InDels can be used as a genetic marker in forensic sciences [12, 16]. They are very common in the genome, provide successful results in degraded samples due to their small amplicon size, and have a lower mutation rate than STRs. The methods used for the analysis of STRs can also be used for InDels analysis. Therefore, InDels have been observed to be more expedient than STRs [13, 16, 24].

Loss of heterozygosity is characterized by mutation of one allele followed by deletion of the remaining allele [15]. In addition, the loss of heterozygosity also differs according to the amount of peak intensity. To be considered as complete loss of heterozygosity (cLOH), the peak intensity should be less than 0.5 and higher than 2, while partial loss of heterozygosity (pLOH) occurs when the peak size of an allele decreases by more than %50 [2, 25].

Effect of the paraffinization process on DNA analysis has been investigated in various studies before. In one recent example, Vitosevic showed that 3 gene regions, which are often used as reference genes in genetic analysis studies, can be amplified in paraffin blocks for up to 30 years [26]. Indeed, the fixation and embedding process causes degradation of DNA due to its fragmentation and chemical modifications [27, 28]. These modifications may occur due to mechanisms such as tissue aging over time in the fixative, hydrolysis of DNA with formic acid, and formation of intramolecular methylene bridges [29]. Covalent cross-links formed during fixation can interfere with extraction and downstream applications, impacting PCR, sequencing, and molecular analyses. Additionally, the fixation process often reduces overall DNA yield, posing challenges in high-throughput applications or when working with limited tissue samples. These limitations highlight the complexity and constraints associated with using FFPE tissues for DNA studies [30, 31]. In our study this degradation effect manifested itself as a randomly distributed reduction in peak sizes or complete loss of alleles in some of the InDel loci both in tumoral and non-tumoral blocks, as well.

In many studies conducted with paraffin embedded blocks, it is reported that there is a decrease in the amount of DNA as well as in the quality of DNA [27, 28]. In order to cope with the disadvantages of using tissues embedded in paraffin blocks, procedures such as adjusting the coverage numbers, the amount of DNA template analyzed, pretreatment methodologies, optimizing proteinase K digestion conditions or simplifying DNA extraction procedures can be implemented [32, 33]. Kerick et al. showed that there was no difference between FFPE and snap frozen tissues in terms of detection of InDel mutations because of increasing the coverage levels to 40 × [34]. In one study, they reported that successful results were obtained by adding 30 ng of DNA template to the PCR reaction [35]. In our study, this problem is solved by increasing the amount of DNA. In certain samples, diluting DNA to 1 ng/µL, following Filoğlu’s study [22], yielded a complete profile, while in other cases, profiles with numerous missing alleles persisted even with 2 ng/µL or 3 ng/µL DNA. In these cases, the PCR and electrophoresis protocol was repeated by adding the DNA undiluted to the PCR mixture to obtain a complete profile. This adjustment proved effective because, even during fixation, certain DNA chains endure in an intact state. The increase of template DNA quantity enhances the likelihood of amplifying intact DNA chains, thereby achieving a more comprehensive and accurate profiling of the genetic material. In these cases, a complete profile could be obtained by adding DNA without dilution. As a result, an overall profile of 88.7% could be obtained in tumor and non-tumor tissues.

From another perspective, although they show a certain degree of degradation, FFPE tissues are attractive tools for clinical studies because they are much easier to obtain than frozen tissues. However, this degradation effect may not cause a limitation as expected [19]. Although Oh et al. obtained lower yielding and mapping results in FFPE samples compared to frozen samples in their exome sequencing studies, they showed that this did not have a statistically significant effect. Moreover, they showed that frozen samples could lead to higher off-target rate determinations compared to FFPEs, thus pointing to an advantageous aspect for FFPE tissues. They attribute this to DNA fragments that shorten with fragmentation and say that this will increase on-target coverage [36]. This study’s primary aim is to determine whether the identification success of the InDel multiplex panel exhibits any difference between tumor tissues and normal tissues. In this retrospectively conducted study, the use of paraffin blocks as controls could have quickly demonstrated differences between normal and tumoral tissues. Indeed, in a study similar to ours, Oliveira and colleagues utilized tumor-free tissue within the tumor tissue block as a control [35].

There was no significant difference between the degradation rates in terms of two tumor types we studied or whether the tissue contains tumoral or non-tumoral tissue. These ratios, which were between 6.2% and 7.5% in both cases (See Table 2). Similarly in a study by Soo et al., small-sized DNA or RNA fragments were amplified successfully in formalin-fixed paraffin-embedded samples after they were kept for several years [37]. Conversely, in a study by Guyard et al., they show that after 4 to 6 years the strong decrease in the amount of amplifiable DNA is due to fragmentation of DNA [38]. In our study, there were medium-long waiting times distributed between 1 and 38 months compared to these studies, and more than 90% matching was possible (Supplementary Table 1).

As reported at "Results" section, the mutations were detected in only six of 21 thyroid cancer cases. Moreover, in these six cases, mutations were detected at only 1 or 2 loci. This finding suggests that archival FFPE tissues of thyroid tumor can be used in cases of forensic genetic identification. However, the presence of mutations in 21 of the 26 breast cancer cases examined suggests that paraffin blocks, in which this cancer type is detected, should be used carefully in forensic genetic identification. The most mutations observed in the ID24(rs1610919), ID26(rs2308163), and ID16 loci(rs16458), along with the prevalence of cLOH, results from these loci should be treated with suspicion. Particularly in malpractice cases, it is essential to acknowledge that such variations in these loci can be dismissed. However, as reported by Nam SK et al., depending on the nature of the case, it is still possible to make a reliable comparison in cases with a small number of mutations [37]. In a study where Pereira et al. used 38 non-coding bi-allelic indel markers on different populations, they found random match probabilities (RMP) to be 1 in 10-17 billion even for the 25 most informative markers. Accordingly, even if an incomplete profile is obtained, the possibility of identification is high [19].

Turajlic et al. reported that mutations at renal cell carcinomas are twice as much as all other cancer types and breast cancers have higher averages than thyroid cancers in terms of both InDel numbers and InDel ratio [39]. Similarly, we observed higher instability rates in breast cancer cases than in thyroid cancer cases. On the other hand, we found that the number of mutations did not show a relationship with the grade for breast cancers or the stage for thyroid cancers. Moreover, no difference was detected according to the storage time of the blocks and the age of the cases. In this instance, it does not seem possible to say with certainty, type of malignancy may play an important role in the rate of mutational changes. Indeed, Wu et al. suggested that the number of InDel mutations which is described as ‘Tumor Mutational Burden (TMB)’ may differ between cancers and associated with prognosis and treatment response [40]. Unlike this study, which took into account the coding regions, our study showed the effect of cancers on the analysis in terms of forensic identification, as it was performed on non-coding loci.

An additional significant result indicated by our findings is that, when identification is required using an InDel panel from FFPE tissues, it is preferable to extract material from a non-tumor block rather than a tumor block. No similar study has been reported in the literature. In the closest study to ours, Oliveira et al. detected InDel mutations in the CDH1 gene that were not present in normal tissue in cases of Hereditary Diffuse Type Gastric Carcinoma [35]. In their study, they dissected and compared both tumor and non-tumor tissue from the same FFPE block. Although their study focused on a gene that codes differently from ours, our findings underscore the preference for selecting these blocks whenever normal tissue is available. Tumorous FFPE tissues may be utilized in samples such as needle biopsy where no normal tissue block is present. It is essential to determine the mutation dynamics in cancer types for this to be possible.

Cancerous transformation in tissues may adversely affect the success of forensic identification studies. It has been shown by previous studies that cancer can accompany disruptions in STR loci [17, 41, 42]. For example, Peloso and his colleagues worked with archival FFPE tissues taken from 24 people with lung cancer as well as corresponding normal tissues obtained from lymph node sections of the same patients. Although allele drop-out was observed in at least one STR in 20 of 24 samples, allelic imbalance was observed in the STR loci of all samples. Additionally, a small portion of the samples showed loss of heterozygosity [13]. Also, Anaian et al. observed DNA profiles from 12 gastric,12 breast and 10 colorectal formalin-fixed paraffin-embedded tissue (FFPET) samples, revealing 55 cases of partial loss of heterozygosity (pLOH), 15 cases of complete loss of heterozygosity (cLOH), and 13 cases of microsatellite instability (MSI) [2]. On the other hand, other identification markers (e.g. SNPs, InDels) have been also tested in tumor samples [25, 43]. In the study of Tozzo et al., 61 (92.4%) of 66 frozen tumor samples (hepatic, gastric, breast, and colorectal cancer) were found to have at least one mutation in InDel loci [25]. Majority of these mutations were LOHs of which 41.7% were only partial (pLOH) and MSI events constituted only 20.6% of all the mutations. These authors argued that because of these ratios, the use of STR loci for identification purposes would be more appropriate than InDel loci. On the contrary, in a study by Zhao et al., only pLOH and cLOH mutational events of InDels loci were observed in fresh tumoral tissue samples (colorectal and gastric cancer). The total mutation rate of InDels was 0.25% in tumoral tissues [43]. Authors suggested that InDels might be more powerful than STR in source identification; unlike the results reported by Tozzo et al.

Our results support the view that MSI is the least common type of InDel mutations in tumoral tissues. While only 22,35% of the mutations are of this type, the vast majority are LOH mutations. Moreover, in 27 of 47 cases, a mutation was detected in at least one of the loci compared to non-tumoral tissues (See Table 3).

A problem we are dealing with was more missing alleles in the profiles of the non-tumoral breast tissue samples which can be due to the high density of adipose tissue that leads to losses in terms of DNA yield. Similar to our study, in a study conducted by McDonough et al., it is stated that there is less DNA in non-tumoral breast tissues than the corresponding tumoral part [28]. In the study conducted by Mee et al., it was observed that since non-tumoral breast tissue consists of almost completely fat, it has less DNA, RNA and protein amount compared to tumoral breast tissue [44].

A limitation of our study is that it was carried out retrospectively on archival FFPE tissues. The degradation effect of paraffinization will be able to be determined more clearly in future studies using fresh biological tissues as the control group. In prospectively planned studies, the effects of prognostic variables regarding the tumor on both degradation and detection of mutations can be determined. The effectiveness of the panel can be increased by determining tumor-specific mutation distributions (Table 5).

As a result, complete profiles of 36 InDel loci were obtained in the majority of paraffinized tissues, both tumoral and non-tumoral. Multiple mutations were seen in some of the cases, while no mutations were found in almost half of the cases. In the vast majority of cases with mutations, only one or two mutations such as loss of heterozygosity, partial loss of heterozygosity and InDels instability were observed. These low InDel mutation rates compared to STR instability, make InDel analysis from paraffin blocks suitable for forensic genetic identification. However, researchers should keep in mind that there may be differences between the profiles of the tumoral tissues taken as reference and the actual case. In addition, by incorporating additional markers such as Single Nucleotide Polymorphisms (SNPs) and microhaplotypes with low mutation rates into the study alongside Indels, researchers can significantly enhance the discrimination power in identification processes. Low mutation rates reduce the likelihood of errors or inconsistencies in the analysis, ensuring the accuracy of the results over time. The combination of Indels, SNPs, and microhaplotypes creates a multi-marker approach that significantly boosts the discrimination power in identification processes. This increased power allows for more precise differentiation between individuals, particularly in forensic applications or population genetics studies.