were taxane and anthracycline-based protocol, adding transtuzumab in the HER2 positive subtypes. Surgical treatment was decided as to treatment response. Radiotherapy and endocrine therapy were given in accordance with institutinal guidelines. Inoperable patients following neoadjuvant therapy were excluded of this study. The patients were categorized according to molecular subtypes as triple negative (TN), HER2 positive and luminal type. All patients in each subtypes were distributed into two groups according to TILs, with 10 % of cut-off value. In order to determine TILs value all patients’ paraffin sections stained hematoxilyn and eosin (H&E) from core biopsies were retrospectively assessed by an experienced pathologist (S.A.), blinded of clinical information. Briefly, all mononuclear cells (including lymphocytes and plasma cells) in the stromal compartment within the borders of invasive tumour were evaluated and reported as a percentage value. pCR was defined as ypT0/Tis ypN0.

We analysed associations of TILs with other clinicopathological factors such as menopausal status, tumour molecular type, ypTNM classification, histology, grade, lymphovascular and perineural invasion. In order to evaluate the predictive role of TILs, we investigated pathological responses to neoadjuvant therapy in high and low TILs groups of each subpopulation. To determine the prognostic role of TILs we recorded the patients’ locoregional recurrence and systemic metastasis in all groups and detected overall survival and disease free survivals. Statistical analysis was carried out using NCSS 11 package program. For categorical variables the frequency and percentage values were calculated and for continuous variables, average and standard derivation values were calculated. Standard distribution sort of variables was made with Kolmogorov-Smirnov test. Student t test was used in the comparison of two groups and single direction variance analysis was used in the comparison of more than two groups for variables exhibiting standard distribution. Disease-free survival (DFS) and overall survival (OS) were obtained using Kaplan-Meier survival analysis. The relations between categorical variables were examined with Chi square test, Fisher exact probability test and Fisher-Freeman-Halton test. P<0,05 was accepted as statistically significant.

Results

The study consisted of 89 breast cancer patients who underwent neoadjuvant chemotherapy. The mean follow-up duration of all patients was 33,8 (15-84) months. According to molecular subtypes, 39 patients had luminal tumour, and 25 patients each triple negative and HER2 positive.

TILs values in all groups and its relation with other clinicopathological factors

The mean TILs values were 10.8%, 15.4%, 6% in TN, HER2 positive and luminal groups, respectively. Among the patients of high TILs levels, HER 2 positive group was the most frequent molecular subtype (50 %), followed by triple negatives (32%) (Table 1). Sixty percent of patients with TILs less than 10 % had luminal tumour. There was statistically significant relationship between TILs value and both molecular type of tumour (p=0.0001) and presence of lymphovascular invasion (p=0.048). No association was detected between TILs and other variables such as menopausal status, ypTNM classification, histology, grade, and perineural invasion (Table 2).

TILs and prognosis

No association was seen in any subtypes between TILs levels and presence of either locoregional recurrence or systemic metastasis (Table 3). Neither OS nor DFS were significantly different between high and low TILs groups in both TN and HER2 positive groups. Because there was no death in luminal group, we could not detected survival analyses in this subtype.

Predictive role of TILs

In the neoadjuvant setting, no interaction was found between TILs level and complete pathological response according to each subtypes (Table 4). But, there was non-significantly better pathological response rates in high TILs group of HER2 (+) population.

Discussion

The constant interaction between tumour and host immunity influences patient’s outcome. Innate and adaptive immune response in the tumour milieu require functional cytotoxic T-cells in order to achieve efficent tumour elimination [1]. The impact of TILs on the prognosis and treatment prediction suggests its importance on the reflection of individual immunity [13]. Various studies of TN and HER2 (+) breast cancer have pointed consistent results that the increased TILs value the patients have, the better OS and DFS in the adjuvant setting [6,14]. Moreover, the highest TILs also show the highest expression of T-cell checkpoint receptors which found on T cells [1]. Antitumour immunity is enhanced by monoclonal antibodies against checkpoint inhibitors. In addition to standard therapeutic options, immunotherapy in breast cancer is a new and promising implication especially in selected patient population. Therefore, TILs evaluation may be accepted as a biomarker for immunotherapeutics in patients with breast cancer at near future.

In the light of these rapidly evolving field, in 2014 international TILs working group has recommended TILs evaluation as a immune parameter along with standard histopathological practise to facilitate its use in research setting and clinical trials [4]. They reported TILs measurement as a percentage of stromal TILs on a continuous scale by visual assessment of H&E-stained sections of primary tumour specimens similar to any other quantitative histological biomarker. Several studies have evaluated lymphocyte subtypes by using immunohistochemical analysis, yet added value of immunohistochemistry on to TILs evaluation remains to be demonstrated [4,15]. Moreover, digital image analysis of TILs can provide more exact measurement and eliminate inter-pathologists differences in TILs assessment [13]. Visual measurement of TILs is prefered at many trials like our study [4,6]. Because this route is stated as rapid, easly available, less expensive and adequate tool in TILs assessment. There is no established threshold for high TILs. Various studies used different cut-off values [7,9,16,17]. Moreover, 50-60 % of TILs value for lymphocytes-predominant breast cancer had been arbitrarly chosen [17]. In this study, ROC Curve was applied for the estimation of pathological response of TILs values, but a significant breakpoint could not be found (unreported data). Therefore, we accepted 10 % of TILs level as a cut-off value similar to many studies [6,18].

In our study, we investigated the relation of TILs value with other clinicopathological parameters and its impact on patients with all molecular subtypes of breast carcinoma who were operable following neoadjuvant treatment. The positive relation of higher quantities of TILs and both negative steroid receptor status and presence of HER2 positivity is striking in the literature like in our study [6]. Also LPBC is commonly found in HER2 positive and triple negative subtypes [4,19]. Similarly, in our study HER2 positive group was the main molecular subtype (50%) that harbours the high percentage of TILs. But, interestingly unique patient with LPBC was seen in our series. In this study, patients with luminal type breast cancer had lower TILs value, with the median of 6%. Similar to litreture, no significant prognostic role of TILs was found in patients of our luminal group [1]. According to the trial by Huszno [7] higher grades of TILs were present more frequently in younger patients than older women (47% vs. 24%). Moreover, higher TILs levels was appeared to be associated with higher histological grades. On the other hand, TILs value also was significantly correlated with lymphovascular invasion in our study.

The positive association between TILs levels and favourable clinical outcomes was based on Sistrunk’s cohort trial at 1922 [20]. Eastern Cooperative Oncology Group’s phase III trials showed that each 10 % increase in TILs levels significantly predicted better DFS and OS in TNBC with a median follow-up of over 10 years [21]. A recent meta-analysis of twenty-five trials including more than twenty thousands patients confirmed the survival benefit of TILs in TN and HER2 positive breast cancer patients [18]. Moreover, according to secondary analysis of the NeoALTTO trial reported by Salgado [17] patients with high TILs levels at baseline had better outcomes for event free survival independent of whether they achieved pCR in the neoadjuvant phase. The association between higher levels of TILs and decreased distant recurrence rates in TN breast cancer was found in FinHER trial [22].

Contrary to obvious results as to prognostic impact of higher TILs levels on the adjuvant and neoadjuvant settings, we could not detected any significant relationship between TILs and survival analysis. Another issue related to TILs is its utility in prediction of adjuvant and neoadjuvant therapy response [6]. At the adjuvant setting, the association between higher levels of TILs and increased transtuzumab benefit in HER2 (+) subtype was detected in phase III trial including 1010 early stage BC patients [22]. Positive correlation between TILs levels and pCR rates was prominent in HER2 (+) tumours. According to the study of Carbognin [12] pCR rates were increased by nearly 30 % in the presence of TILs reported as LPBC. Another trial has also indicated that the level of TILs greater than 5% was associated with higher pCR rates [17]. Although we could not detected significant correlation between TILs value and pCR in our study, there were non-significantly higher pCR rates in HER2 (+) group. The minority of our patient population explains the limited power of the statistical analysis.

Conclusion

Host immunity controls cancer and influences patient’s outcome. How can a host enhance anti-tumor immune response and which biomarker provides better treatment approach are the main questions. Although which level of TILs should be used to determine optimal treatment strategy for breast cancer patients has not been certain yet, many trials indicate that TILs is of prognostic effect in long-term disease control and predictive effect of a better local response to treatment. But, further resaerchs are warrented before the utility of TILs as an immune biomarker for routine clinical practise of breast cancer patients.

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One in a thousand people have an acute gastrointestinal (GI) hemorrhage per year [1]. There are around 300,000 hospitalizations for GI bleeds, costing an estimated $2 billion per year [2-4]. Compared to lower gastrointestinal bleeding (LGIB), upper gastrointestinal bleeding (UGIB) is associated with a much higher mortality rate, with some studies suggesting a 30-day mortality rate of up to 14% [2,3]. A majority of these UGIB (67 - 80%) are attributed to gastric erosions/ulcers 6,17,18. However, of this morbid group of bleeds, a rare (1% or less), yet more serious cause is a Dieulafoy’s lesion (DFL). DFL, is an obscure type of bleeding that can cause life threatening hemorrhages with a mortality rate ranging from 28-67% [5,6].

DFL was first described by MT Gallard [7] in 1884 as a type of aneurysm and later clarified by P. G. Dieulafoy in 1898 who believed this was an early stage of ulceration [7-9]. DFL’s are a collection of large tortuous arterioles of the gastrointestinal vessels. These are often compared to aneurysms, however, DFL’s are caused by genetic malformations rather than degeneration. While the exact mechanism of rupture and subsequent hemorrhage is still poorly understood, several studies have suggested mucosal erosion and ischemic injury, related to aging and/or cardiovascular disease, as possible causes [10]. DFLs are predominantly seen in elderly patients (mean age 69.7 years) though they can be seen in younger patients as well. A vast majority of patients also will have underlying comorbidities such as renal failure, diabetes, or coronary artery disease. Additionally, there have been a few isolated cases associated with chronic immunosuppression whether from underlying malignancies or medication induced [11].

More than 70% of these rare lesions are found in the stomach, usually near the lesser curvature. The discovery of extragastric DFL’s are infrequent, with the duodenum (14%) and colon (5%) being the most common locations [12-14]. The most unusual site is the jejunum, which accounts for 1% of all DFL’s [12-14]. Historically, there have been a few case reports worldwide of jejunal DFL’s, however, of these reported cases, the lesions were found by advanced imaging (CT angiogram or Bleeding scan). We present a case of a jejunal DFL that was unable to be found by advanced imaging but was diagnosed on push enteroscopy.

Assessment

The patient was a 79-year-old African American female with a known history of end stage renal disease and large granular lymphocytic leukemia. Over the course of 8 months, the patient had 3 admissions for gastrointestinal bleeds. She received a total of 16 units of blood with 4 EGDs, 2 colonoscopies, 1 bleeding scan and 1 CT angiogram. Of the listed procedures performed, all were negative for active bleeding and there was no solid evidence of a bleeding source. She was presumed to have bled twice from erosive gastritis and the most recent admission was from an unknown etiology. An outpatient small bowel capsule study was done after these admissions showing no findings.

She subsequently presented to the ER 24 days after her most recent admission for melena and orthostasis, where she was found to have a hemoglobin of 4.4 g/dL (previous hemoglobin was 10.2 g/dL). EGD and colonoscopy did not show any findings or source of bleeding. The patient continued to have melena and required 8 units of blood. A CT angiogram and bleeding scan were again inconclusive. A push enteroscopy was performed on day 6 of admission, showing an actively oozing area in the jejunum with no surrounding ulceration or malformations (Figure 1). Bipolar cauterization and 2 hemoclips were placed ceasing the actively bleeding Dieulafoy’s lesion. The patient’s hemoglobin stabilized, and the melena resolved 1 day after the enteroscopy.

Management

Our patient had a history of large granulocytic lymphocytic leukemia (LGL) and end stage renal disease. Though the usual course of LGL presents with neutropenia and anemia, thrombocytopenia can be seen in up to 20% of cases [15]. Thrombocytopenia combined with immunosuppression from underlying malignancy and ESRD put this patient at an increased risk of developing hemorrhagic complications such as DFLs. On multiple admissions, our patient was found to pancytopenia, likely as a result of her bleeding and immunosuppression. To date, there have been a few case reports citing immunosuppression, immunotherapy, and thrombocytopenia all being associated with GI bleeds due to a DFL [16,17]. While the mechanism is not yet established, it is thought to be related to impaired tissue remodeling and repair.

The current endoscopic modalities to manage a DFL include mechanical treatment (with endoclips or band ligators), injection therapy (with diluted epinephrine), thermal coagulation therapy, or in some cases, a combination of different modalities. Two controlled trials suggested that mechanical hemostasis with endoclips can control acute bleeding and may reduce recurrent bleeds compared to injection therapy alone [18]. There have been no studies comparing the efficacy of thermal coagulation alone or in combination with other methods. A second trial comparing endoclips to injection therapy with epinephrine showed equal rates of initial hemostasis but significantly lower rates of rebreeding in the endoclip arm (0%) vs epinephrine arm (35%) [19].

Post endoscopic management depends on whether the patient is at high risk or low risk for rebleeding. One method used to calculate risk of recurrence is the Glasgow - Blatchford bleeding score (GBS score). A GBS score of 0-1 is considered low risk for rebleeding, and in this case the patient may be discharged from inpatient care with plans for outpatient endoscopic intervention. Inpatient treatment is recommended for patients with GBS scores of 2 or greater [20]. The rate of recurrence of bleed for a Dieulafoy’s lesion ranges from 9-40% [21]. There is a higher rate of recurrence with endoscopic monotherapy compared to combined endoscopic interventions [22]. The rate of rebleeding is not associated with gender or location of DFL or past medical history [23]. Inpatient treatment recommendation is to treat with IV pantoprazole for 72 hours in order to keep gastric pH above 6 in order to maintain intact coagulation process, followed by oral pantoprazole therapy. While mortality is lowest in patients with no significant medical history or comorbidities, overall longterm prognosis of a DFL is favorable once primary hemostasis is achieved [24]. Following push enteroscopy our patient did well without any further complications or signs of rebleeding [25-30].

Conflict of Interest

All authors have read and approved the submission of this manuscript. The manuscript has not been published and is not being considered for publication elsewhere, in whole or in part. The authors declare that there is no conflict of interests regarding the publication of this paper

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One in a thousand people have an acute gastrointestinal (GI) hemorrhage per year [1]. There are around 300,000 hospitalizations for GI bleeds, costing an estimated $2 billion per year [2-4]. Compared to lower gastrointestinal bleeding (LGIB), upper gastrointestinal bleeding (UGIB) is associated with a much higher mortality rate, with some studies suggesting a 30-day mortality rate of up to 14% [2,3]. A majority of these UGIB (67 - 80%) are attributed to gastric erosions/ulcers 6,17,18. However, of this morbid group of bleeds, a rare (1% or less), yet more serious cause is a Dieulafoy’s lesion (DFL). DFL, is an obscure type of bleeding that can cause life threatening hemorrhages with a mortality rate ranging from 28-67% [5,6].

DFL was first described by MT Gallard [7] in 1884 as a type of aneurysm and later clarified by P. G. Dieulafoy in 1898 who believed this was an early stage of ulceration [7-9]. DFL’s are a collection of large tortuous arterioles of the gastrointestinal vessels. These are often compared to aneurysms, however, DFL’s are caused by genetic malformations rather than degeneration. While the exact mechanism of rupture and subsequent hemorrhage is still poorly understood, several studies have suggested mucosal erosion and ischemic injury, related to aging and/or cardiovascular disease, as possible causes [10]. DFLs are predominantly seen in elderly patients (mean age 69.7 years) though they can be seen in younger patients as well. A vast majority of patients also will have underlying comorbidities such as renal failure, diabetes, or coronary artery disease. Additionally, there have been a few isolated cases associated with chronic immunosuppression whether from underlying malignancies or medication induced [11].

More than 70% of these rare lesions are found in the stomach, usually near the lesser curvature. The discovery of extragastric DFL’s are infrequent, with the duodenum (14%) and colon (5%) being the most common locations [12-14]. The most unusual site is the jejunum, which accounts for 1% of all DFL’s [12-14]. Historically, there have been a few case reports worldwide of jejunal DFL’s, however, of these reported cases, the lesions were found by advanced imaging (CT angiogram or Bleeding scan). We present a case of a jejunal DFL that was unable to be found by advanced imaging but was diagnosed on push enteroscopy.

Assessment

The patient was a 79-year-old African American female with a known history of end stage renal disease and large granular lymphocytic leukemia. Over the course of 8 months, the patient had 3 admissions for gastrointestinal bleeds. She received a total of 16 units of blood with 4 EGDs, 2 colonoscopies, 1 bleeding scan and 1 CT angiogram. Of the listed procedures performed, all were negative for active bleeding and there was no solid evidence of a bleeding source. She was presumed to have bled twice from erosive gastritis and the most recent admission was from an unknown etiology. An outpatient small bowel capsule study was done after these admissions showing no findings.

She subsequently presented to the ER 24 days after her most recent admission for melena and orthostasis, where she was found to have a hemoglobin of 4.4 g/dL (previous hemoglobin was 10.2 g/dL). EGD and colonoscopy did not show any findings or source of bleeding. The patient continued to have melena and required 8 units of blood. A CT angiogram and bleeding scan were again inconclusive. A push enteroscopy was performed on day 6 of admission, showing an actively oozing area in the jejunum with no surrounding ulceration or malformations (Figure 1). Bipolar cauterization and 2 hemoclips were placed ceasing the actively bleeding Dieulafoy’s lesion. The patient’s hemoglobin stabilized, and the melena resolved 1 day after the enteroscopy.

Management

Our patient had a history of large granulocytic lymphocytic leukemia (LGL) and end stage renal disease. Though the usual course of LGL presents with neutropenia and anemia, thrombocytopenia can be seen in up to 20% of cases [15]. Thrombocytopenia combined with immunosuppression from underlying malignancy and ESRD put this patient at an increased risk of developing hemorrhagic complications such as DFLs. On multiple admissions, our patient was found to pancytopenia, likely as a result of her bleeding and immunosuppression. To date, there have been a few case reports citing immunosuppression, immunotherapy, and thrombocytopenia all being associated with GI bleeds due to a DFL [16,17]. While the mechanism is not yet established, it is thought to be related to impaired tissue remodeling and repair.

The current endoscopic modalities to manage a DFL include mechanical treatment (with endoclips or band ligators), injection therapy (with diluted epinephrine), thermal coagulation therapy, or in some cases, a combination of different modalities. Two controlled trials suggested that mechanical hemostasis with endoclips can control acute bleeding and may reduce recurrent bleeds compared to injection therapy alone [18]. There have been no studies comparing the efficacy of thermal coagulation alone or in combination with other methods. A second trial comparing endoclips to injection therapy with epinephrine showed equal rates of initial hemostasis but significantly lower rates of rebreeding in the endoclip arm (0%) vs epinephrine arm (35%) [19].

Post endoscopic management depends on whether the patient is at high risk or low risk for rebleeding. One method used to calculate risk of recurrence is the Glasgow - Blatchford bleeding score (GBS score). A GBS score of 0-1 is considered low risk for rebleeding, and in this case the patient may be discharged from inpatient care with plans for outpatient endoscopic intervention. Inpatient treatment is recommended for patients with GBS scores of 2 or greater [20]. The rate of recurrence of bleed for a Dieulafoy’s lesion ranges from 9-40% [21]. There is a higher rate of recurrence with endoscopic monotherapy compared to combined endoscopic interventions [22]. The rate of rebleeding is not associated with gender or location of DFL or past medical history [23]. Inpatient treatment recommendation is to treat with IV pantoprazole for 72 hours in order to keep gastric pH above 6 in order to maintain intact coagulation process, followed by oral pantoprazole therapy. While mortality is lowest in patients with no significant medical history or comorbidities, overall longterm prognosis of a DFL is favorable once primary hemostasis is achieved [24]. Following push enteroscopy our patient did well without any further complications or signs of rebleeding [25-30].

Conflict of Interest

All authors have read and approved the submission of this manuscript. The manuscript has not been published and is not being considered for publication elsewhere, in whole or in part. The authors declare that there is no conflict of interests regarding the publication of this paper

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A Case Report on a Rare Cervical Polyp

Authored by Ayman Aboda

Abstract

Background: Giant cervical polyps are rare. They are commonly asymptomatic and diagnosed incidentally on pelvic examination.

Case: This is a 59-year-old multiparous who had a giant cervical polyp diagnosed on speculum examination after presenting to her general practitioner reporting symptoms of pelvic pressure and heaviness. The polyp was successfully resected with hysteroscopy.

Conclusion: A giant cervical polyp in a multiparous female which was successfully resected during hysteroscopy.

Keywords: Giant cervical polyps; Gynecologic pelvic examination; Hysteroscopy; Submucous leiomyoma; Pelvic pressure; Vaginal resection

Introduction

Giant cervical polyps are rarely seen in routine clinical practice. They are defined as being greater than 4 cm in size [1]. Giant cervical polyps protruding outside the vaginal canal can cause diagnostic confusion. They are rarely encountered in gynecologic practice. They are uncommon though can appear in all age groups and in both nulliparous and multiparous women. The incidence is 4-10%. They are most commonly asymptomatic, discovered incidentally at routine gynecologic pelvic examination. Cervical polyps originate most commonly from the ectocervix and are called cervical polyps. More rarely, they arise from the endocervical canal and are called endocervical polyps. Most are less than 2cm in diameter.

Case Presentation

This case report is about a giant cervical polyp in a 59-year-old female, gravida 4 para 3 with three previous normal vaginal deliveries. Her menarche was at the age of 16 and her menstrual interval was 28-30 days with a duration of 3 days. She was menopausal at the age of 48. She became aware of a lump in her vagina 5-6 months prior to presentation to her general practitioner (GP). She reported no vaginal or post coital bleeding and had not been sexually active for some time. The sensation of the lump was most noted when both walking and standing however there were no concerns with bowel or urinary function. She never had cervical screening. She was otherwise in good health apart from hypertension not requiring medications. Her mother had ovarian cancer but there was no other significant family history.

Her GP noted a cervical polyp protruding from the cervix and extending to the introitus. Clear views of the cervix were not possible and cervical screening though attempted, could not be successfully completed. The patient was referred to our service. On speculum examination, a large polyp was again visualized and noted to obstruct clear views of the cervix. Differential diagnosis included cervical polyp or prolapsed fibroid. She was booked for diagnostic examination under anesthesia with hysteroscopy and vaginal resection if possible.

At the procedure, a polyp extending 5 cm beyond the external cervical os was noted, a pedicle was located within the endocervical canal. The cervix was dilated to Hagar number 10 followed by hysteroscopic resection with tissue sent to histopathology. Cervical screening test was completed at the same time. The patient recovered easily and was discharged safe and well the same day.

The histopathology reported a tissue measuring 50x35x25mm with no atypia, necrosis or significant mitotic activity. The conclusion was that the cervical polyp was a submucous leiomyoma. The cervical screening test was negative with recommendation for rescreening in five years.

Discussion

Giant cervical polyps are rare. In this case, the polyp was a fibroid or leiomyoma which had arisen from the endocervix. This is a rare presentation occurring in only 0.6% of cases where pathology has been identified.

Classification of fibroid is according to the involvement of uterine wall, being intramural, sub-serosa and transmural [2]. Cervical fibroids are grouped as type 8, an additional category for leiomyomas not related to myometrium [2].

Diagnosis of fibroid usually start with pelvic examination followed by confirmation on ultrasound which is the gold standard test [3]. MRI could be used to further assess the fibroids to provide information on size, vascularity, location in relation to endometrial cavity and serosa [3]. Hysteroscopy is used to differentiate myoma with polyps, and sometimes an endometrial biopsy is performed at the same time [3]. No radiological investigation was used in this case which is not common. The lesion in this case was intracervical which was found in 22% of the patients in a recently published systematic review [4].

Management of fibroid usually depends on symptoms and preference on fertility preservation. The above-mentioned systematic review on cervical leiomyoma showed that they are most commonly associated with symptoms of abnormal uterine bleeding (44%), followed by volume-related symptoms (20%) such as pressure or the sensation of a lump. Chronic pelvic or lower back pain (14.6%) and chronic urinary complaints (11%) may also occur [4]. Only 7.7% of the patients were asymptomatic [4]. The patient described in this case report presented with bulk-related symptoms only with no other related concerns.

Medical management for symptom relief may include progesterone receptor modulators, tranexamic acid and gonadotropin-releasing hormone (GnRH) agonist [5]. These tend however to be limited by the duration of treatment, with symptoms often returning once intervention has ceased. Surgical treatment may be more successful in providing long term improvement. Procedures include myomectomy, embolization, hysterectomy and hysteroscopic myomectomy [5,6]. Most patients with cervical leiomyoma undergo surgical treatment (88%) with myomectomy, hysterectomy or trachelectomy. In the case described, fertility preservation was not an issue. It was still important however to provide surgical management in the least provocative manner and thus safeguard against risk of iatrogenic morbidity. Hysteroscopic resection was thus successfully performed. There are no citations in the literature reporting similar success in patients presenting in this manner [4].

Conclusion

In conclusion, this was a case of a patient with finding of a large cervical fibroid with mass-related symptoms successfully treated with minimally intrusive, hysteroscopic resection.

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An Uncommon Localization of a Hydatid Cyst Presenting with Pulmonary Embolism

Authored by Francesco Finizola

Abstract

Human echinococcosis is an important infection in undeveloped and developing countries, caused by larval forms of the genus Echinococcus. In western countries this disease is sporadic. The organs mostly involved by the cysts are the liver (70%) and the lungs (20%) [1]. Cardiac hydatid cyst is a rare condition, and the location of a hydatid cyst in the interventricular septum is exceptional. Cardiac echinococcosis is not frequent, only 0.01%-2% of all hydatid infestations [2]. The left ventricle is the heart chamber most frequently involved (55-60%) and the involvement of the interventricular septum is reported in 4% of cardiac cases. Chest pain, palpitations, and dyspnea are the most frequent symptoms associated with cardiac echinococcosis. We describe a case of an unusual presentation of this disease.

Keywords: Echocardiography; Interventricular septum; Hydatid cyst; Echinococcosis

Case Report

A 39-year-old Caucasian female was admitted to our department for the evaluation of syncope and dyspnea. Two years before, the patient presented with an abdominal pain. An abdominal echography revealed the presence of two liver hydatic cyst. Treatment with Albendazole was undertaken for one year with improvement of symptoms. Physical and neurological examination revealed nothing unusual and routine laboratory tests were normal. Chest CT scan showed pulmonary embolism involving principal arteries and a mass in the right ventricle, initially interpreted as a tumor or blood clot. A transthoracic echocardiography was done and revealed the presence of a 40x25mm cystic mass in the apical part of interventricular septum with protrusion to the right ventricle cavity. ELISA test was positive for echinococcosis antibodies. Albendazole was started for 6 months with an improvement of pulmonary symptoms. Then,cystectomy was performed and the remaining cyst contents and germinative membrane were removed. Histopathological exam of cystic material confirmed the diagnosis of hydatid cyst. The postoperative period was uneventful, and the patient was discharged from the hospital.

Discussion

Echinococcosis or hydatid disease is caused by larval stage of Echinococcus granulosus. Humans are only intermediate and accidental hosts. ELISA is the most specific serologic tests that can be used and a positive result for echinococcus antibodies confirms the diagnosis. Cardiac hydatidosis is a rare condition potentially fatal or leading to severe complications without treatment being undertaken. The most common cardiac locations are left ventricular wall (60%), right ventricle (10%), pericardium (7%), left atrium (6-8%), right atrium (4%), and the interventricular septum (4%). Right ventricle is rarely involved and cysts are more prone to rupture, as compared to cysts on the left ventricle. This condition may lead to pulmonary embolization, a lethal complication. Such a condition should be taken into consideration as other complications (arrhythmia, angina, dysfunction of valvular and ventricular functions) may initially give the same symptoms. Echocardiography is the most efficient method in the diagnosis of cardiac hydatid cyst [3]. CT scan and MRI can be used to detect the involvement of other organs, but they have a low sensitivity and a low sensitivity in the diagnosis of intra-ventricular hydatidosis. Transesophageal ecography can provide a more accurate definition of the mass. Surgical excision with removing germinal layer and albendazole therapy showed successful results in treating cardiac hydatid cysts [4] (Figure A-C).

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