Introduction

With an estimated 2.3 million new cases every year, breast cancer is currently the most frequent non-cutaneous malignant tumor worldwide (National 2021). Breast cancer currently accounts for one in four new cancer cases and one in six cancer deaths among women worldwide (National 2021), and one in eight women born in developed countries are expected to develop the disease in their lifetime (Early 2021). The burden of breast cancer continues to increase worldwide, particularly in developing countries, notwithstanding the great achievements of the past decades in terms of mammographic screening, increased understanding of genetic and environmental risk factors, and treatment (National 2021) (Guide 2019) (Royal 2021). Like many countries, Brazil faces an increasing challenge in providing health care to cancer patients; in this country, breast cancer is now the most frequent non-cutaneous malignant tumor in both sexes combined (Allison et al. 2020), but several barriers need to be overcome in the attempt to provide comprehensive diagnosis and treatment for our patients at the national level (Assis 2020) (Brasil. 2011) (Brasil. 2020). Moreover, Brazil has a dual health-care system, whereby nearly 75% of the population relies on medical care provided by a government-funded public system, and the remaining 25% has access to private health insurance (Burstein et al. 2021). Despite the attempts of the public system to provide full and comprehensive care to all citizens, access to health care in Brazil is very heterogeneous.

One of the greatest recent changes in our understanding of breast cancer has been the creation of a molecular taxonomy with diagnostic and therapeutic implications (Royal 2021) (Buzaid et al. 2020) (Cardoso et al. 2016). As a result, systemic treatment for molecularly defined subtypes of breast cancer has led to an increasingly complex decision tree for the management of patients with early-stage, locally advanced and metastatic disease (Cardoso et al. 2019) (Cardoso et al. 2020) (Compton et al. 2019) (da Cunha et al. 2021) (D'Angelo and Mejabi 2016) (De Las Casas and Hicks 2021) (Denduluri et al. 2021). This approach to treatment has paved the way to precision oncology, marked by the development of monoclonal antibodies and signal-transduction inhibitors of several relevant pathogenic alterations found in breast cancer and other tumor types. Thus, therapeutic decisions are now guided by comprehensive analysis of such alterations, and the molecular profile of each patient’s tumor now routinely accompanies histopathological assessment (Ellis 2021) (Fitzgibbons 2020). Moreover, biological features (tumor grade, estrogen and progesterone receptors [ER and PR] and HER2 expression) and gene expression-based assays with prognostic relevance are now included in the 8thedition of the American Joint Committee on Cancer staging manual for breast cancer (Gundisch et al. 2015). Finally, reliance on genotypic and molecular phenotypic features is only likely to increase in the future, as a result of the increasing role played by precision oncology in the treatment of patients with breast cancer (Harbeck et al. 2019) (Hatzis et al. 2011) (Heer et al. 2020) (Hewitt et al. 2012).

For all these reasons, timely and correct assessment of histopathological, immunohistochemical (IHC) and molecular features of biopsy and surgical specimens is of paramount importance in the provision of care to patients with breast cancer. As a result, a concerted effort needs to be continuously undertaken by institutions and individuals in order to ensure that tissue samples are obtained, processed, analyzed and reported in an optimal way that takes into account state-of-the-art scientific and technical knowledge and circumvents logistic and operational constraints. This may be particularly challenging in some settings due to several sources of inefficiency in terms of economic, structural, organizational and communication features that preclude optimal pathological assessment of tumor specimens. In the current article, we present the issues related to the journey of breast cancer samples in Brazil that were identified and discussed by a working group convened in an expert panel and review important recommendations selected by the group based on the current literature and guidelines and also on their professional experience to address these issues. This paper is part of a larger initiative that aims to improve the health-care journey of breast cancer patients in Brazil (Assis 2020). The article was developed through a collaboration between members of the Brazilian Society of Pathology, Brazilian Society of Mastology, Brazilian Society of Histotechnology, and Brazilian Society of Operating-Room Nurses, and has been published jointly by invitation and consent in both, theSurgical and Experimental Pathology and Mastology journals.

Composition, objectives and funding of the working group

The multidisciplinary working group was composed of two pathologists (HG and FMC), one breast surgeon (RMSR), one oncology nurse (MIK), and one histotechnologist (DLP) from Brazil with experience or professional focus on breast cancer. The five members work in large hospitals/services located in four states of two different regions of the country. The working group convened in May 2021 in an expert panel upon invitation from Roche Produtos Químicos e Farmacêuticos, Brazil, who also had representatives attending the meeting with the aim of organizing it. The working group attempted to identify the most salient issues related to the breast cancer tumor-tissue journey in Brazilian public and private medical institutions, based on their experience, since all of them actively work at both sectors, and discussed the current scientific literature, with the main objective of selecting and reviewing recommendations that may mitigate and prevent preanalytical, analytical, and post-analytical issues that may affect diagnostic and therapeutic decisions. The financial sponsor had no influence on the discussions during the expert panel. Hence all the recommendations reviewed here and the writing of this article rest under the entire responsibility of the authors.

Issues identified and discussed by the working group

The preanalytical, analytical, and post-analytical issues discussed by the working group members were grouped under the four headings presented below and summarized in Table 1.

Table 1 Categories and issues identified as critical for optimizing the tumor-tissue journey
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Professional education and awareness

Adequate knowledge on the part of the various individuals impacted by the tumor-tissue journey is a prerequisite for all the procedural steps required in this process. Each individual needs to understand the process as a whole and in its different steps, their own role, and the roles of others. Table 1 displays the specific issues identified by the experts based on their professional experience; the prevention or resolution of these issues can be accomplished with continued education, the creation of standardized operating procedures, and participation in external quality assurance programs. Moreover, institutional buy-in is paramount, because the process cannot rely simply on the goodwill of a few key persons. Institutions need to recognize their role in fostering professional education and awareness, as well as enforcing operating procedures.

Communication and integration within teams

In addition to awareness of their roles in the process, individuals must establish adequate communication with other team members; likewise, adequate communication among institutional sectors or departments is vital, and managers should work to ensure the necessary procedures and infrastructure. This may be particularly critical in publicly funded institutions, where the organization of roles and structures may depend on several layers of administration. Importantly, there must be a two-way communication between the pathologist and the rest of the team, in the sense that the relevant medical and practical information needs to be provided to the pathologist, who in turn must provide feedback to the team about sample quality and issues that may arise. There is often insufficient provision of relevant details, even on the part of surgeons, and this may preclude optimal interpretation of findings. Table 1 summarizes the communication issues identified by the task force members.

Procedures in the operating room and sample transportation

Table 1 also summarizes the key issues identified by the working group members regarding the procedures required in the operating room with the aim of optimizing the quality of the sample. A key issue in some institutions is the unduly long time taken before the sample reaches the laboratory, sometimes due to internal organization of the operating room or due to the physical distance between the hospital and the laboratory where samples will be processed and analyzed. In some cases, insufficient technology, e.g., lack of electronic medical records and barcode system for digitizing information, may increase that time. Other issues may also contribute to that increase, including individual institutional features that may create additional bottlenecks. Once again, institutional will is of paramount importance toward ensuring adequate and streamlined procedures that may ensure the minimum possible time between sample collection and delivery to the laboratory, and the best possible handling of the sample during that journey.

Issues related to sample identification, labeling, conditioning and transportation may occur from sample removal to its delivery to the pathology laboratory (Table 1). Incorrect or incomplete labeling of the specimen or filling of forms accompanying the sample are unfortunately frequent occurrences. Individuals and the institution play an important role in devoting attention to the design of the forms and the choice of packaging and labeling materials. Of particular concern is the frequent lack of awareness about the importance of buffered formalin and of swift transportation of the sample to the pathology laboratory.

Procedures in the pathology laboratory

The pathology laboratory plays a central role in minimizing issues that may compromise correct and timely information required for diagnostic and therapeutic decisions (Table 1). In addition to standardization and proper implementation of techniques related to sample processing, including those involving conditioning, specimen cleavage and fixation, laboratory personnel must ensure that sufficient information has been provided upon receipt of samples. Very often, forms accompanying samples are incompletely filled. In publicly funded institutions, the practice of outsourcing pathology services is not uncommon, and frequent change in the providers of such services may represent an important hurdle for adequate patient management.

Recommendations to mitigate the identified issues and optimize pathological assessment of tumor specimens

Breast specimens obtained from outpatient procedures or from procedures performed in the operating room for the diagnosis of breast cancer require attention from collection to reporting of histological results. In this journey, several factors may interfere with the quality of the final diagnosis in terms of the disease definition, type, characteristics of greater or lesser biological aggressiveness, presence of hormone receptors, and HER2 expression. These factors guide the selection of the best therapeutic option for each case and, when incorrectly evaluated, may negatively affect patient prognosis.

The tumor-tissue journey of breast specimens involves the participation of physicians, nursing team members, biomedical professionals, biologists, lab technicians, and administrative personnel. As part of the task and based on the current guidelines and the published literature, the experts discussed the steps involved in each of the three phases of the tissue processing journey to review important recommendations. Figure 1 summarizes the steps comprising the pre-analytical, analytical, and post-analytical phases of the tissue journey, although variation may exist in how the steps are grouped (Hortobagyi 2017).

Fig. 1
figure 1

Flowchart of the main steps of the preanalytical, analytical, and post-analytical phases of the tissue-journey, adapted from the WHO document (WHO 2005).

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Based on the issues identified (Table 1), the working group selected and discussed recommendations to address each aspect. The recommendations reviewed here were based on the current guidelines and orientations published by international organizations, such as World Health Organization (WHO) (Hortobagyi 2017) and the College of American Pathologists (CAP) (Khoury 2018), and Brazilian Society of Pathology (SBP) (Laboratory 2005), among other documents (Lee et al. 2012) (Loi 2019), as well as on the professional knowledge and experience of the multidisciplinary members of the working group, especially considering the local scenario.

Recommendations are summarized in Tables 2, 3 and 4 and discussed below, according to the three phases, following the criteria adopted by the WHO guidelines (Hortobagyi 2017).

Table 2 Summary of actions and recommendations for the pre-analytical phase
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Table 3 Summary of actions and recommendations for the analytical phase
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Table 4 Summary of actions and recommendations for the post-analytical phase
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General recommendations

In all the steps, samples must be identified with the name of the responsible person, the date and time, to ensure traceability. The experts recommend that the sample be accompanied throughout its journey, not only by the medical request form, but also by a document listing all the steps, with the name of the person responsible for each step, date and time, either on paper or electronically. Important information includes:

  • Time of sample collection

  • Time of sample placing in the fixative

  • Cold ischemia time

  • Time of sample delivery to the person responsible for transferring it to the pathology laboratory (intra- and inter hospital transport)

  • Time of entry at the pathology laboratory

  • Time of macroscopic evaluation

Pre-analytical phase

Table 2 displays actions and recommendations for the different steps of the pre-analytical phase (Cardoso et al. 2020), (D'Angelo and Mejabi 2016), (Hortobagyi 2017), (Khoury 2018), (MacGrogan et al. 2014), (Perou et al. 2000).

Sample collection and conditioning

Sample collection is under the responsibility of the physician, surgeon, or radiologist, who is also responsible for filling in the exam request form with clinical information. Information about the time of specimen collection and the time of cold ischemia (defined as the time between removal of the tissue from patient until placement into the fixative) are under the responsibility of the nursing team (operating room) or the radiology assistant (radiology services). The cold ischemia time is an important variable to be emphasized as it can alter the gene expression and protein characteristics, thus interfering with the results of IHC and molecular tests (Khoury 2018). Regarding this, a cold ischemia time of less than 1 h is recommended.

The excised material must be clearly detailed in the request form and should be checked by the nursing team before placement in the containers with fixative. Regarding the handling of the specimens before placement in the fixative, there are specific recommendations for outpatient procedures and for surgical specimens, as detailed in Table 2. Large tumor specimens require preparation for adequate fixation. Recommendations regarding sectioning before fixation, including the thickness of the sections, type of fixative and fixation time are provided in Table 2. This is an important topic, as inadequate fixation impairs the histological diagnosis (differential diagnosis between benign and malignant, histological typing and grading, and the immunoreactivity of target molecules, especially those of cytoplasm or membrane localization, such as programmed death 1 ligand [PD-L1], HER2, etc.) (MacGrogan et al. 2014)–(Najjar and Allison 2022).

Recommendations regarding sample identification, which is an attribution of the nursing team, characteristics and labeling of containers, fixation registry, duration, and fixative solutions are also detailed in Table 2. 10% neutral buffered formalin is the fixative solution most frequently preferred for routine histological preparations of surgical specimens. Monitoring the fixation time is critical. For hormone receptors and HER2, a fixation time of 6–72 h is recommended (Cardoso et al. 2020), (D'Angelo and Mejabi 2016).

Exam request

As previously mentioned, the medical team is responsible for completing the request form with clinical data and specimen information. The precise and complete filling of this form is of crucial importance to the tissue journey.

Transportation

The last step of the pre-analytical phase is the transportation of the sample to the pathology laboratory, which may be located at the same hospital/service involved in the specimen resection or may be in a different, distant location. Special care must be taken when transporting surgical specimens from the operating room to outside pathology laboratories. Specimens must be transported in rigid containers, with an adequate volume of buffered formalin (WHO 2019). Information regarding current recommendations in guidelines for specimen transportation is also detailed in Table 2.

Analytical phase

The analytical phase comprises the sample/specimen reception at the pathology laboratory, sample/specimen macroscopic examination, tissue processing, paraffin embedding, sectioning/microtomy of the paraffin blocks, routine staining, special staining, IHC, and other molecular techniques such as in situ hybridization (Fig. 1) (Hortobagyi 2017). To be performed with safety and quality, this phase requires the establishment of standardized procedures and efficient channels of communication between the pathology laboratory and the clinical-surgical and imaging services where the samples were obtained. In the analytical phase, only a few steps are automated, with several steps in the process being manual, relying on the care and skill of the pathologist (gross examination, specimen cleavage and selection of samples for microscopy) and the laboratory technicians (inclusion and microtomy) (Laboratory 2005).

Factors that are determinant to the analytical phase include the criteria adopted for sample acceptance or rejection, the thickness of tissue section into cassette, tissue processor fluid maintenance, paraffin type and temperature, and validity tests and controls (Hortobagyi 2017).

A summary of the actions and recommendations for the main steps of the analytical phase is presented in Table 3and briefly described below (Hortobagyi 2017), (Laboratory 2005)–(Loi 2019), (WHO 2019), (Santana and Ferreira 2017).

Sample reception

The reception of the pathology laboratory is where the samples are received. Upon receipt, it must be guaranteed that each specimen received is accurately labeled with the patient identification and accompanied by the examination request containing clinical information and previous laboratory tests, date and time of collection. The date and time of receipt of the material must be registered in the laboratory, confirming whether the tissue was received fresh or fixed and the type of fixative used. Predetermined rules previously established by the pathology laboratory receiving the samples should be followed for rejecting inadequate specimens whenever needed. These rules must be communicated to all physicians and healthcare professionals who send the materials. Situations in which specimens must be rejected include: unlabeled sample with no information regarding patient name and material identification; insufficient patient information; and information provided in the sample label not matching the patient name on the pathology request form (Hortobagyi 2017), (Schettini et al. 2021). Additionally, there are situations that do not imply rejection of material, but can interfere with the quality of the specimen, exam and results, including: damaged or leaking tube/container; inadequate volume of fixative for the amount of material; material partially dried up due to inadequate volume of fixative; and extended transportation time or other improper handling during transportation (Hortobagyi 2017), (Laboratory 2005).

It is important that the laboratory communicates to the physician who requested the pathology exam any problem related to the rejection of the sample or the identification of situations that interfere with the quality of the exam.

Sample registration

Upon receipt, one important step is checking if the received specimens match the information and description provided for the case in the container labels and in the request form. Once the correspondence is confirmed, sample registration proceeds with the attribution of a unique identification number to facilitate sample tracking during the process. To improve traceability of materials (sample fragments, paraffin blocks, histological slides, routine and special staining, etc.), the use of barcode labels is recommended wherever possible.

Macroscopic examination of specimens

Gross examination is performed by the pathologist or laboratory technicians. This step involves the description of the specimen in terms of shape, color, texture, consistency, and delimitation of the adjacent tissue, the measurement (size and weight) of the specimen, and its dissection. Lesions should be described and measured with information about their topography. More detailed recommendations are provided in Table 3. It is highly recommended to follow protocols and guidelines for testing and sampling established by pathology scientific societies and international institutions (Lee et al. 2012), (Loi 2019), (Santana and Ferreira 2017).

Histological processing

Tissue processing is performed using an automated tissue processor prior to microtomy. This equipment is maintained by lab technicians for the control of reagents used (formaldehyde, alcohols, xylene, paraffin). The time of tissue processing should be adequate to each type of specimen (Table 3).

Paraffin embedding

After processing, the tissue samples are embedded in paraffin wax. Monitoring paraffin temperature is crucial to avoid excessive heat. Samples should be carefully oriented, handled and positioned in the inclusion blocks. Specific recommendations selected by the working group based on the current guidelines and literature are listed in Table 3.

Microtomy

Sectioning the tissue block with the use of a microtome is the following step. Specific recommendations on sections thickness, quality and positioning of blades were reviewed and are provided (Table 3).

Tissue floatation in warm water bath, placement of the paraffin embedded tissue sections on slides, and dehydration of sections

As part of the process, the tissue slices are placed in a warm water bath. Precautions need to be taken to avoid cross contamination and damage of sections (Table 3). Tissue sections should be carefully selected and placed on slides. Before proceeding to staining, histological sections should be dehydrated. More detailed recommendations are displayed in Table 3 and in the original publication of the cited guidelines.

Routine and complementary stainings

Hematoxylin and eosin (H&E) are the stains routinely used in histopathology. Table 3 displays recommendations for this step. Special stainings (histochemistry) or, more often, IHC stainings, can be used to provide complementary information for diagnosis or for predictive tests for therapeutic response.

Immunohistochemical stain

It can be performed on specific equipments (autostainers) or manually using standardized procedures and specific reagents. Positive-charged or silane coated glass slides are recommended to ensure adherence of the histological sections and avoid loss of material during the different stages of the IHC technique. The choice of reagents (primary and secondary antibodies, detection system, and counterstaining) is of paramount importance and determines the quality of the reactions together with the standardization of procedures. The equipment used must be routinely calibrated. Antibodies should be chosen with care and used following the manufacturers' technical specifications, using antigen retrieval in the appropriate medium when necessary. Use an appropriate detection system, standardize washing steps and optimize counterstaining. An appropriate positive tissue external control should be included on all reactions. The WHO, the College of American Pathologists and the American Society of Clinical Oncology recommend that all primary breast tumors should be tested for hormone receptors (ER and PR) and HER2 (Cardoso et al. 2020), (D'Angelo and Mejabi 2016), (Siegel et al. 2020).

In situhybridization

In situ hybridization should follow the same precautions recommended for the IHC method using properly fixed tissue and silane coated or positive-charged slides to avoid detachment problems and loss of material. Specific and standardized reaction protocols should be followed. Probes must be carefully chosen for each diagnostic indication, and appropriate controls used for all reactions.

Post-analytical phase

The post-analytical phase involves the interpretation of the slides and the preparation of pathology reports to describe the results. The use of synoptic reports is highly recommended to improve data reporting, as they provide a structured and standardized documentation (Hortobagyi 2017).

As emphasized in the guide published by WHO in 2019, the post-analytical phase also includes the retention and disposal of all the materials containing patient tissues/samples (paraffin blocks and glass slides) and data archiving, with specific recommendations being attributed to these steps (Hortobagyi 2017).

The quality of the sample must be assessed and the reasons for a sample to be considered of limited or inadequate quality must be notified, as described in the recommendations listed in Table 4 (Cardoso et al. 2020), (D'Angelo and Mejabi 2016), (Hortobagyi 2017). Parameters used to attest the quality of a sample include the cold ischemia time, type and volume of fixative, fixation time, presence of technical artifacts, and factors affecting the IHC reaction/interpretation (e.g., the use of internal and external controls).

Recently, new categories of tumors, based on low expression of the traditional biomarkers ER and HER, have shown important prognostic and predictive differences (Sotiriou and Pusztai 2009). HER2-negative 2018 ASCO/CAP group includes tumors with no staining (score 0), incomplete and faint/barely perceptible staining in up to 10% of tumor cells (score 0), incomplete and faint/barely perceptible staining in > 10% of cells (score 1 +), and those with weak/moderate complete membrane staining in more than 10% of cells (score 2 +) with no amplification by in situ hybridization (D'Angelo and Mejabi 2016), (Sparano et al. 2018). Breast cancer with low HER2 expression, particularly the group denominated HER2-low (score 1 + or 2 + without gene amplification), has shown response to new generation of antibody–drug conjugates, capable of delivering drug to tissues by binding to target cells (Start et al. 1992). However, reproducibility of the correct classification among pathologists is suboptimal, with discordance of 35% of the cases, in part because of influence of preanalytical artifacts (Sung et al. 2021). Pathologists should follow the specimen fixation, processing, and interpretation guidelines proposed by the 2018 ASCO/CAP HER2 test recommendations to ensure the reliability and reproducibility of classifying tumors into different expression categories of this biomarker.

Discussion

The importance of pathological preanalytical and analytical issues to the adequate provision of contemporary cancer care cannot be overemphasized (Assis 2020), (Cardoso et al. 2020), (D'Angelo and Mejabi 2016), (Tutt et al. 2021), (Venetis et al. 2022). Most issues affecting timely and correct assessment of specimens occur in the preanalytical phase of processing (Fitzgibbons 2020), (Tutt et al. 2021), (Venetis et al. 2022). Studies suggest that about 60–70% of laboratory errors are due to preanalytical factors (Khoury 2018). Adequate handling of surgically removed specimens involves labeling, packaging, transportation, fixation and storage, as well as the collection and reporting of administrative, demographic and medical information. Attention to specimens at all these steps may mitigate errors and optimize histopathological, immunohistochemical, and molecular testing in breast cancer.

The relevance of the issues outlined here is only likely to increase, as a result of the increasing role played by precision oncology in the treatment of patients with breast cancer. The time from tissue removal to formalin fixation (cold ischemic time) and temperatures during fixation are crucial (Cardoso et al.2020), (D'Angelo and Mejabi 2016), (Williams et al. 1997). These parameters are particularly critical for the analysis of ER, PR, and HER2 expression (Williams et al. 1997). Among other problems, antigen loss in formalin-fixed tissue sections is sufficient to preclude optimal diagnostic histopathology and IHC studies (Venetis et al. 2022). Even though we focus our attention on handling of samples for histopathological and IHC assessment, the problem is broader when one considers the increasing role of newer molecular-biology technologies that rely on the quality of tissue RNA in the assessment of gene expression (Wolff et al. 2018). Prognostic gene expression-based assays play an increasing role and have been increasingly used for decision-making regarding the indication of chemotherapy (Xie et al. 2011).

If the preanalytical phase is optimized, errors in the analysis or interpretation of results by the pathologist are minimized. Nevertheless, attention is needed to the frequent communication issues identified in Table 1, particularly with regard to insufficient provision of the relevant clinical information to the pathologist. Unfortunately, the pathology laboratory is also place for some of the preanalytical issues that can compromise correct and timely acquisition of information required for diagnostic and therapeutic decisions in oncology (Ellis2021). In Brazil, many hospitals do not have their own pathology laboratory, but rather outsource this service, which creates an additional layer of complexity in the attempt to minimize errors. Of note, there is frequent concern about the quality of the services provided by some of these laboratories, which are usually contracted on the basis of public procurement.

Ideally, patients with breast cancer should be under the care of a multidisciplinary team involving the various specialized professionals required for optimal results (Assis 2020), (Cardoso et al. 2019), (Ellis 2021). Although there is overlap between the function of individuals, departments and institutions in terms of their contribution to a seamless tumor-tissue journey, each participant in the process needs to be aware of their contribution and of the overall process. Education, communication, standardization of procedures, and creation of adequate infrastructure are the keys to success, and are ideally achieved in institutions motivated and with the required administrative will. These institutions are further embedded in larger publicly funded or private systems, which must recognize the importance and foster implementation of the issues highlighted here. We hope the recommendations reviewed here can play a role in that goal, and potentially inform public policy related to these issues.