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Concurrent loss of MLH1, PMS2 and MSH6 immunoexpression in digestive system cancers indicating a widespread dysregulation in DNA repair processes

Immunohistochemical analysis of mismatch repair (MMR) protein expression is widely used to identify tumors with a deficient MMR (dMMR). MMR proteins (MLH1/PMS2 and MSH2/MSH6) work as functional heterodimers, which usually leads to the loss of expression in only one functional MMR heterodimer. Recently, there have been studies showing the simultaneous loss of immunoexpression in proteins of both heterodimers. Yet, this phenomenon has been rarely investigated. In this study, we retrospectively considered cases of different digestive system cancers (gastric cancer, ampullary cancer, small bowel cancer, colorectal cancer), which were immunohistochemically tested for dMMR within a 4-year period at our university hospital (n=352). Of the 103 cases showing dMMR, 5 cases (1.4% of all, 5.1% of dMMR cases) showed a concurrent loss of MLH1, PMS2 and MSH6 immunoexpression, whereas in the other 98 dMMR cases only one MMR heterodimer was affected. MLH1-/PMS2-/MSH6- cancer cases almost arose throughout the entire digestive tract: from the gastric antrum to the left colic flexur. To provide a comprehensive molecular characterization of this MLH1-/PMS2-/MSH6- immunophenotype, tumors were analyzed for microsatellite instability, MLH1 promotor hypermethylation and BRAF exon 15 status. Furthermore, we performed next-generation sequencing focusing on genes related to DNA repair. Here, we could detect pathogenic germline variants as well as multiple sporadic mutations in different genes involved in MMR and homologous recombination repair (HRR) respectively. The affected MMR/HRR-related genes were: ATM, BARD1, BRCA1, CDK12, CHEK1, CHEK2, FANCA, MLH1, MSH6, PALB2, TP53. Considering the biologic function of HRR/MMR proteins as potential drug targets and the low frequency of most of these mutations in digestive system cancers in general, their common occurrence in our MLH1-/PMS2-/MSH6- cases seems to be even more noteworthy, highlighting the need for recognition, awareness and further investigation of this unusual IHC staining pattern.


Introduction
DNA mismatch repair deficiency (dMMR) is routinely studied by immunohistochemical staining via analyzing the expression of MMR proteins (MLH1/PMS2 and MSH2/ MSH6). As MLH1/PMS2 and MSH2/MSH6 work together as functional heterodimers (1) and as PMS2 and MSH6 are unstable in absence of MLH1 and MSH2 respectively, there is usually only one DNA MMR subsystem affected in malignancies with dMMR. However, there exist also rare unusual cases with defects in both MMR subsystems, which have been described in recent studies (2)(3)(4)(5)(6) and coincide with our clinical practice. Furthermore, there are also digestive system cancer cases that show a heterogenous loss of MMR protein expression, which is also a rarely studied phenomenon (7)(8)(9).
With MLH1 promoter hypermethylation being the most common reason for dMMR (10), the most widespread immunohistochemical pattern is the loss of MLH1 and PMS2 expression (11). Simultaneous loss of MLH1 and PMS2 expression is also the most common pattern in Lynch syndrome based on MLH1 germline mutations, followed by a MSH2/MSH6 loss due to MSH2 germline mutations. In much lower frequency, isolated losses of PMS2 (12) or MSH6 (8,13) occurred in patients with LS due to the corresponding germline mutations. As cells that are unable to correct DNA replication mistakes accumulate errors, dMMR induces microsatellite instability (MSI). Typically, dMMR and MSI-high (MSI-H) are used nearly interchangeably, as there is a high level of consistency (14). Plenty is known about the frequency, aetiology, prognostic (15,16) and predictive (17, 18) implications of dMMR/MSI-H status. However, there are only few studies focusing on the special subgroup of malignancies with an unusual immunophenotype, namely the loss of expression in both MMR functional heterodimers (MLH1/ PMS2 and MSH2/MSH6) (2)(3)(4)(5)(6). In general, MSI is a frequent phenomenon in different digestive tract cancer entities like colorectal carcinoma (CRC) or gastric cancer (GC), which both contribute extensively to the burden of disease globally (19). Thus, we included four different digestive tract cancers: small bowel carcinoma, CRC, GC and ampullary cancer. These cancer entities have very different prevalences but all display a significant proportion of MSI. For small bowel carcinoma widely differing MSI rates exist in the literature: from approximately 23% (20) up until 45.5% (21). GCs display a MSI frequency of 9-19% and CRCs of 6-19% (22). Finally, ampullary cancers possess a MSI frequency of about 6% (23) to 10.4% (24). Whereas MMR acts during DNA replication to correct for polymerase errors, MMR is also closely linked to homologous recombination repair [HRR (25)], a mechanism, that constantly repairs double-strand breaks in DNA. Considering this overlap of MMR and HRR and with the advent of drugs targeting DNA repair in clinical practice for other cancer entities, especially PARP-inhibitors for BRCAmutated carcinomas (26,27), we proposed that our subgroup with a dysregulated MMR immunophenotype may be also a suitable cohort to search for additional defects in DNA repair processes. As novel therapies targeting other HRR pathway components than BRCA1/2 like ATM, ATR or CHEK1/2 are now under clinical investigation (28, 29), working out subgroups likely to benefit from such drugs will be crucial.
We aim to shed light on features of this peculiar phenotype with an involvement of both MMR subsystems. Therefore, the aim of this study was a comprehensive molecular characterization of digestive system cancer cases with concurrent, immunohistochemically detectable deficiencies in both DNA MMR subsystems. Besides analyzing the frequency, providing clinical characteristics and performing conventional histology, immunohistochemistry, PCRbased MSI testing, testing for MLH1 promotor hypermethylation and BRAF exon 15 mutation, we additionally applied nextgeneration sequencing (NGS) focusing on genes related to DNA repair to further understand the molecular basis of dysregulated MLH1/PMS2 and MSH2/MSH6 expression among different digestive system cancer entities.

Ethical approval and patient cohort
The study was approved by the ethical committee of Ludwig Maximilian University (LMU) of Munich (reference: project number 22-0381) and was performed in accordance with the Declaration of Helsinki. Prior to enrolling cases with an involvement of both MMR subsystems into the study and performing further molecular testing, the affected patients gave written informed consent.
The entire cohort to identify cases of interest consisted of 352 cases, that underwent immunohistochemical testing for dMMR at the University Hospital Augsburg between January 2018 and February 2022 (4-year period) with the two-stain method, initially only testing for PMS2 and MSH6 expression (30,31). In case that both markers were negative initially (PMS2 -, MSH6 -), the two missing MMR markers (MLH1, MSH2) were always stained additionally. The cohort was assembled by a retrospective database search of our institution's internal laboratory information system. As stated, we included different digestive system cancer entities each with a considerable MSI frequency: 300 CRCs, 41 GCs, 9 ampullary cancers and 2 small bowel carcinomas. As the simultaneous deficiency in both MMR subsystems is a rare eventpresumably about 1-5% (3, 5, 6), we tried to study a broad variety of cases in order not to miss any cases. Hence, we included a) primary cancers as well as metastases, b) biopsies as well as resected specimens, c) patients with and without neoadjuvant chemotherapy, d) different tumor stages (T1 to T4), and e) different histologic subtypes e.g. in CRC mucinous, medullary or signet-cell, in GC intestinal or diffuse and in ampullary cancer pancreatobiliary as well as intestinal type. Patients had a median age of 70 years (25% quantile: 61; 75% quantile: 79) with a balanced male/female-ratio of 175/177. After informed consent, we completed molecular testing (if missing: MSI-PCR, MLH1 promotor methylation and/or BRAF status) on the formalinfixed paraffin-embedded (FFPE) material from these peculiar cases with a dysregulated expression in both MMR subsystems, and additionally performed NGS with a focus on HRR genes. Mannheim, Germany). Nuclear staining throughout the whole tumor that is predominantly stronger in intensity than that of the internal control was considered as proficient MMR (pMMR). Any deviation from this staining pattern constitutes an abnormal pattern; especially the loss of nuclear expression of MSH6 or PMS2 is typical for dMMR cases. Partial or heterogeneous loss of expression was defined according to the criteria published by Joost et al. as tumors showing either zonal loss or so-called intraglandular heterogeneity (9). A strong homogeneous nuclear staining of non-neoplastic cells served as an internal control. Adequate controls were used for quality control of staining. All IHC slides as well as conventional H&E stains were digitized using a 3D Histech Pannoramic Scan II.

MLH1 promotor methylation status
DNA was extracted from paraffin-embedded tissue by using the Maxwell ® FFPE Plus DNA Kit (Promega, AS1135 [Madison, Wisconsin, USA]) following the manufacturer's protocol. DNA methylation patterns in the CpG islands of hMLH1 gene was determined by chemical treatment with sodium bisulfite (Zymo EZ DNA Methylation Kit #D5001) and subsequent methylationspecific polymerase chain reaction (MSP, MS-PCR) as described (32). Primer sequences for hMLH1 for unmethylated reaction were 5'-AGAGTGGATAGTGATTTTTAATG-3' (forward) and 5'-CCTCATACTCACATTCTTCCT-3' (reverse), and for the methylated reaction were 5'-AGCGGATAGCGATTTTTAACG-3' (forward) and 5'-AAACGTCTAAATACTCAACGAAA-3' (reverse). All PCRs were performed with positive controls for both unmethylated and methylated alleles and no template control. The amplified fragments were separated on a 1.5% agarose gel. Here, PCR yields a 202 bp amplicon in the presence of a hypomethylated MLH1 promoter. In the presence of a hypermethylated MLH1 promoter, a 248 bp product is amplified.

Microsatellite analysis
For MSI molecular test was performed on normal and tumor DNA extracted from paraffin-embedded tissue by using the Maxwell ® FFPE Plus DNA Kit (Promega, AS1135) following the manufacturer's protocol. Corresponding normal and tumor DNA were investigated with a set of 5 microsatellite markers (BAT25, BAT26, D5S346 [APC locus], D17S250 [p53 locus] and D2S123) by multiplex amplification. These were subsequently separated by capillary electrophoresis using a SequStudio Genetic Analyzer (Applied Biosystems, Waltham, Massachusetts, USA), and analyzed by GeneMapper software v4.1 (Applied Biosystems). The performed examinations and analysis of the data were performed according to the guidelines of the study protocol of the collaborative project "Familial Colorectal Cancer" of the German Cancer Aid and correspond to point 5 of the guidelines for HNPCC (Hereditary Non Polyposis Colorectal Carcinoma) clarification (www.krebshilfe. de). MSI was defined as any marker with the highest peak shifted more than two base pairs when compared to the same marker in the normal sample. MSI-H (MSI-high) is present if at least 40% of the microsatellites of the biomarkers (2 or more) of the NCI panel (BAT25, BAT26, D2S123, D5S536, D17S250) show microsatellite instability (33).

Sanger sequencing
Sanger sequencing was performed on tumor samples using 50 ng of genomic DNA with 0.83 µM of each primer, 0.6 U Ampli-Taq GOLD (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 10 × Buffer II (Thermo Fisher Scientific) an 0.2 mM each dNTP. Cycling conditions were denaturation an 95°C for 10 min, followed by 40 cycles of 30 s 94°C, 30 s of 60°C and 30 s of 72°C, with final elongation at 72°C for 10 min.

NGS HRR Panel
NGS was performed on normal and tumor tissue. For DNA extractions from FFPE samples, tumor areas were marked on hematoxylin-eosin (H&E) stained slides by a pathologist. The corresponding area was macrodissected from two to three 10 µm sections with at least 20% tumor cellularity. For the library preparation of this study the multiplex PCR based AmpliSeq for Illumina Custom Panel (HRR IPMD; Illumina [San Diego, CA, USA]) was used (Custom designed panel, this panel is available for research use only). The panel consists of 1294 primer pairs for the detection of hot-spot regions and full-genes in 25 HRR-related genes with a targeted size of 101654 bp. Amplicon library preparation was performed using approximately 10 to 100 ng of DNA, as recommended by the manufacturer. In brief, the DNA was mixed with a primer pool 1 and 2 containing all primers for generating the amplicons and with the AmpliSeq HiFi master mix. The mastermix was transferred to a PCR cycler. PCR cycling conditions were initial denaturation at 99°C for 2 minutes, followed by 21 cycles of 99°C for 15 seconds and 60°C for 4 minutes. After the end of the PCR reaction, primer end sequences were partially digested using FuPa reagent according to the manufacturer's instructions; this step was followed by the ligation of barcoded sequencing adapters (AmpliSeq CD Indexes Set A, for Illumina Technologies). The final library was purified using AMPure XP magnetic beads (BeckmanCoulter, Krefeld, Germany) and was quantified using QuantiFluor ® ONE dsDNA System (Promega) The individual libraries were diluted to a final concentration of 11 pmol/L, and libraries were pooled and processed for sequencing by synthesis using a MiSeq reagent kit V2 (300 cycles) on a MiSeq System (Illumina).
Data analysis: Secondary analysis was performed using the application Generate FASTQ (Version 2.0.01.17; RUO) and IAA26013167 manifest (Version 1; RUO) on the Local Run Manager (Version 1.0.0.7; Illumina). The reads were aligned to the human reference sequence build hg19. Detection of SNVs and indel polymorphisms, relative to the human reference sequence, was performed using the BaseSpace Variant Interpreter (Illumina). Filtering was performed for nonsynonymous and non-polymorphic alterations. The detection limit of variant allele frequency is 1%. In addition, the data were analyzed using the BaseSpace Knowledge Network and the variants were documented accordingly. Interpretation of the alterations was performed using different databases according to ACMG guidelines (34), such as cbioportal.org, ClinVar, Varsome, and CiVIC. Classification of BRCA1/2 mutations is based on the six public databases ARUP (Association of regional and University Pathologists Inc.), BRCA Exchange

Results
Frequency of concurrent loss of MLH1/ PMS2 and MSH6 expression among different digestive system cancer entities A total of 352 cases of digestive system cancers from January 2018 to February 2022 from Augsburg University Hospital, for which immunohistochemical testing for MMR proteins was routinely performed in clinical practice, were retrospectively analyzed. Whereas 249 (70.7%) cases showed a proficient DNA MMR (pMMR), 103 cases (29.3%) displayed deficiencies in DNA MMR. In the vast majority of dMMR cases (n=98), only one MMR heterodimer, either MLH1/PMS2 or MSH2/MSH6, was affected. However, overall five cases (1.4% of all cases, 5.1% of cases with dMMR) presented with a concurrent loss of MLH1/ PMS2 and MSH6. Interestingly, we could only observe cases with a concurrent loss of MLH1/PMS2 and MSH6 expression and no other immunophenotype with an involvement of both functional MMR heterodimers. The whole study sequence is shown in the patient flow chart in Figure 1.

Clinicopathological findings
The clinicopathological features of the five MLH1 -/PMS2 -/ MSH6cases are summarized in Table 1. Most patients (4 out 5, 80%) were female, with ages at diagnosis ranging from 63 to 82 years. The sites of cancer origin were distributed throughout the digestive tract (gastric antrum, ampulla vateri, caecum, colon transversum, left colic flexur). We could not identify any rectal cancer with a concurrent MLH1/PMS2 and MSH6 loss. Only one included case (case 5) received neoadjuvant therapy prior to resection. Regarding TNM classification, the five cases were heterogenous with T-stages ranging from 2 to 4a, one case with nodal involvement and two cases with distant metastases. As all patients gave informed consent, all patients were still alive at the time of the study. Cases 2, 4 and 5 were initially diagnosed in 2019, case 3 in 2020 and case 1 in 2021. Except the adenocarcinoma of the caecum of case 3 (low-grade, formerly moderate differentiation, G2), all cases were high-grade (formerly poorly differentiated, G3). Cases 4 & 5 were medullary carcinomas of the colon, whereas cases 1 to 3 were adenocarcinomas of different subtypes. The histology (H&E stain) of the MLH1 -/PMS2 -/MSH6cases is shown in   Study flow chart. 352 digestive system cancer samples underwent immunohistochemical testing for MMR deficiencies over a 4-year period at our hospital (University Hospital Augsburg, Germany). Only, five MLH1 -/PMS2 -/MSH6could be identified. Consecutively, additional extensive molecular testing including a NGS panel focusing on genes involved in MMR/HRR was applied on these cases.

Immunohistochemistry and conventional molecular pathology
As already depicted in the study flow chart (Figure 1), we could detect five cases with a concurrent loss of immunostaining for MLH1, PMS2 and MSH6 ( Figure 2  H&E as well as MMR IHC of MLH1 -/PMS2 -/MSH6cancer cases arising in the digestive tract (100x magnification). Whereas MSH2 staining was preserved, MLH1 & PMS2 were completely absent in all cases. Cases 1&5 presented with a heterogeneous staining pattern for MSH6 immunoexpression with only a partial loss. In all IHC slides, nuclear staining of stromal cells as well as infiltrating lymphocytes served as internal positive control. Genes with variants that have not only be identified in tumor but also in normal tissue (germline) are highlighted in bold. Interpretation as well as molecular consequences of the variants are depicted in Table 3 Reitsam et al.  Table 3.
We further evaluated the genetic content of our observed variants regarding the involvement of mononucleotide repeats (Table 4).
In case 1 an accumulation of mutations in microsatellitestypical for dMMR cancerscould be observed. To put our findings into a broader context, we extracted the frequency of the detected mutations from publicly available datasets using cBioPortal [https://www.cbioportal.org (36, 37)]. The involved genes that have been mutated in our five cases have already been described for the corresponding malignancies (Table 5). Except TP53, these genes are mutated in only a small proportion of the respective cancer entities, making their common occurrence even more special (38,39).

Discussion
Immunohistochemistry for MMR proteins (MLH1, PMS2, MSH2, MSH6) is widely used in clinical practice and does not only bear potential implications for the whole family of patients in cases of underlying germline variants but is also of predictive value for response to immune-oncology (IO) therapy (17, 18). Besides usual staining patterns for MMR proteins, there further exist rare staining patterns, which are known to be a peculiar phenomenon, on which there is only limited literature (2)(3)(4)(5)(6)(7)(8)(9).
To shed light on the simultaneous loss of expression in both functional MMR heterodimers (MLH1/PMS2, MSH2/MSH6), we retrospectively analyzed 352 cases of different digestive system cancer entities with differing dMMR frequencies (CRC, GC, small bowel carcinoma & ampullary cancer). We could identify five cases with a concurrent loss of MLH1/PMS2 and MSH6 expression. Our study demonstrates that MLH1 -/PMS2 -/ MSH6digestive system cancer cases are a rare subgroup of dMMR cancers, that account for 1.4% of all cases in our cohort and do not only occur in CRCs but also in ampullary as well as gastric cancer. As far as we know, this staining pattern has not been described for ampullary or gastric cancer so far. Interestingly, we could only find the concurrent loss of MLH1/ PMS2 and MSH6 staining, and no other combination affecting both heterodimers. Even though there are few case studies describing a so-called 'null-phenotype' (2, 5), we could not observe a single case with a loss of expression in all four MMR proteins in our cohort. Furthermore, MSH2 expression was retained in all our dMMR cases, which is in line with other studies investigating the exact pattern of MMR expression in dMMR cancer cases (3)(4)(5). Besides the rarely occurring immunohistochemical 'null-phenotype', the concurrent loss of MLH1/PMS2 and MSH6 expression seems to be the prevalent expression pattern in dMMR cases with an involvement of both MMR subsystems (3)(4)(5).
As the concurrent loss of immunoexpression of both MMR heterodimers has not been under much investigation yet, the reasons and the implications of this phenomenon remain mostly unclear. It is already known that neoadjuvant treated tumors can show a scanty MSH6 staining (4,40,41), which may explain the partial loss of MSH6 expression in case 5 of our cohort. Moreover, Shia et al. could impressively show that frameshift mutations in the C8 [NM_000179.2 (MSH6) c.3254_3261] tract of the MSH6 gene coding region are responsible for scanty MSH6 staining in dMMR cases (4). The same group could not detect this particular MSH6 exon 5 mutation in neoadjuvant treated pMMR and MSH6negative CRC cases (4), indicating a different mechanism behind the scanty MSH6 staining in pretreated pMMR CRCs. As the MSH6 gene contains a microsatellite of eight mononucleotide repeats (42), it is likely that a secondary somatic mutation in these microsatellites of the MSH6 gene is a potential mechanism for the complete or partial loss of MSH6 immunoexpression in already MMR-deficient cancers (4). The additional MMR/HRR mutations we could detect in our MLH1 -/PMS2 -/MSH6cases could play a mechanistic role in the loss of MSH6 expression in already MLH1/PMS2-deficient digestive system cancers by paving the way for further mutations in the MSH6 gene. We also could detect MSH6 exon 5 mutations in this specific coding region with an involvement of mononucleotide repeats, that have already been described by Shia et al. (4), in three out of our five cases (60%; cases 1, 3 & 5). We additionally evaluated the genetic content of our observed variants regarding mononucleotide repeats as repeats of single bases are the most abundant type of microsatellites in the human genome (43). This was particularly interesting as dMMR cancers are known to be hypermutated (44), and accumulation of mutations in microsatellites is the general main feature of dMMR/MSI-H cancers. Here, the mutations indeed show an involvement of microsatellites -but by far not in all cases (Table 4). This phenomenon was particularly pronounced in case 1. Together with the interpretation of the variants and their respective molecular consequence in Table 3, this evaluation with regard to the mononucleotide repeats complements the mutation profile of our MLH1 -/PMS2 -/MSH6cases. Together with the MLH1 promotor hypermethylation, the MSH6 mutation explains the concurrent MLH1/PMS2 and MSH6 loss in cases 1 and 4. Case 1 presented with an additional sporadic MLH1 mutation. Besides the MSH6 mutation, the neoadjuvant treatment in case 5 could be in some part responsible for the partial loss of MSH6 expression in this colon cancer case. In cases 2, 3 and 5, the staining pattern can only be partially explained by our molecular findings. In case 2, we could detect a germline mutation in MLH1, that is associated with Lynch Syndrome according to ClinVar. It is already known that ampullary cancers can arise in the setting of Lynch syndrome (45). However, the reported MLH1 missense mutation only explains for the simultaneous loss of PMS2 but not for the complete loss MSH6 expression. Even though we cannot clarify the loss of MSH6 expression in this case without detected MSH6 mutations or pre-treatment, it is already known that IHC findings do not always completely overlap with NGS results (46,47), which may be due to methodical issues like the used NGS panel or the amount of tumor tissue for NGS but also due to biologic reasons, such as epigenetic regulation of expression. The opposite phenomenon would be easier to explain: It is known that some MLH1 missense mutations lead to catalytically inactive but antigenically active proteins (48,49). In our subgroup, we could not identify this phenomenon.
To the best of our knowledge, we are the first group to specifically focus on the abnormalities in DNA repair of MLH1 -/ PMS2 -/MSH6digestive system cancer cases. Our study provides first evidence that digestive system cancers with this peculiar and rare immunophenotype display widespread dysregulation of DNA repair. Although MMR acts during DNA replication to correct polymerase errors and HRR is responsible for the constant repair of double-strand DNA breaks, these two mechanisms are closely linked to each other as the proteins involved in these processes overlap (25). Cells that have defects in MMR accumulate other mutations such as mutations in HRRrelated genes more easily, and vice versa. Therefore, it seems plausible that the loss of expression in multiple MMR proteins may be associated or even rely on more widespread defects in DNA repair. We could detect multiple mutations in HRRrelated genes with impressive high allele frequencies in our subgroup. That is why, drugs targeting DNA repair like PARP-inhibitors, that have already found their way into clinical practice in other cancer entities (26,27), seem to be a logic therapeutic strategy in our MLH1 -/PMS2 -/MSH6digestive system cancer cases as all five cases showed simultaneous mutations (up to 6) in MMR as well as HRR-related genes. We firmly believe working out subgroups likely to benefit from novel therapies, that are currently under investigation and aim to tackle HRR pathway components like ATM or CHEK1/2 (28)we could detect mutations in all underlying genes -, will be crucial. The successful off-label use of PARP inhibitors in a patient with an ATM-deficient CRC has already been described (50). As IHC for MMR is widely available, cost-effective and can give a first hint to a more widespread dysregulation of DNA repair according to our data, our results seem to be of potential clinical significance. The sequence variants we found in genes associated with DNA repair (Table 2) are already described in the corresponding cancer entities even though in rare frequency as shown in Table 5 and further supported by different studies (51-54). Therefore, the potential clustering of such hopefully soon to be treatable mutations in MLH1 -/PMS2 -/MSH6cases is even more noteworthy. Even though it is already known that dMMR CRCs show a distinct gene mutation profile compared to pMMR CRCs harboring significantly more often BRCA2 or ATM mutations (55), which we also could observe in our MLH1 -/PMS2 -/MSH6cases, we could detect further mutated HRR genes such PALB2, RAD51D or RAD54L. These HRR gene mutations, whichas far as we knowhave not been linked to dMMR cases previously, seemingly even clustered in our peculiar subgroup. Furthermore, it is known that MSI-H CRCs harbor significantly more mutations than MSS CRCs (44). However, some of our observed mutations have not been linked to dMMR CRCs yet (53,55) and we could detect up to six synchronous MMR/HRR mutations in our MLH1 -/PMS2 -/MSH6cases, which is something we rarely observe in clinical practice in other cancer entities. Therefore, we believe our findings suggest MLH1 -/PMS2 -/MSH6cases to be a potential special subgroup that that is biologically distinct from dMMR cases with a loss of expression in only one functional heterodimer.
Whereas much is known about the occurrence of germline pathogenic variants in MMR genes in the setting of LS [see case 2, MLH1 (NM_000249.3); Exon 16; c.1745T>C], there is recent evidence that also other cancer susceptibility genes, many of them associated with DNA repair, harbor germline variants in individuals affected by CRCs (51, 52, 56). In case 3, we could observe a pathogenic FANCA germline variant as well as a VUS in the ATM gene, which are both known to be associated with CRC cases (51, 52,56). Interestingly, VAFs of the observed germline variants indicate that the MLH1 pathogenic variant (VAF: 81%) is likely a loss of heterozygosity (LOH) event, while the ATM VUS (VAF: 50%) and FANCA pathogenic variant (VAF: 52%) do not show evidence of LOH. Yurgelun et al. could show in their large cohort study that more than 6.5% of CRC patients harbor pathogenic germline variants in non-LS cancer susceptibility genes, of which many are involved in DNA-repair processes (52). Even though we only could observe one MLH1 -/PMS2 -/ MSH6case with such pathogenic germline variant in a non LSrelated gene (case 3, FANCA), it would be highly interesting to correlate these germline variants with immunohistochemical MMR expressions. After all, patients with pathogenic germline variants in DNA-repair related genes are more likely to accumulate additional mutations in MMR proteins, which could eventually lead to a loss of expression in both MMR subsystems. Unfortunately, due to the still relative low occurrence of such germline variants, large numbers of CRC cases, that undergo extensive multigene panel testing as well as immunohistochemical staining for all four MMR proteins, would be extremely beneficial to study this relationship. Such pathogenic germline variants in HRR genes like BRCA1 should always be considered especially in young CRC cancer patients (57). However, all our patients with MLH1 -/PMS2 -/MSH6cancers were over 60 years of age at time of diagnosis and did not have a familiar history suspicious for cancer predisposing syndromes. Therefore, germline testing would not have been performed outside for research use in these cases.
Our findings seem to be promising considering that dMMR/ MSI cancers benefit from IO-therapy and as there is emerging evidence that combining PARP-inhibitors with IO-therapy is reasonable, especially in metastatic disease when there are often few other therapeutic options available: PARP-inhibitors induce double-strand breaks, which promotes neoantigen generation and tumor mutational burdenboth enhancing the impact of IOtherapy (58). Furthermore, it has been already shown in a Chinese cohort that CRC patients with HRR mutations show a better survival upon IO-therapy than HRR-wildtype CRC patients (53). To exemplify this idea with regards to our cohort: Case 4 benefits greatly from Pembrolizumab therapy at the moment. In case of tumor progress, the combination of PARP-inhibitors like Olaparib with Pembrolizumab could be a promising therapeutic approach considering the detected PALB2 mutation in this case because response to PARP inhibition has already been reported for PALB2mutated breast cancer (59). By evaluating pathogenic HRR mutations among different cancer entities in primary as well as metastatic lesions, the already cited study by Heeke et al. could show that most HRR gene mutations displayed either similar (e.g. CHEK2, PALB2) or even higher frequencies (e.g. BRCA1/2) in metastatic than in primary lesions (54). Additionally, responsiveness to PARP-inhibitors has already been shown for CRC cell lines as well as patient-derived organoids (60). This further supports the potential future role of combining IO-therapy with PARP inhibitors in a metastatic setting in MMR/HRRmutated digestive tract cancers. An interesting still remaining question is whether or not cancers with multiple HRR mutations as our MLH1 -/PMS2 -/MSH6cases are more likely to benefit from PARP-inhibitor therapy than those with only a single HRR mutation. Considering the biologic mechanism of action of PARP-inhibitors, this is indeed conceivable. Apart from that, it should not be neglected that HRR-deficient cancers show an increased sensitivity to conventional DNA-damaging chemotherapy (61,62), which are often part of the therapeutic strategy in metastatic digestive system cancers depending on the molecular alterations of the tumor as well as patient's general condition.
The authors are aware of some methodical issues and limitations within this work, namely the absence of copy number variant (CNV) germline analysis for MMR genes and the possibility of mosaicism for some MMR variants. Additionally, non-coding germline or somatic MMR variants that are not covered by the used NGS panel would be also of scientific and potentially clinical interest. Further studies performing CNV analysis as well as whole exome or even whole genome sequencing of MLH1 -/ PMS2 -/MSH6digestive system cancers are necessary to fully understand the mutational landscape of these cases.
Of course, our findings are limited by the rarity of MLH1 -/ PMS2 -/MSH6digestive system cancers. It would be highly interesting to study more cases in larger cohorts to confirm our findingsespecially the accumulation of mutations in HRR genes. Larger case numbers would also be very important to check whether the level of mutations remains stable, as even in our small cohort we found a large range regarding the amount of mutations (two to six simultaneous mutations in HRR genes)indicating that our subgroup is a heterogenous group as well. Considering the high incidence of cancers arising in the digestive system (19) and the frequency of 1.4% of MLH1 -/PMS2 -/MSH6cases across the included entities, a considerable number of patients could fall into this subgroup overall.
If further studies with larger cohorts can prove the clustering of mutations in DNA-repair related genes in MLH1 -/PMS2 -/MSH6digestive system cancers, this peculiar immunophenotype could immediately trigger additional NGS-testing for HRR mutations as basis for therapy. NGS-based multigene testing is, as far as we know, not routinely performed for all digestive system cancer patients but only occasionally in the specialized setting of molecular tumorboards or in case of suspected hereditary tumor predisposition syndromes. As such extensive multigene testing is still time-as well cost-consuming, a preselection by IHC seems extremely valuable and could be easily implemented into the diagnostic workflow.
To sum up, our study gives first evidence for MLH1 -/PMS2 -/ MSH6digestive system cancer cases to be a rare but extremely interesting subgroup that is easy detectable by IHC and may be associated with widespread dysregulation of DNA repair.

Data availability statement
Further information on the datasets can be obtained from the corresponding author on reasonable request and in restricted form due to privacy reasons. However, all relevant data generated during this study is included in this article.

Ethics statement
The studies involving human participants were reviewed and approved by the institutional review boards of the Ludwig Maximilian University of Munich (reference: 22-0381). (Chair: Prof. Dr. R. M. Huber Telefon+49 (0)89 440055191 Telefax +49 (0)89 440055192 Ethikkommission@ med.uni-muenchen.de www.ethikkommission.med.uni-muenchen.de, Anschrift: Pettenkoferstr. 8a D-80336 München). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.