Diagnostic Methods Used in Detecting Multiple Myeloma in Paleopathological Research—A Narrative Review

Abstract

Objective: This study aims to analyze and evaluate the diagnostic methods used to detect multiple myeloma in paleopathological research. As a malignancy characterized by bone lesions, multiple myeloma presents unique opportunities for study through the paleopathological analysis of human skeletal remains. Methods: A literature review was conducted across PubMed, ScienceDirect, Embase, Scopus, Web of Science, and Google Scholar, focusing on macroscopic, radiological, and microscopic methods. A total of 43 original peer-reviewed studies published over six decades were selected. Results: The most commonly used diagnostic technique was macroscopic analysis of bone material, focusing on the characteristics of the lesions. Radiological methods, including X-ray, magnetic resonance imaging, computed tomography (CT), and micro-CT, provided complementary insights. Various microscopic techniques, chemical analyses, and fluoroscopy provided additional diagnostic detail. The diagnostic process is shaped by factors such as preservation, context, and access to technology; despite these variables, characteristic features of lesions were consistently recognized. Conclusion: This review highlights how macroscopic analysis remains central to diagnosis in paleopathology, with radiological and microscopic methods increasingly enhancing accuracy and interpretive depth. A multidisciplinary approach, combining macroscopic, radiological, microscopic, and chemical analyses where feasible, continues to strengthen paleopathological research and offers new insights into the historical presence of multiple myeloma.

1. Introduction

According to a WHO report from 2021, cancer is currently among the top ten leading causes of death globally [1]. Due to the rising number of cancer cases, researchers have been working for years to improve diagnostics, accelerate detection, develop effective treatments, and implement preventive measures. However, the question of when neoplastic diseases first appeared in human history remains unanswered. Studies conducted by paleopathologists, who analyze skeletal remains, may offer valuable insights into the diseases that affected humans in the past, including their timing and frequency. In this paper, we will focus on multiple myeloma, a condition that causes characteristic bone changes, which can be studied by paleopathologists and physical anthropologists in human skeletal remains from various historical periods.

1.1. Myeloma in Contemporary Times

Myeloma is currently estimated to account for 1% of all cancers and 10% of hematological cancers. It most commonly affects individuals around the age of 65–70, making it the second most common hematological cancer in adults [2]. It is an incurable, malignant cancer of the plasma cells in the bone marrow, with an unknown etiology. In this disease, plasma cells transform into cancerous cells that proliferate uncontrollably, producing abnormal proteins called paraproteins (monoclonal proteins or M proteins) [3]. The symptoms of multiple myeloma include anemia, hypercalcemia, kidney failure, hyperglobulinemia, increased infection risk, and bone lesions [4]. Myeloma-related bone disease, a focus of paleopathological research, affects approximately 80% of patients at the time of diagnosis and nearly all patients as the disease advances [5]. The mentioned symptoms, although characteristic of the disease, also occur in many other conditions, making accurate diagnosis significantly more challenging. As a result, patients must undergo a comprehensive diagnostic process to accurately determine the cause of their symptoms [3]. The diagnosis of multiple myeloma is based on the revised criteria of the International Myeloma Working Group. It requires either the presence of clonal plasma cells in the bone marrow exceeding 10% or a biopsy-confirmed plasmacytoma (either bone-based or extramedullary), along with at least one of the following: CRAB features (hypercalcemia, renal failure, anemia, or bone lesions) or high-risk biomarkers [6,7].

Recently, the survival rate for patients with multiple myeloma has significantly improved due to the introduction of pharmacological therapies that help manage the symptoms of the disease. However, these treatments do not result in a complete cure [3].

The treatment of multiple myeloma involves several stages. Induction therapy, typically based on multi-drug regimens, prepares the patient for further procedures. In patients with good overall health, autologous stem cell transplantation (auto-PBSCT), usually preceded by chemotherapy, is often used. After initial treatment, maintenance therapy is applied to prolong remission. In cases of relapse or resistance to treatment, additional therapeutic lines are introduced and tailored to the patient’s condition. For asymptomatic myeloma, regular monitoring is conducted, and localized treatments, such as radiotherapy, are used for plasmacytomas. Treatment approaches are individualized and continue to evolve in response to ongoing medical advances [6].

1.2. Myeloma in Paleopathological Research

Paleopathology is a multidisciplinary field that focuses on reconstructing the health of past populations by examining pathological changes in human remains. This research enables the identification of diseases that have affected individuals throughout history, providing insights into their impact on societies. Cancer research, in particular, holds significant value in this context, as its presence in archaeological remains, though rare, offers unique perspectives on the history of medicine and the evolution of diseases over time [8,9].

Although paleopathologists face unique challenges due to the condition and context of archaeological remains, many have successfully adapted well-established diagnostic criteria from the clinical literature to suit the constraints of their field [10,11]. Myeloma is a disease of particular interest to paleopathologists due to the distinctive bone changes it produces, which can be observed in skeletal remains even in the absence of soft tissues. These characteristic osteolytic lesions, resulting from the abnormal proliferation of plasma cells, are present in approximately 90% of patients. These bone defects typically affect highly vascularized skeletal areas, such as the vertebral column, ribs, pelvis, and skull. This distribution reflects the disease’s tendency to involve areas with rich blood supply.

Previous research has underscored the relevance of multiple myeloma in paleopathological research. In a systematic review conducted by Hunt et al., based on data from the Cancer Research in Ancient Bodies database, which comprises skeletal remains dated between 1.8 million years ago and 1900 CE, multiple myeloma was identified as the second most frequently observed primary bone malignancy in the archaeological record, following metastatic carcinoma [12]. Although neoplastic diseases are often considered modern “diseases of civilization”, archaeological evidence suggests that such conditions have affected humans throughout history. Some of the earliest documented cases of neoplasm in anatomically modern humans were found in skeletal remains dated to approximately 4000 BCE in Austria [13] and around 3300 BCE in Kentucky, USA [14], both diagnosed based on characteristic myeloma-related bone lesions.

The osteolytic bone lesions characteristic of multiple myeloma can be difficult to distinguish from those caused by other conditions due to similarities in their distribution and typical age of onset. Such lesions may overlap with those observed in metastatic cancer and other hematologic disorders, making differential diagnosis essential for accurate identification [15]. In the study by Hunt et al., the authors acknowledged that, in some cases, distinguishing multiple myeloma from metastatic bone disease was nearly impossible. As a result, for the purposes of quantifying cancer prevalence, certain lesions were classified as both myeloma and metastatic carcinoma [12].

To address the diagnostic challenges presented by skeletal remains, researchers have employed a range of complementary methods—beginning with macroscopic analysis and, where possible, incorporating radiological and microscopic techniques. These methods are applied according to the preservation of remains, institutional resources, and ethical considerations [16,17,18]. While technological advancements have expanded new possibilities for detecting pathological changes in archaeological materials, challenges in interpreting the results of these findings persist.

1.3. Study Objectives

The aim of this study was to review the literature on diagnostic methods used in paleopathology for detecting multiple myeloma, with a focus on describing the approaches employed and identifying their limitations. A clearer understanding of these research methodologies can broaden knowledge about the prevalence and diagnosis of cancer in ancient populations.

2. Materials and Methods

Articles published between June 1964 and July 2024 were reviewed. The databases searched included PubMed, ScienceDirect, Embase, Scopus, Web of Science, and Google Scholar, using the following search terms:

• Myeloma AND differentiation AND anthropology;
• Myeloma AND molecular AND anthropology;
• Myeloma AND markers AND anthropology;
• Myeloma AND imaging AND anthropology;
• Myeloma AND diagnostics AND anthropology.

The review was performed manually by members of the research team. After removing duplicates, 84 original scientific papers remained (as of 30 July 2024). Of these, 43 were included based on the following criteria:

• The study was original, peer-reviewed, and published;
• The full text was available;
• The full text was in English;
• The content was relevant to the topic of our review.

Our paper discusses how the described diagnostic methods are currently, or could potentially, be used to detect osteological lesions or molecular markers associated with multiple myeloma in anthropological samples, as well as their applicability in the differential diagnosis of such lesions.

To minimize bias during data extraction, each article was reviewed by at least two researchers.

Table 1. List of analyzed studies of multiple myeloma in paleoanatomical materials, categorized by diagnostic techniques used, sample age, and the types of bones examined.

Article	Used Diagnostic Techniques	Age of Samples	Examined Bones with Myeloma
Abegg and Desideri, 2018 [19]	Macroscopic analysis, radiological examination	XIX/XX century CE	Cranium, scapulae, manubrium, ribs, vertebrae, humeri, femora
Bauduer et al., 2014 [16]	Macroscopic analysis, radiological examination	V–VII century CE	Cranium
Biehler-Gomez et al., 2019b [18]	Macroscopic analysis, radiological examination	XX century CE	Cranium, mandible, clavicles, humeri, femora, manubrium, ribs, vertebrae, sacrum, pubis, ilia, scapulae
Binder et al., 2014 [20]	Microscopic methods	ca. 1200 BC	Almost complete skeleton
Dabbs and Zabecki, 2015 [21]	Macroscopic analysis, radiological examination	1300–1400 BC	Scapulae
De La Rua et al., 1995 [22]	Macroscopical analysis, microscopic methods	ca. 3100 BC	Iliac blade, sacrum, vertebrae, scapula, femur
Dittmar et al., 2020 [23]	Macroscopic analysis, radiological examination	1750–1400 BC	Humeri, radii, phalanges, clavicles, scapulae, sternum, ribs, vertebrae, pelvis, femora, ulnae, fibulae
Gróf et al., 2015 [24]	Macroscopic analysis, radiological examination	XIII–XVI century CE	Cranium, ulnae, femora, tibiae, humeri, radii, fibulae, vertebrae
Grupe, 1988 [25]	Macroscopic analysis, radiological examination	Middle Ages	Cranium, mandible, manubrium, scapula, humerus, ulna, radius, iliac bone
Haidle, 1995 [26]	Macroscopic analysis,	Middle Ages	Cranium, mandible, vertebrae, ribs, pelvis, sacrum, scapulae, coccyx, humeri, femora, radii, ulnae
Isidro et al., 2019 [27]	Macroscopic analysis, radiological examination	ca. 2160–2000 BC	Complete skeleton
Khwaileh, 2016 [28]	Macroscopic analysis	Islamic period	Cranium, vertebrae, ossified trachea, sternum
Klaus, 2017 [29]	Macroscopic analysis	X–XVII century CE	Cranium, vertebrae, sacrum,
Klaus, 2016 [30]	Macroscopic analysis	ca. 1533–1620 CE	Clavicle, humerus, ulnae, ribs, scapulae, vertebrae
Loveland et al., 1992 [31]	Macroscopic analysis, radiological examination	400–1300 CE	Complete skeleton
Luna et al., 2008 [32]	Macroscopic analysis, radiological examination, microscopic methods	1030–370 BP	Vertebrae, ribs, scapula, humerus, ulna, radius, femur, tibia, fibula, tarsal and metatarsal bones, phalanges
Manchester, 1983 [33]	Macroscopic analysis, radiological examination	After 650 CE	Cranium, humeri, radii, ulnae, femora, tibiae, clavicles, scapulae, fibula, calcanei, atlas and axis vertebrae
Marcsik et al., 2001 [34]	Macroscopic analysis	VIII–IX century CE	Cranium, mandible, vertebrae, manubrium, rib, sacrum, iliac bone
Marks and Hamilton, 2007 [35]	Radiological examination	XX century CE	Cranium, vertebrae, pelvis, ribs, scapula
Marques et al., 2013 [36]	Macroscopic analysis, radiological examination	XX century CE	Complete skeleton
Matos et al., 2011 [37]	Macroscopic analysis, radiological examination	XIII/XIV–XIX century CE	Vertebrae, ribs
Molnár et al., 2009 [38]	Macroscopic analysis, radiological examination, microscopic methods	III–XVI century CE	Cranium, mandible, pelvis, ribs, vertebrae
Molto and Sheldrick, 2018 [39]	Macroscopic analysis	1100–680 BC	Complete skeletons
Morgunova et al., 2022 [40]	Macroscopic analysis, radiological examination	3300–3100 BC	Cranium, mandible, vertebrae, sacrum, femur, radius, tibia, rib, scapula, sternum, iliac bone
Morse et al., 1974 [41]	Macroscopic analysis	200–1200 CE	Cranium, teeth, mandible, ribs, vertebrae, scapulae, pelvis, humeri, ulnae, radii, femora, tibiae, fibulae, bones of hand and foot
Němečková and Strouhal, 2010 [42]	Microscopic methods	After 4000 BC	Cranium, iliac bone,
Nerlich et al., 2006 [43]	Macroscopic analysis, Radiological examination	3200–500 BC	Complete skeletons
Nerlich et al., 2000 [44]	Macroscopic analysis, radiological examination	1550 BC–400 CE	Cranium, teeth, ulnae, radii, clavicles, vertebrae, pelvis, humeri
Rothschild et al., 1998 [45]	Macroscopic analysis, radiological examination	Unspecified	Complete skeleton
Rothschild and Rothschild, 1995 [46]	Macroscopic analysis, radiological examination	XX century CE	Complete skeletons
Rothschild et al., 1997 [47]	Macroscopic analysis, radiological examination	Unspecified	Complete skeletons
Schats et al., 2018 [48]	Macroscopic analysis, radiological examination	1382–1598 CE	Scapula, clavicle, ribs, one thoracic vertebral arch
Siek et al., 2021 [17]	Macroscopic analysis	XII–XVI century CE	Cranium, mandible, humeri, ulnae, radii, femora, tibiae, fibulae, vertebrae, clavicles, sacrum, iliac bones, bones of hand
Strouhal, 1993 [49]	Macroscopic analysis, radiological examination	VI–XI century CE	Almost complete skeleton
Strouhal, 1976 [50]	Macroscopic analysis, Radiological examination	III–IV century CE	Cranium, pelvis, calcified tumor,
Strouhal and Kritscher, 1990 [13]	Macroscopic analysis	After 4000 BC	Cranium, vertebrae, ribs, scapulae, clavicles, humeri, ulnae, radii, iliac bones, sacrum, femora, patella, tibiae, fibulae, bones of feet
Strouhal, 1991 [51]	Macroscopic analysis	VI–XI century CE	Complete skeletons
Suzuki, 1981 [52]	Macroscopic analysis, radiological examination	1709–1810 CE	Cranium
Vargová et al., 2013 [53]	Macroscopical analysis, microscopic methods	XVIII–XIX century CE	Complete skeletons
Wada et al., 1987 [54]	Macroscopic analysis	Unspecified	Almost complete skeleton
Wahba et al., 2021 [55]	Macroscopic analysis, radiological examination	1570–332 BC	Cranium, iliac bones
Wakely et al., 1998 [56]	Macroscopic analysis	1300–1540 CE	Almost complete skeleton
Wasterlain et al., 2011 [57]	Macroscopic analysis	XV–XX century CE	Almost complete skeleton

CE, common era; BC, before Christ; BP, before present.

presents the list of studies included in the final review.

3. Results

We divided our discussion into three sections to provide a comprehensive overview of each diagnostic method. The sections include macroscopic methods, microscopic and advanced chemical methods, and radiological methods.

3.1. Macroscopic Methods

Gross analysis is the most fundamental method for assessing paleo-anatomical samples and serves as the basis for determining whether human remains are suitable for further examination using techniques such as microscopy, endoscopy, or radiology [18,44,57]. Macroscopic descriptions of lesions typically include an assessment of their size, shape, margin characteristics, presence of osteolysis or sclerosis, and anatomical location. Lesions may also be evaluated collectively by examining their distribution and symmetry across the skeleton [29,36].

Multiple myeloma and metastatic cancer are among the most commonly identified neoplastic conditions in paleo-oncology material from Ancient Egypt [27,39]. Evidence suggests that neoplastic diseases have affected humans throughout various historical periods, including modern times, the Middle Ages, antiquity, and prehistory. Traces of such conditions have been identified in paleontological material from nearly all regions of the world, with notable findings reported in Egypt, Poland, Hungary, Peru, the United States, Japan, and China [17,23,30,38,41,52,55]. Accurately assessing the historical increase in the incidence of neodysplastic chondrosis is complicated by multiple factors. In addition to the advancements in diagnostic capabilities, changes in human lifespan, lifestyle, and environmental conditions over the centuries must also be considered. Hou et al., in their analysis of approximately 30 years of data, showed that as populations have aged and life expectancy has increased, the number of cancers detected, including multiple myeloma, has also risen [58]. Shifts in occupational and living conditions are similarly correlated with changes in cancer incidence. Studies have shown that the risk of developing neoplastic diseases, such as myeloma, increases with unhealthy lifestyles (e.g., use of stimulants, chronic stress) and after prolonged exposure to toxic substances, such as solvents [59,60]. However, available sources indicate that neoplastic diseases were significantly less prevalent in populations from the time period corresponding to the age of the analyzed samples, approximately 4000 BC to the 20th century CE.

Lesions caused by multiple myeloma have a distinctive morphology: they are round, with smooth, well-defined margins and a flat surrounding area. These lesions are usually numerous, uniform in size (typically ranging from 5 to 20 mm), and are purely osteolytic. In the literature, they are often described as “punched out” or “eaten by moths” due to their characteristic appearance. Multiple myeloma originates in bone marrow cells and initiates bone destruction from within, progressing outward to the cortical layer and potentially infiltrating surrounding tissues, such as the periosteum. The lesion may go undetected during visual examination if it has not yet reached the cortical layer of the bone. The disease progresses rapidly, and in advanced stages, individual lesions may merge, forming larger, irregularly shaped areas of bone destruction [13,17,41,48,53].

The lesions are most commonly found in the axial skeleton, including the cranium, vertebral column, pelvis (especially the iliac plate), ribs, and sternum. In the appendicular skeleton, they typically occur in the scapula and the proximal regions of the humerus and femur [19,43].

Multiple myeloma lesions can lead to pathological fractures, which may be identified in paleopathological skeletal remains. Reports from the literature describe such pathological fractures occurring in the ribs and vertebrae. The process of osteolysis within the trabecular layer of the vertebral body can increase the risk of compression fractures. While these lesions are non-specific, they may indicate the presence of multiple myeloma [26,31,56].

Multiple myeloma is more commonly observed in male populations over the age of 40, and according to Aufderheide et al. [61], this pattern is also reflected in skeletal remains. The epidemiology of the disease can aid in the differential diagnosis of other conditions with similar morphological features. Women between the ages of 25 and 29 years can be considered an exception, as multiple myeloma is the most probable diagnosis in this age group [16,21,22,28,40].

Differentiating multiple myeloma from metastatic cancer can be challenging due to their similar radiological features [62]. As knowledge in this field advances, the previously held belief that these conditions cannot be distinguished is increasingly being questioned. However, the diagnostic process becomes even more complex in cases of incomplete skeletal preservation and may be impossible when only a single element, such as the cranium, is available. Rothschild has critically reassessed previous research, concluding that, when modern diagnostic criteria are applied, none of the previously reported archaeological cases diagnosed as multiple myeloma meet the criteria for this disease; instead, they are more consistent with metastatic cancer [63]. Walter concludes that distinguishing metastatic carcinoma from multiple myeloma is possible by integrating information on the individual’s age and sex with detailed macro- and microscopic analysis of lesion appearance and distribution [64]. The authors proposed the following criteria summarized in

Table 2. Macroscopic criteria for differentiating multiple myeloma from metastatic bone cancer [49].

Characteristic	Multiple Myeloma	Metastatic Cancer
Shape	Round	Irregular with denticulation
Size	Constant	Variable
Distribution	Numerous and mostly symmetric	Less numerous and asymmetric
Surrounding area of lesions	Smooth	Pitted
Character of lesions	Pure osteolytic	Mixed (osteolytic and osteoblastic)
Localization on the spine	More often on the body of the vertebra	More often on the arc of the vertebra

.

Multiple myeloma primarily localizes in areas containing bone marrow in adults, which is why lesions found in the dorsal part of the spine, particularly in the pedicles, raise suspicion of a secondary malignancy. In modern clinical practice, the most common primary cancers that lead to bone metastases, in decreasing order of frequency, are breast cancer, prostate cancer, thyroid cancer, and lung cancer [32,34,47,49,50,51,54,65].

Other conditions to consider in the differential diagnosis include leukemia (more often in pediatric cases), primary bone tumors, Paget’s disease, trauma, thanatological changes, and infectious diseases, such as tuberculosis, syphilis, brucellosis, and fungal infections [24,25,33,37,45,46]. Advances in research techniques and the discovery of new paleo-oncological specimens allow us to better understand the causes of neoplastic development and highlight the need for further investigation into this topic.

3.2. Microscopical and Advanced Chemical Methods

Among advanced methods beyond macroscopic analysis and X-ray imaging, anthropologists use chemical and histological analyses, as well as fluoroscopy and microscopic methods, such as SEM and laser scanning microscopy (LCSM).

In the study of bone lesions, determining the trace element content can be useful, for which an Atomic Emission Spectrometer with Plasma Excitation (ICP-AES) is commonly used. Microscopic and histological methods are sometimes used to analyze changes present on skeletal remains. Němečková and Strouhal [42] investigated the usefulness of SEM, as well as confocal LCSM. In addition, they also applied toluidine blue staining to semi-thin sections, which were then analyzed by light microscopy (LM), and utilized histological methods using acridine orange staining. Their study focused on osteolytic lesions of the hip bone and skull [22,42,66].

Performing SEM requires electrical grounding of human remains. In addition, to prevent the accumulation of electrostatic charge during electrical irradiation, a conductive coating must be applied to the surface of the specimen. For this purpose, human remains are sometimes coated with a thin layer of conductive material, such as gold, platinum, or osmium. Once prepared, the specimen is placed in a scanning microscope for analysis.

LM is a widely used method in cancer diagnosis. One of the more technically demanding steps in this method is the precise cutting of tissue samples. Semi-thin sections, typically 1–2 μm thick, allow for a detailed view of the analyzed tissues. Before placing the sections onto a microscope slide, the tissue blocks are embedded in resin (Epon resin was used in the referenced study). The sections are transferred to the slide with a drop of water, and then heated to 60 °C. While on the heat source, a histological strain (toluidine blue) is applied. After rinsing with distilled water and drying, the prepared section is analyzed using a light microscope [42,66].

Another microscopic method for analyzing tissue lesions is confocal LCSM. This technique enables so-called optical sectioning, allowing for the visualization of structures at various depths within the sample without the need for physical slicing. LCSM also allows for the 3D reconstruction of selected elements. Compared to traditional physical cutting of bone in paleontology, LCSM offers easier sample preparation, reduces the risk of damaging delicate specimens, and produces high-quality images. In their research, Nemeckova and Stroughal utilized this method and stained bone samples with acridine orange to enhance visualization [42,67].

The use of microscopic methods in combination with histological methods in paleopathology enables the visualization of morphological changes in bones and the analysis of lytic lesions. Specific imaging methods, such as SEM and LCSM, allow for detailed examination of the bone destruction process in the course of conditions such as multiple myeloma. The examination of hip bone and skull fragments using these methods has made it possible to visualize changes in both the compact and spongy layers of bone. Using these methods, researchers were able to describe osteolytic changes characterized by brittle trabeculae and numerous irregularly shaped lacunae, which were attributed to a neoplastic process, specifically multiple myeloma [42,67].

Czech researchers used SEM to visualize lytic openings (of irregular shapes) and newly formed bone trabeculae in the skeletal samples (skull and fetal bones) from the 18th and 19th centuries [53]. According to the researchers, these changes may correspond to the progression of multiple myeloma, and their detection was made possible by this advanced analytical method [53].

The use of chemical analysis in the context of osteolytic changes has not been sufficiently described in the literature. However, such analysis can provide insight into the organic components of calcified tissues, which may be useful in anthropological studies [50].

Fluoroscopy (both anterior-posterior and lateral views) is sometimes used in conjunction with conventional X-ray imaging to enhance the visualization of bone lesions. In cases of multiple myeloma, such lesions are characterized by the obliteration of trabecular bone and the cortical layer. The resulting bone defects usually exhibit smooth surfaces and sharply defined edges. Researchers have described these types of lesions in various bones of the human skeleton, including the skull, ribs, vertebrae, tibia, femur, and fibula [45].

Molnar et al. mentioned the use of histological and immunohistological methods in paleopathological studies [38]. However, the authors did not provide a detailed description of the methods used, as they considered them supplementary to macroscopic analysis and radiographic imaging [38].

Advanced chemical analysis, such as ICP-AES, can be used to determine the degree of mineralization in cancerous tumors (e.g., metastatic prostate cancer). This technique enables both qualitative and quantitative determination of the presence of specific elements, including phosphorus, calcium, cadmium, and zinc [22]. It can be a useful tool for differentiating bone lesions from various cancers, including distinguishing multiple myeloma from metastases of other cancers.

Analytical methods using modern-generation microscopes are likely to play a significant role in the future of anthropological research. These techniques allow for the detection of bone lesions that may be invisible in macroscopic or radiological methods, especially those that are small or located deep within the bone. Broader access to chemical and histological analysis methods, as well as fluoroscopy and microscopic methods (SEM and LCSM), would enable more efficient and detailed identification of osteolytic lesions and facilitate more precise differentiation of their underlying causes.

3.3. Radiological Methods

Radiology is a valuable tool in the diagnosis and differentiation of multiple myeloma. Nowadays, there is a continual effort to simplify and expedite diagnostic processes. While radiology, as an advanced imaging technique, might be assumed sufficient on its own, it is not used as a standalone method in paleopathology. As a rule, relying on a single diagnostic approach is not considered good practice unless no other alternatives are available. Wasterlain et al. [57], Abegg and Desideri [19], and other researchers indicated that radiological imaging was preceded by a macroscopic evaluation of human remains [18,49]. X-ray imaging often reveals bone lesions that are smaller and more subtle than those identified solely through macroscopic observation; although, in some cases, macroscopic examination alone may be sufficient. According to the research by Strouhal, the accuracy of radiographic techniques in identifying bone changes is evident; of 65 bone lesions detected via radiography, only 45 were visible macroscopically [49]. It appears that trabecular changes can go unnoticed during visual inspection and can also be missed in X-ray imaging due to limitations in contrast resolution [18,49]. Unfortunately, the study by Biehler-Gomez and colleagues indicated that both of the aforementioned methods may sometimes fail to detect changes characteristic of multiple myeloma [18].

Radiology is a powerful diagnostic tool, and technological advancements have led to the development of increasingly accurate imaging methods, such as computed tomography (CT), magnetic resonance imaging (MRI), and micro-CT. However, their role in detecting bone-related conditions is not yet as widespread or established as traditional X-ray imaging. The accurate interpretation of radiological images requires a minimum bone density of about 40%, which can pose diagnostic challenges when this threshold is not met [18].

Many studies on bone cancers, including multiple myeloma, highlight the challenges of differential diagnosis. The main difficulty lies in distinguishing multiple myeloma from lytic metastases of solid tumors, especially those originating from the breast, lung, thyroid, and prostate. These conditions often show similar features in bone remains, although prostate cancer is easier to differentiate, as it typically presents with osteosclerotic (bone-forming) lesions, in contrast to the osteolytic (bone-destroying) lesions characteristic of multiple myeloma and other metastatic cancers [19,23,32,33,40,45,57].

Osteolytic metastases present a pattern of bone loss often described as resembling a “moth-eaten garment.” This appearance results from the degeneration of bone tissue and the rapid growth of metastatic lesions [40,49].

Additionally, the regions most commonly affected by these diseases are those containing active bone marrow, primarily in the following order: the vertebrae and sacrum, ribs and sternum, skull and pelvis, and the proximal ends of the femur and humerus [32,49]. However, this does not mean that the two conditions cannot be distinguished. Skeletal lesions in multiple myeloma tend to be more regular in shape and smaller in size, typically ranging from 5 mm to 2 cm in diameter. Multiple myeloma is also characterized by a greater number of lesions that are round or oval, with sharply defined, clear edges, surrounded by smooth, intact bone. In contrast, metastases are typically larger, with irregular or “ragged” edges. Additionally, in X-ray imaging, vertebrae affected by metastases may exhibit an “ivory bone” appearance [19,23,45,49,57].

Moreover, the location of the lesions within the bone itself is also diagnostically significant. Multiple myeloma rarely affects the vertebral pedicles, especially in the thoracic and lumbar regions, due to the lack of active bone marrow in these areas. The analysis of the type and distribution of lesions, combined with information about the individual’s sex and age, can facilitate the diagnostic process [32].

Based on our review of the literature, we found that researchers, drawing on both published sources and their own experience in pathology, most commonly assess nine criteria when differentiating between metastatic lesions and multiple myeloma [28]. These criteria are not limited solely to radiography but also involve various methods of bone evaluation. The criteria include lesion density, shape and size of the lesions, macroscopic assessment, the appearance of lesion margins, the presence or absence of sclerotic reactions, the sex of the individual, and lesion location. One criterion specifically pertains to radiography: the assessment of lesion margin morphology. In multiple myeloma, the margins are sharply defined, appearing as if “cut out”, whereas in lytic cancer metastases, the image is cloudy and obscure, resembling a “moth-eaten” pattern [49].

Lloret et al. developed diagnostic and differentiation guidelines for multiple myeloma that include the following criteria: site of origin (extraosseous versus intracalvarial), lesion size, pattern of bone destruction, and characteristics of the lesion margins [68].

In conclusion, the use of radiological methods is highly beneficial in the diagnosis of multiple myeloma. However, diagnosing skeletal remains from ancient times poses significant challenges for researchers due to several complicating factors. Foremost among these is the incomplete and often deteriorated condition of the skeletons, which makes it difficult to image lesions in specific anatomical regions. Furthermore, taphonomic processes can cause alterations and distortions in the bone, complicating radiological interpretation [23].

Thanks to increasingly advanced radiographic techniques, researchers frequently rely on these methods in paleopathological investigations. Bauduer et al. consider CT to be the most accurate tool for assessing bone material. However, newer radiological imaging methods have not been sufficiently described in the literature [16].

After reviewing the studies, it is evident that while X-ray imaging provides more details than macroscopic examination, CT offers even greater precision, which could aid in the differentiation and diagnosis of multiple myeloma. However, the literature does not indicate that radiology has become a standalone method for diagnosing this condition. As the field continues to develop, radiological techniques, particularly CT, with its capacity to generate 3D models, show significant diagnostic potential. Once implemented, the use of radiological methods is likely to become increasingly common.

4. Discussion

Diagnosing multiple myeloma in paleopathological research involves a variety of established and emerging techniques, each contributing to a more nuanced understanding of skeletal pathology within the constraints of archaeological contexts. Macroscopical analysis of skeletal remains is the most widely used method, focusing on the evaluation of lesion size, shape, margins, presence of osteolysis or sclerosis, and anatomical location of lesions. In multiple myeloma, lesions are typically round, have smooth margins, are osteolytic, and mostly commonly occur in the axial skeleton. An important challenge is distinguishing between multiple myeloma and metastatic cancer, as their skeletal manifestations can be similar. Differentiating multiple myeloma from non-neoplastic conditions is also an important aspect of the diagnostic process. Ragsdale et al. [69] listed several conditions that can be easily mistaken for cancer, including cysts, developmental abnormalities, hyperostosis or Paget’s disease of bone, rheumatic diseases, infectious diseases affecting the skeleton, and trauma with its subsequent healing. Equally important is differentiation from pseudopathologies, which result from post-mortem changes that alter the condition of the bone [61].

Other methods, such as radiography, are invaluable for detecting lesions that are not visible macroscopically, particularly those that are smaller or located deeper within the bone. X–ray, MRI, CT, and micro-CT provide high-resolution imaging that complements macroscopic findings and offers critical insights into lesion morphology. Among those, X-ray is the most commonly used radiological method, with multiple myeloma lesions often displaying characteristic “moth-eaten” patterns. Radiological have significantly enhanced diagnostic accuracy in paleopathology and are most effective when integrated with macroscopic and contextual analysis.

Advanced microscopic and chemical techniques, although used less frequently, offer an additional level of diagnostic accuracy. LM, despite its relatively complex preparation process, is frequently used to analyze bone microstructure. In paleopathology, combining microscopic and histological methods allows for the detailed visualization of morphological changes in bones and the analysis of lesions. Scanning electron microscopy (SEM) and confocal LCSM enable visualization of detailed morphological changes and microlesions that may not be visible through macroscopic or radiological methods. Fluoroscopy, often used alongside X-ray imaging, provides further depth in lesion assessment. Although chemical analysis is less frequently reported in the literature, methods such as ICP-AES have demonstrated promising potential in differentiating lesions, illustrating the growing role of interdisciplinary approaches in paleopathological diagnosis.

This review has several limitations. Although predefined inclusion and exclusion criteria were applied, the review followed a narrative rather than a systematic approach, which may introduce a degree of bias. A comprehensive search strategy was not employed, potentially limiting the reproducibility and transparency of the review process. Additionally, a formal risk of bias assessment was not conducted, as many of the included studies were single-case descriptions. Considerable heterogeneity among the studies reflects the diversity of approaches in paleopathological research, which is shaped by the availability of methods, preservation conditions, and ethical guidelines. While this variability complicates direct comparisons, it also highlights the adaptive and interdisciplinary nature of the field. Moreover, the search was not restricted by geographical region or chronological period, which may have introduced additional variability and skewed the interpretation of the historical prevalence and presentation of multiple myeloma.

Finally, paleopathological research faces a number of inherent limitations that significantly impact the scope and depth of diagnostic investigations. Access to advanced imaging and analytical technologies—such as micro-CT, 3D modeling, or biochemical assays—is often limited or entirely unavailable, particularly in regions where medical and research infrastructure is underdeveloped or underfunded. Financial constraints further restrict the feasibility of using high-cost diagnostic tools, which are rarely prioritized in archaeological research budgets. Legal and regulatory barriers also play a critical role; in many cases, laws prohibit the transport or sampling of skeletal remains across institutions, cities, or national borders. Time constraints imposed by excavation permits or contractual obligations may also limit the extent of analysis that can be performed. Moreover, ethical considerations—especially those concerning the destructive nature of certain analyses, such as histological or biochemical sampling—necessitate careful deliberation and often preclude invasive methods. These constraints help explain why macroscopic evaluation remains the primary diagnostic tool in paleopathology, despite the recognized benefits of advanced techniques. Acknowledging these limitations is essential for a realistic understanding of the field and its methodological choices.

5. Conclusions

Compared to previous reviews, this study offers a broader chronological range and employs a more comprehensive methodological approach to the detection of multiple myeloma in paleopathological research. It emphasizes that macroscopic analysis remains the foundation of diagnosis in paleopathology, with radiological and microscopic techniques increasingly contributing to more nuanced interpretations and comparative assessments of historical cases. Incorporating advanced diagnostic techniques through a multidisciplinary approach, including macroscopic, radiological, microscopic, and chemical analyses, is essential for advancing research in this field. By promoting an integrated, multidisciplinary methodology, this study contributes to the growing body of work guiding future paleopathological investigations of multiple myeloma in diverse archaeological contexts.