Romain Cohen

Alex Duval

Magali Svrcek

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

We thank Jian Xu and colleagues for their interest in our work. We totally agree that, besides 3 misdiagnosing of the MSI/dMMR status, specific mechanisms including those described by 4 Xu et al. may underlie primary resistance of true MSI tumors to immunotherapy. Although it 5 is not per se a resistance mechanism, it is likely that the majority of primary resistance to 6 immunotherapy results from such misdiagnoses in clinical trials today. From a clinical point 7 of view, the medical oncologists, pathologists and molecular biologists must be aware of this, 8 so that patients experiencing immediate disease progression after ICKi initiation should be 9 quickly re-assessed for their MSI/dMMR status, even more so since ICKi might be continued 10 potentially until the next radiological evaluation (with the hypothesis of a pseudo-11 progression). 12 Routine double mismatch repair testing for checkpoint inhibitors. Authors's reply 1 Kotoula and colleagues express concern about the small sample size of our study and also the 2 financial cost of a routine double MSI/dMMR status screening using both 3 immunohistochemistry (IHC) and PCR. 4

Though our findings were also based on the screening of a large retrospective tumor 5 collection of MSI colorectal cancers, we totally agree that they have to be further confirmed 6 in the future due to the small size of the study population we prospectively examined in this 7 study. 8

It is true that MSI/dMMR misdiagnosis was mainly related to misinterpretation of pentaplex 9 PCR rather than IHC in this study. Nevertheless, we would like to respectfully remind 10 Kotoula and colleagues that IHC alone has been shown to be associated with several pitfalls 11 (for review see 1 ). Consistent with the literature 2 , we have observed in our cohort 12 approximately 5% of false negatives by IHC, probably related to false sense mutations which 13 did not lead to total inactivation of the corresponding MMR protein in cancer cells. 14

Considering the major and durable clinical benefit of immune checkpoint inhibitors for 15 patients with MSI cancer, we do believe that a 5% false-negative rate is ethically not 16 acceptable and that hence dual screening with both IHC and pentaplex PCR is absolutely 17 necessary. The complementarity of IHC and pentaplex PCR to reach maximum sensitivity 18 and specificity have been already well documented in the literature. 1 The cost of a combined 19 analysis is of course higher, and we agree that this point has to be taken into consideration, 20 especially as the screening of non-metastatic patients may become more and more 21 indispensable also in pan-cancer in the future. 22

Regarding HSP110 T17 assay, we show here (and in previously published studies 3 ) that it is 23 a highly sensitive, helpful microsatellite marker. One might use it as a third confirmatory 24 assay specifically for cases with discrepant results between IHC and pentaplex PCR with a 1 very small extra cost (5 to 10% of all tested specimen). An other possibility would be to 2 include this marker directly in the routine tested panel with almost no additional cost. In any 3 case, the strategy will remain cheaper than commercial next-generation methods that have 4 recently obtained FDA approval for MSI testing and other actionable therapeutic targets 4

*

  Université, INSERM, Unité Mixte de Recherche Scientifique 938 and SIRIC 6 CURAMUS, Centre de Recherche Saint-Antoine, Equipe Instabilité des Microsatellites et 7 Cancer, Equipe labellisée par la Ligue Nationale contre le Cancer, F-75012 Paris, France 8 2: Sorbonne Université, department of medical oncology, AP-HP, hôpital Saint-Antoine, F-9 75012 Paris, France 10 10: Sorbonne Université, department of pathology, AP-HP, hôpital Saint-Antoine, To whom correspondence should be addressed. Tel: +33 1 49 28 66 80 ; Fax: +33 1 49 28 14 28 78 ; Email: alex.duval@inserm.fr 15 Alex Duval, Team 'Microsatellite Instability and Cancer', UMRS938 -Centre de Recherche 16 Saint-Antoine, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75012 Paris, 17 France; email: alex.duval@inserm.fr 18 Manuscript word count: 149 words + 334 words =

  Buhard O, Lagrange A, Guilloux A, et al. HSP110 T17 simplifies and improves the 13 microsatellite instability testing in patients with colorectal cancer. J Med Genet. 14 2016;53:377-384. doi:10.1136/jmedgenet-2015-103518 15 4. Prasad V. Why the US Centers for Medicare and Medicaid Services (CMS) should have 16 required a randomized trial of Foundation Medicine (F1CDx) before paying for it. Ann 17 Oncol. 2018;29(2):298-300. doi:10.1093/annonc/mdx786 18

	5	.
	6
	7	1. Svrcek M, Lascols O, Cohen R, et al. MSI/MMR-deficient tumor diagnosis: Which
	8	standard for screening and for diagnosis? Diagnostic modalities for the colon and other
	9	sites: Differences between tumors. Bull Cancer (Paris). February 2019.
	10	doi:10.1016/j.bulcan.2018.12.008
	11	2. Hampel H, Frankel WL, Martin E, et al. Screening for the Lynch syndrome (hereditary
	12	nonpolyposis colorectal cancer). N Engl J Med. 2005;352(18):1851-1860.
		3.