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Table of Contents
Year : 2021  |  Volume : 4  |  Issue : 1  |  Page : 49-55

Kaleidoscope beyond the microscope: Colorectal cancer

1 Department of Laboratory, Molecular and Transfusion Services, Rajiv Gandhi Cancer Institute and Research Center, New Delhi, India
2 Department of Pathology, Rajiv Gandhi Cancer Institute and Research Center, New Delhi, India
3 Department of Molecular Diagnostics, Rajiv Gandhi Cancer Institute and Research Center, New Delhi, India

Date of Submission03-Jun-2021
Date of Acceptance05-Jun-2021
Date of Web Publication31-Jul-2021

Correspondence Address:
Dr. Divya Bansal
Department of Pathology, Rajiv Gandhi Cancer Institute and Research Center, New Delhi.
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jco.jco_21_21

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How to cite this article:
Mehta A, Bansal D, Kumar D. Kaleidoscope beyond the microscope: Colorectal cancer. J Curr Oncol 2021;4:49-55

How to cite this URL:
Mehta A, Bansal D, Kumar D. Kaleidoscope beyond the microscope: Colorectal cancer. J Curr Oncol [serial online] 2021 [cited 2023 May 27];4:49-55. Available from: http://www.https://journalofcurrentoncology.org//text.asp?2021/4/1/49/322892

  Case Presentation Top

A 53-year-old male, chronic smoker and an alcoholic, presented with weakness, history of bleeding per rectum, and weight loss since three months. There was a family history of endometrial cancer (EC) in the mother (who died 20 years ago), gastric cancer in the elder sister (who died eight years ago), and urothelial cancer in the elder brother (who died five years ago). Routine lab investigations revealed severe anemia, and stool for the occult blood test was positive. CEA and CA19.9 were within normal limits. Contrast-enhanced computed tomography (CECT) whole abdomen revealed circumferential thickening, heterogeneously enhancing soft tissue lesion measuring 5.6 × 5.2 × 4.3 cm in the cecum with mild perilesional fat stranding, and a few local subcentimetric nodes. A clinicoradiological diagnosis of carcinoma cecum was made. No significant abnormality was noted on CECT thorax and ultrasonography (USG) abdomen.

The patient underwent right hemicolectomy, which revealed a circumferential, gray white tumor in the cecum measuring 4.5 × 3.8 × 1.6 cm. A single sessile polyp measuring 0.5 cm in the greatest dimension was also identified at the ileocecal junction. Histopathological examination revealed a tumor with poorly differentiated histology (solid with <50% gland formation) invading muscularis propria [Figure 1]A. The tumor cells were positive for CK20 and Special AT-rich sequence-binding protein 2 (SATB2) [Figure 1]B. Tumor budding score was low in one hotspot field. No lymph node metastasis, tumor deposits, and lymphovascular and perineural invasion were seen. The polyp at the ileocecal junction revealed features of villous adenoma with high-grade dysplasia [Figure 1]C.
Figure 1: The scanner view shows a poorly differentiated adenocarcinoma of the colon with a predominant solid pattern of growth and pushing margins. No “dirty or garland necrosis” was observed (A). Tumor cells show diffuse positivity for special AT-rich sequence-binding protein 2 (SATB2) (B). Villous adenoma with high-grade dysplasia (C). The advancing edge of the adenocarcinoma shows peritumoral rosary bead-like lymphoid infiltrate (D). Lymphocytes infiltrating between tumor cells are counted as tumor infiltrating lymphocytes (E and F). Also note a low tumor budding score

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  Q1. What Is Your Diagnosis and Pathologic Staging on the Basis of Clinicoradiological Features and Laboratory Investigations? Top

Answer: a) Poorly differentiated adenocarcinoma, cecum

b) Pathologic staging: pT2N0

Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of all cancer-related deaths as per Global Cancer Incidence, Mortality and Prevalence (GLOBOCAN) 2018.[1] The role of pathologists is not only to provide accurate diagnosis, but also to assess pathologic staging, surgical margins, and prognostic parameters such as lymphovascular invasion, perineural invasion, tumor deposits, and tumor budding.

  Q2. Any Interesting Feature(s) You Noted in Histopathological Examination? Top

Answer: a) Peritumoral rosary bead-like (Crohn like) lymphoid infiltrate [Figure 1D]

b) Tumor had pushing margins with marked tumor infiltrating lymphocytes (TILs) [Figure 1E] and F].

The pathologists play an important role in looking for histopathological features that are suggestive of microsatellite instability (MSI), selection of tumor sections for molecular testing, and interpreting their results. Certain histologic variants such as mucinous, medullary, and signet ring cell carcinoma can be associated with high MSI (MSI-H) and hence, behave as low-grade biologically.[2] The TILs and Crohn-like lymphoid reaction can be also associated with MSI-H and a better outcome.[2] It is noted that in the updated cancer protocols and checklists by the College of American Pathologists, the reporting of TILs has been removed.

  Q3. From a Family History of Multiple Affected First-degree Relatives and Histopathological Features, Which Syndrome Would You Think of? Top

Answer: Lynch syndrome (LS)

As per the affected case and genealogical tree of the family [Figure 2] (four family members affected, high penetrance, autosomal dominant), it gives an impression of hereditary cancer syndrome. Approximately 70%–80% of CRC are sporadic cancers, whereas 20%–30% have genetic factors that are responsible for them.[3] LS and familial adenomatous polyposis are the two most common culprits for hereditary CRC, accounting for 3%–4% and nearly 1%, respectively, of the total CRC burden.[4]
Figure 2: Genealogical tree of the family showing a case of colorectal cancer (proband marked with black arrow head). Note three of first- degree relatives being affected by various cancers and succumbed to the disease

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LS (formerly called hereditary nonpolyposis CRC) is possibly the most common of all hereditary cancer syndromes, and it is transmitted in an autosomal dominant manner with a potentially high penetrance.[5] The LS is characterized by predisposition to cancers in a variety of organs, including colorectum, endometrium, ovaries, stomach, small bowel, urinary tract, hepatobiliary tract, prostate, breast, brain (usually glioblastoma), skin (sebaceous adenomas, sebaceous carcinomas, and keratoacanthomas), and pancreas.[5] LS is divided into two types: LS I (site-specific colon cancer) and LS II (extracolonic cancer).[6] The patients with LS are also at an increased risk of developing synchronous and metachronous cancers.[5]

Usually, the CRC in LS occurs at a younger age (<50 years of age), on the right side of the colon and preceded by a single/few colonic villous adenoma.[7] The tumors in LS show commonly histopathological features that are suggestive of MSI-H (as described earlier), but they are not specific enough to distinguish from microsatellite-stable (MSS) cases.[2]

LS is caused by a germline loss-of-function mutations in one of the pathogenic variants of DNA mismatch repair (MMR) genes MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), and PMS1 Homolog 2 (PMS2) or a deletion in the 3′ region of the EpCAM gene.[8] The National Comprehensive Cancer Network recommends criteria that can be used to select patients for evaluating LS under three domains, as shown in [Table 1].[9]
Table 1: Criteria to select patients for evaluating LS

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  Q4. What Next Investigation Will You Advise? Top

Answer: a) Universal tumor screening for MMR deficiency

b) The MMR status can be determined by two methods: immunohistochemistry (IHC) for MMR proteins on tumor tissue or by MSI testing on tumor DNA.

The relatively low performance of clinical screening criteria (Amsterdam criteria 1, Amsterdam criteria 2, Bethesda guidelines, and revised Bethesda guidelines) and clinical prediction models (MMRpredict, MMRpro, and Prediction Model for Gene Mutations) for LS have led to an alternative strategy “universal tumor screening for Lynch syndrome” for all newly diagnosed CRC or EC by MSI testing or IHC for MMR proteins.[9],[10] This screening method is not only cost-effective but also has a sensitivity of 100% and specificity of 93%.[8] However, there is no consensus as to whether MMR by IHC or MSI is the preferred test, and they can be used in combination.

MMR genes (MLH1, PMS2, MSH2, and MSH6) code for proteins that identify and correct DNA mismatches, which occur especially in microsatellites during replication. Defective MMR status in tumors leads to the accumulation of replication errors in microsatellites called as MSI. More than 90% of patients with LS are MMR-deficient (MMR-D)/MSI.[11] The MMR deficiency testing is an important test not only for LS screening but also for prognosis, decision making of adjuvant chemotherapy in stage II CRCs. Recently, the use of immune-checkpoint inhibitor therapy in chemoresistant metastatic MMR-D/MSI tumors has shown groundbreaking success.[12]

The MMR by the US Food and Drug Administration approved Ventana IHC testing was performed on tumor tissue sections in our case [Figure 3]. The MSI testing of tumor tissue was performed by fluorescent polymerase chain reaction (PCR)–based assay by using Promega MSI Analysis System (Promega Corporation, USA) and capillary electrophoresis resolution using an Applied Biosystems SeqStudio Genetic Analyzer (ThermoFisher Scientific, USA) [Figure 4].
Figure 3: Immunohistochemistry of mismatch repair (MMR) proteins. MMR-deficient tumor with loss of (A) MLH1 and (B) PMS2 nuclear expression in tumor cells. Note the intact nuclear expression in stromal cells and lymphocytes that acts as an internal control. MSH2 (C) and MSH6 (D) showed intact nuclear expression in tumor cells. MLH1 = MutL homolog 1, MSH2 = MutS homolog 2, MSH6 = MutS homolog 6, PMS2 = PMS1 Homolog 2

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Figure 4: A microsatellite-stable profile of five stable mononucleotide repeats using Promega microsatellite instability (MSI) analysis system (A). MSI profile with five unstable mononucleotide repeats indicated by red arrows (B)

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  Q5: How Do You Interpret MMR and MSI Results? Top

Answer: a) The tumor was MMR-D with loss of both MLH1 and PMS2 expression [Figure 3].

b) The tumor was MSI-H, with five of five microsatellites being unstable [Figure 4].

A tumor is MMR-D[13] if IHC shows loss of nuclear expression in any one of the proteins of MMR genes and implies that an inherited pathogenic variant may be present in the abnormal gene [Table 2]. A normal IHC pattern with intact nuclear expression of all four proteins of MMR genes is called MMR-proficient. Nuclear staining of lymphocytes, stromal cells, and epithelial cells is considered a positive internal control.
Table 2: Interpretation of IHC staining of mismatch repair proteins

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For MSI testing,[13] microsatellite loci, either mononucleotides or a combination of dinucleotides and mononucleotides repeats, are analyzed by multiplex PCR or by next-generation sequencing (NGS) more recently. Five microsatellites are usually tested by the National Cancer Institute panel comprising two mononucleotide loci (BAT-25 and BAT-26) and three dinucleotide loci (D2S123, D5S346, and D17S250) or Promega panel comprising five mononucleotides (BAT-25, BAT-26, NR-21, NR24, and NR27) and two pentanucleotide loci (Penta C and Penta D for specimen identification). The quasi-monomorphic nature of these microsatellites facilitates their assessment. Instability in one locus is termed MSI-low, and instability in ≥2 loci is termed MSI-H.

  Q6. How to Proceed Further with MMR-D/MSI-H? Top

Answer: a) BRAF V600E mutation analysis and MLH1 promoter methylation studies.

b) If negative, germline mutation testing for LS is performed.

MMR-D/MSI-H CRC tumors are seen in LS and sporadic tumors of CpG island methylator phenotype (CIMP; 15%). The CIMP-associated CRCs are due to sporadic somatic biallelic hypermethylation of MLH1 gene promoter, via the serrated pathway.[14] Nearly 75% of CIMP CRC is associated with BRAF V600E mutation, which can be used to distinguish them from LS-associated CRCs.[14] In cases with defective MMR and BRAF wild-type status, MLH1 methylation study is conducted to determine the sporadic trait of CRC. Thus, additional tumor testing for somatic BRAF V600E mutation analysis and MLH1 promoter methylation should be done in MSI-H/MMR-D with lack of expression of both MLH1 and PMS2 to rule out sporadic CRC.

If there is no hypermethylation of MLH1 gene promoter, the germline mutation testing for LS is conducted by NGS assays, which detect pathogenic MMR variants. When no point mutation is detected, it is recommended to use methods that identify large genomic rearrangements such as multiplex ligation-dependent probe amplification.[13]

It should be noted that individuals with MMR-D/MSI-H (without BRAF V600E mutation or MLH1 hypermethylation) who do not have germline pathogenic MMR variants are termed as having Lynch-like syndrome.[15] They reveal biallelic somatic mutations in MMR genes.[15]

Subsequent BRAF V600E mutation and MLH1 methylation studies were negative in our case; hence, germline DNA MMR gene mutation testing for LS was carried out by NGS using Ion Ampliseq plus libraries with 160 amplicons (in two pools 81 and 79). The result is shown in [Figure 5].
Figure 5: The integrative genomics viewer image of MLH1 nucleotide sequencing reads of proband. Mutated nucleotide (c.2041G>A) is shown in green color, whereas the nucleotide sequence and coding amino acids of wild-type MLH1 are shown at the bottom

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  Q7: Which Gene DID the Patient Have Germline Mutation in and What Is Your Final Diagnosis? Top

Answer: Pathogenic mutation was detected in the MLH1 gene; hence, a final diagnosis of LS was established.

A germline missense variant in the MLH1 gene annotated as c.2041G>A, p.Ala618Thr at a variant allele fraction of 53.29% was revealed by NGS. This was predicted as pathogenic as per American College of Medical Genetics and Genomics (ACMG) guidelines[16]; hence, a final diagnosis of LS was established in the patient.

The patients with LS have a risk of developing CRC by age 70 for 40%–50% for MLH1 and MSH2 carriers, 20% for MSH6 carriers, and even lower for PMS2 carriers.[17] For EC in patients with LS, the risk by age 70 is 35%–46% for MLH1, MSH2, and MSH6, and it is 13% for PMS2.[17]

There are emerging studies on direct NGS germline testing panels for all patients with CRC regardless of their tumor screening results.[18] These tests are widely available, are cost-effective, facilitate the detection of CRC predisposition syndrome beyond LS, and offer opportunities for personalized medicine.

  Q8. What Would You Do Next After Identifying LS in a Patient? Top

Answer: Genetic counseling and predictive testing if desired for at-risk family members.

Identifying LS is critical for optimizing the clinical management and future risk for individuals with CRC (for recurrences/second cancer) by cancer surveillance and prevention strategies. When the LS pathogenic variant is found in the patient, predictive (site specific) testing can be done for at-risk family members after genetic counseling. It offers an opportunity for the early detection and prevention of cancer in an LS gene mutation carrier in the family. A surveillance scheme for LS gene mutation carriers is summarized in [Table 3].[9]
Table 3: Surveillance schemes for LS gene mutation carrier

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  Conclusion Top

The identification and management of individuals and families with LS has evolved and continues to rapidly improve. The diagnosis of LS is not only important to reduce the cancer-associated morbidity and mortality but it also offers innovations in treatment, such as immunotherapies. MMR/ MSI testing is important not only for LS screening but also for therapeutic management, as a prognostic and predictive biomarker. Recently, NGS has revolutionized the diagnosis of LS by facilitating both MSI screening and direct germline multigene testing. However, there is also a need to focus on the identification of LS in healthy cancer-free individuals apart from individuals with newly developed tumors.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394-424.  Back to cited text no. 1
Shia J, Schultz N, Kuk D, Vakiani E, Middha S, Segal NH, et al. Morphological characterization of colorectal cancers in The Cancer Genome Atlas reveals distinct morphology–molecular associations: Clinical and biological implications. Modern Pathology 2017;30:599-609.  Back to cited text no. 2
Müller MF, Ibrahim AE, Arends MJ. Molecular pathological classification of colorectal cancer. Virchows Arch 2016;469:125-34.  Back to cited text no. 3
Rustgi AK. The genetics of hereditary colon cancer. Genes Dev 2007;21:2525-38.  Back to cited text no. 4
Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP. Milestones of Lynch syndrome: 1895-2015. Nat Rev Cancer 2015;15:181-94.  Back to cited text no. 5
Lynch HT, Schuelke GS, Kimberling WJ, Albano WA, Lynch JF, Biscone KA, et al. Hereditary nonpolyposis colorectal cancer (Lynch syndromes I and II). II. Biomarker studies. Cancer 1985;56:939-51.  Back to cited text no. 6
Lynch HT, Lynch JF, Shaw TG, Lubiński J. HNPCC (Lynch syndrome): Differential diagnosis, molecular genetics and management—A review. Hered Cancer Clin Pract 2003;1:7-18.  Back to cited text no. 7
Sehgal R, Sheahan K, O’Connell PR, Hanly AM, Martin ST, Winter DC. Lynch syndrome: An updated review. Genes (Basel) 2014;5:497-507.  Back to cited text no. 8
NCCN clinical practice guidelines in oncology: Genetic/familial high risk assessment: Colorectal. Version1.2021. https://www.nccn.org/professionals/physician_gls/pdf/genetics_colon.pdf. Published May 11, 2021. Accessed May 30, 2021.  Back to cited text no. 9
Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, et al. Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 2008;26:5783-8.  Back to cited text no. 10
Silva FC, Valentin MD, Ferreira Fde O, Carraro DM, Rossi BM. Mismatch repair genes in Lynch syndrome: A review. Sao Paulo Med J 2009;127:46-51.  Back to cited text no. 11
Battaglin F, Naseem M, Lenz HJ, Salem ME. Microsatellite instability in colorectal cancer: Overview of its clinical significance and novel perspectives. Clin Adv Hematol Oncol 2018;16:735-45.  Back to cited text no. 12
Evrard C, Tachon G, Randrian V, Karayan-Tapon L, Tougeron D. Microsatellite instability: Diagnosis, heterogeneity, discordance, and clinical impact in colorectal cancer. Cancers (Basel) 2019;11:1567.  Back to cited text no. 13
Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, et al. Cpg island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006;38:787-93.  Back to cited text no. 14
Geurts-Giele WR, Leenen CH, Dubbink HJ, Meijssen IC, Post E, Sleddens HF, et al. Somatic aberrations of mismatch repair genes as a cause of microsatellite-unstable cancers. J Pathol 2014;234:548-59.  Back to cited text no. 15
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405-24.  Back to cited text no. 16
Dominguez-Valentin M, Sampson JR, Seppälä TT, Ten Broeke SW, Plazzer JP, Nakken S, et al. Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: Findings from the prospective Lynch syndrome database. Genet Med 2020;22:15-25.  Back to cited text no. 17
Pearlman R, Frankel WL, Swanson B, Zhao W, Yilmaz A, Miller K, et al; Ohio Colorectal Cancer Prevention Initiative Study Group. Prevalence and spectrum of germline cancer susceptibility gene mutations among patients with early-onset colorectal cancer. JAMA Oncol 2017;3:464-71.  Back to cited text no. 18


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3]


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  In this article
Case Presentation
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Q4. What Next In...
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