Received date: May 17, 2016; Accepted date: June 28, 2016; Published date: Jun 30, 2016
Citation: Franzén B, Sennerstam R, Wiksell H, Auer G (2016) Significance of Diagnostic Needle Biopsy for the Development of Inflammation, Tumour Progression and Metastasis. J Mol Biomark Diagn S2:021. doi:10.4172/2155-9929.S2-021
Copyright: © 2016 Franzén B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
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Bo Franzén, Roland Sennerstam, Hans Wiksell, and Gert Auer
Tumours; Fine needle aspiration biopsy; Breast cancer; Core needle biopsy; Surgical biopsy; Cytokine IL-6
In the present review we use breast cancer as a model in discussing biopsy techniques, wounding, inflammation and tumour cell seeding. Breast cancer is the most prevalent female cancer worldwide and despite decades of intense research and development of various therapies, the long term survival in breast cancer has not improved radically .
Therefore, there is a valid reason to once again carefully examine all components of the chain from early detection and diagnosis to treatment and relapse after a long follow up period to examine in detail what can be improved. Diagnostic sampling is an important step of this chain, whose role we do not pay enough attention and which is the main focus of the review presented. It is of course of outmost importance to obtain an accurate and objective analysis of any suspicious lesion in order to generate the best support for the selection of treatment. This is not possible today without a cell or tissue biopsy.
The main question we aim to scrutinize is how the biopsy currently is obtained, if there are any potential risks associated with the procedure, and if so - what can be done to improve current practices and to minimize the risk for complications and at worst distant seeding of cancer cells.
Open surgical biopsy can be subdivided into incisional and excisional biopsy. Incisional biopsy means removal of a part of a suspicious lesion for histological diagnosis, whereas excisional surgical biopsy means intended removal of the entire lesion. Because of the globally rapidly expanding introduction of diagnostic CNB techniques surgical biopsy, especially incisional biopsy is used at a decreased rate. The extension of tissue trauma caused by incisional surgical biopsy is mainly related to the size of the removed tissue and is significantly larger than that of needle biopsy. Complications due to incisional biopsy, that has been the prevailing method in China, has resulted in an increased use of CNB to avoid unnecessarily high frequency of surgery associated problems .
Fine needle aspiration biopsy (FNAB)
Fine needles range from 0.4 to 0.7 mm (26-21 gauge) outer diameter and lengths vary from 1 cm (sampling of e.g. skin lesions) to 20 cm (sampling of e.g. prostate lesions). During several decades, the onehanded syringe pistol handle for 10 to 20 ml disposable plastic syringes designed in the 1960s by Sixten Franzén is generally in use . The course of action regarding aspiration of cellular material from solid lesions varies significantly dependent on configuration and firmness of the lesion and the experience and skill of the investigator . When the needle is inserted into the lesion, the investigator guides the needle in average ten times back and forth through the lesion. The total number of sampling procedures varies but ranges in average between two and five. Palpable lesions are generally biopsied directly whereas non-palpable lesions are biopsied guided with help of various modalities of imaging techniques e.g. ultrasound, stereotactic devices and MRI . The advantages of FNAB is that the sampling procedure causes a minimum of tissue trauma (explained by the diminutive amount of absorbed energy), is extremely patient friendly and that conclusive diagnostic information may be obtained within less than half an hour performing quick staining of the aspirated cells on cytological smears. The main disadvantages are that the method does not produce a similar level of diagnostic results as compared to CNB  and that diagnostic accuracy is highly related to the proficiency of an experienced cytopathologist. Since many years, the lack of experienced cytopathologists hamper the utilization of FNAB. On the other hand, development of ultrasensitive molecular analysis may increase future FNAB based diagnostics. This opportunity will be discussed below.
Core needle biopsy (CNB)
In core needle biopsy technique, larger hollow needles are used compared to FNAB. The outer diameter of CNB needles ranges generally from 1.3 to more than 3 mm. The sampling instrument is an automated cutting biopsy gun with a spring-loaded sampling device . The total number of sampling procedures varies but ranges in average between three and ten, or more, depending on the size of the lesion. Local anaesthesia is generally used and a little cut in the skin has to be made when sampling from e.g. breast lesions is performed. Vacuum-assisted biopsy (VAB) is frequently used in connection to CNB technique in breast diagnostics and reduces the ballistic springloaded devise effect allowing multiple samplings with only one insertion of the needle . After insertion by VAB the needle will stay almost at the same place but is rotated during the biopsy procedure and the created low pressure will suck tissue material into the needle. This biopsy technique was introduced in the early 1990s and the main advantage of CNB based diagnostics is the ability to sample a sufficient amount of tissue for histological analysis which improves diagnostic accuracy including the differentiation between in situ and invasive cancer. Correct diagnosis compared to pathology analysis after surgery was significantly higher for 8 gauge VA-CNB as compared to 11 gauge regarding benign lesions and VAB reduces the side effect of higher energy used by 8 gauge CNB . Generally, the amount of energy density, the time peak value including the final pressure re-balancing and even cavitation due to ballistic effects can be important factors regarding development of tissue trauma and tumour cell dissemination. 
Potential physical problems associated with current performance of CNB
The potential problems associated with needle biopsy have been debated for a long period of time. One meta-study concludes that no general increase of distant metastasis can be found after CNB . In breast cancer, despite frequently observed cell displacement, no increased morbidity has been observed after CNB . However, very few studies have been designed to exclude the possibility of CNB induced metastasis and strategies have been suggested to prevent tumour cell seeding .
Among the drawbacks of CNB are frequently observed widespread tissue trauma and the difficulty of precision placement of the needle which is a well-recognized disadvantage of core needles equipped with spring-loaded devices. Usually, the core needle is inserted using a spring loaded-device at an accelerating speed of 4-10 meters per second. With high needle velocity several complex phenomena might occur, such as standing wave formation, micro-streaming, energy conversion, pumping effects and time for balancing including transports. In analogy with ballistics of a bullet from a gun, the temporary cavity-channel produced will be considerably larger than the diameter of the needle and the kinetic energy induced by the shot must to a great amount be absorbed by the tissues . In addition, an increased number of shots, sometimes up to about twenty, correlate with an increased frequency of clinical complications .
Therefore, it is likely to assume that the total extent of tissue trauma is related to the deposed energy, which in turn is proportional to the number of shots and the square of both needle velocity and needle diameter.
Finally, damaged blood vessels will cause a semi-static lesion pressure which might include a significant volume having a pressure (in the order of 120 mmHg) which in turn can cause emission of blood and cellular material during the low blood pressure phases, until the pressure is again balanced. These factors are likely to play a crucial role behind the observation that tumour cell displacement is detected in more than one third of the patients after CNB . In contrast to CNB, the diameter of the FNA needle is ~2-5 fold smaller and the average needle velocity is >100 fold slower, and thus offers an attractive, minimal trauma technology for sampling of tumour material.
Potential risks and consequences followed by CNB
Numerous reports confirm needle tract seeding after biopsy, a complication which may be avoided to some extent by applying appropriate precautions . A reasonable consequence of needle tract seeding would be a transient increase of circulating tumour cells (CTC) after CNB. However, few reports have studied this in detail by paired testing, i.e. examination of each patient before and after biopsy. One study reported a significant increase of CTC markers detected by reverse-transcriptase polymerase chain reaction (RT-PCR) after colon biopsy . Haematogenous dissemination of epithelial cellular markers has also been observed only minutes after prostate biopsy .
An increase of CTC should be taken serious and several reports show that the concentration of CTC may be used as predictors of metastatic progression and treatment efficacy . When CTC survive circulation, they may finally reach the bone marrow as disseminated tumour cells, where they may enter a dormant state and escape chemotherapy . It is known that premalignant tumour cells can disseminate and remain dormant for long periods of time and after long time give rise to metastases . It has been reported that a majority (~62%) of breast cancer patients get recurrences several years after the 5-year survival mark, and do not survive . The biology of minimal residual cancer cells and cancer cell dormancy is an emerging area of research and there are many questions waiting for answers. Meanwhile, we may review current biopsy routines to find strategies for the study of biopsy associated complications, with the aim to minimize the number of dormant cancer cells displaced at distant sites.
Mouse models for studies of biopsy associated complications
Mouse models may provide important clues on the possible mechanisms underlying tumour cell dissemination and potential metastasis associated with biopsy. Using a nude mice model, various tumour tissue manipulations were tested, including compression, biopsy, laser treatment and complete resection. It was shown that biopsy caused a ~60-fold increased release of CTC . In contrast, compression causes only a minor release of CTC and therefore the magnitude of CTC released will be one important parameter to record when biopsy methods are compared.
Similar results were obtained using a breast cancer BALB/c mouse model. Mathenge et al. recently showed that CNB not only cause a >3- fold increase of CTC but also enhances the frequency of lung metastasis . In addition, immune cell populations and molecular markers were monitored at various time points after biopsy. After 3 days, a significant decrease of macrophages, CD4+ and CD8+ T-cells were observed, paralleled by an increase of myeloid-derived suppressor cells in the tumour tissue. Already after 3 hours post biopsy, TGF+/ SOX4 associated epithelial-mesenchymal transition (EMT) markers and pro-inflammatory factors like e.g. CXCL2, CCL3, S100A8 and COX2 were increased followed by an increase of TNFα (after 24 hours). Taken together, data presented in this study show that CNB causes an acute inflammatory response followed by tumour infiltration, immune suppression, EMT, CTC release and finally lung metastasis.
An acute inflammatory response induced by 2 mm punch biopsy needles has also been shown by Hobson et al. using the MMTV-PyMT mouse model . In this study, the increase of lung metastasis was followed by an increase of the pro-inflammatory cytokine IL-6. Interestingly, IL-6 knock-out animals did not show an0 increased number of lung metastasis after biopsy and animals receiving antiinflammatory Ibuprofen® treatment pre- and post-biopsy, showed a decreased number of lung metastasis.
Microenvironment, inflammation, tumour progression and metastasis
The important role of tumour microenvironment and inflammation are emerging areas of research  and the tumour promoting role of inflammation in cancer is today well established. Moreover, the inflammatory microenvironment and hypoxia has also been shown to be a driving force in the increase of mutations and genetic instability . Inflammatory breast cancer is very aggressive and the prognosis is poor despite advanced therapy . Interestingly, high levels of CTC which is frequently observed in inflammatory breast cancer, can be reduced radically after treatment by statins, which has an antiinflammatory effect . An increasing number of studies have confirmed that aspirin and non-steroidal anti-inflammatory drugs are associated with improved breast cancer survival . Some of the results from the MMTV-PyMT mouse model discussed above are thereby supported by clinical observations.
Clinical relevance of possible complications after CNB
Taken together, tissue trauma including cell damage, cell translocation, blood vessel destruction and acute inflammation will inevitably have detrimental consequences for the tumour microenvironment. With the background of recent research on mouse models after CNB and increased understanding of the role of tumour microenvironment has to be studied in clinical investigations. Why is there so far no strong evidence regarding complications of CNB in breast cancer.
In prostate cancer diagnostics, several complications have been recorded as a consequence of transrectal CNB. However, the focus has primarily been oriented towards complications associated to bacterial infections . In breast cancer, inflammatory reactions have been observed to support tumour growth and supply tumour stem cells to survive treatment by natural selection . An important factor in the immunological response to tissue wound is the macrophage polarization from M1 to M2, which may accelerate tumour progression as observed in oral squamous cell carcinoma. . Weber et al. conclude in this report that M2 polarisation is a consequence of preoperative tumour biopsies.
Preclinical studies in mouse models support the concept that acute inflammation caused by clinical interventions plays an important role regarding the risk of peripheral metastases. This has been studied in e review using mouse models and hence, biopsy ‘wounding’ induced risk of metastases . However, this devastating side effect may be reduced by prophylactic administration of anti-inflammatory medication (e.g. ibuprofen) at the time of the biopsy .
The way forward: Time to upgrade how we view and do diagnostic biopsy
In spite of the fact that numerous studies have shown that diagnostic biopsy cell- or tissue sampling from solid malignancies is associated with increased risk of tumour cell dissemination and also local and/or distant tumour growth. Tåissue traumatic biopsy methods are still globally used in order to receive a final diagnosis. The main reason is the need of tissue based morphologic, immune histochemical and molecular marker based diagnostic analyses in order to be able to make tumour specific treatment decisions. Another decisive reason is the difficulty to differentiate between spontaneous and biopsy related tumour cell dissemination, the influence of time, type of treatment and grade of malignancy of the tumour. However, in spite of remaining uncertainties it is generally accepted that biopsy related tumour cell dissemination may be observed in highly aggressive cancer types, e.g. malignant melanoma, ovarian cancer and pancreatic cancer. Here it is recommended that biopsy should be avoided. The same may well be true regarding e.g. the subgroup of highly malignant breast or prostate cancers. This fraction of cancer subtypes may vary from approximately 10 - 45% which means that a majority of this patient category with low malignant tumours may develop metastases after 10 - 15 years, making it difficult to reveal correlation to biopsy caused cancer cell dissemination.
At present there is no available reliable objective method to determine tumour aggressiveness and thus to estimate the risk of biopsy induced tumour cell spread except performing analysis of tumour specific cell- or tissue samples. In order to minimize the risk of tumour cell spread caused by biopsy tumour tissue sampling and at the same time guarantee the analysis of tumour growth and grade of tumour malignancy, there is an opportunity to make use of newly developed cell- and tissue sampling methods i.e. imaging guided precision placement of the biopsy needle reducing the number of biopsies combined with anti-seeding equipped biopsy needles significantly diminishing the risk of tumour cell spread . In addition, in view of recent reports discussed above, it seems very reasonable to offer patients anti-inflammatory drugs pre- and postbiopsy.
The present review supports that FNAB is associated with a lower risk of biopsy associated metastasis, so why do we not use FNAB more frequently today? As mentioned, there is a lack of experienced cytopathologists. At the same time, new ultrasensitive molecular methods have been developed and the scarce material obtained by FNAB is not any longer a limiting factor for advanced analysis. Already a decade ago it was possible to analyse gene expression profiles from FNAB samples , but this is not yet routine. Analysis of protein biomarkers would be preferable and also comparable to current lowplex immuno-histochemical based profiling. Recently Ullal et al. showed that FNAB samples can be used for very sensitive 88-plex protein analysis, not only for diagnostic purpose but also to guide therapy . This example of recent development of advanced analysis is very promising and opens new opportunities for longitudinal follow up of therapy response and if needed, rapid adjustments of ongoing therapy. We have recently initiated tests using analysis of FNAB samples by proximity extension assay, an ultrasensitive and highly specific multiplex protein analysis method which also may be more cost effective compared to other profiling methods.
We conclude that we today have new unique opportunities to perform molecular diagnostics in FNAB samples. With these methods, we will have not only an at least equivalent diagnostic information compared to current practices, but also a more objective and extended molecular information to guide therapy selection and support the development of new therapies. At the same time, we get the opportunity to validate new potentially valuable biomarkers in clinical materials with minimal consumption of precious patient material. There are several biomarker candidates that may be used to determine e.g. tumour aggressiveness and sensitivity or resistance to a given therapy. Finally, last but not least, we may open up for the possibility to avoid unnecessary biopsy associated complications, minimize the potential risks of biopsy generated metastases and may also deliver diagnoses more timely and at a lower cost using FNAB based molecular profiling. These factors will be of outmost importance when the number of cancer patients increases worldwide.
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