Human papillomavirus in spontaneous abortion

CORRESPONDENCE A n i m p o r t a n t f i n d i n g o f o u r a n d previous studies ~-~ is that, as a rule, t u m o r s s h o w i n g p53 g o n e m u t a t i o n s do n o t show MI a n d vice versa. I n a d d i t i o n , t u m o r s with MI have b e e n suggested to differ f r o m t h e r e m a i n i n g gastric t u m o r s in various clinical a n d h i s t o p a t h o l o g i c a l aspects, i n c l u d i n g m o r e p r o m i n e n t l y m p h o i d infiltrate, lower D N A index, a n d lower p r e v a l e n c e o f l y m p h n o d e metastases. 6 Thus, they m a y repres e n t a distinct g e n e t i c as well as c l i n i c o p a t h o l o g i c subset o f t u m o r s , distinct, anyway, f r o m t h e p53 m u t a t e d subset. F r o m o u r f i n d i n g s it a p p e a r s t h a t a loss o f g l a n d u l a r s t r u c t u r e d u r i n g t u m o r p r o g r e s s i o n m a y affect t h e p53 positive subset; w h e t h e r this process also involves t h e MI positive subset it r e m a i n s to b e a s c e r t a i n e d . Certainly, m u c h work r e m a i n s to b e d o n e ( i n c l u d i n g b e t t e r s t a n d a r d i z a t i o n o f t e c h n i q u e s for MI assessment a n d o f criteria for t h e i d e n t i f i c a t i o n o f t h e R E R + status) b e f o r e we f o r m a l l y r e c o m m e n d to use t h e MI status in r o u t i n e clinical work. However, we believe t h a t in r e s e a r c h work analysis o f MI as well as p53 a n d c a d h e r i n E 7,s status o f glandular, diffuse, or m i x e d cancers in b o t h early a n d a d v a n c e d stage is i m p o r t a n t for u n d e r s t a n d i n g t h e n a t u r a l h i s t o r y o f gastric cancer.

classification o f gastric c a n c e r s or subtypes for e i t h e r clinical routine or research purposes.

RUGGE,MD Cattedra di Istochimica & Immunoistochimica Patologica Universit~t degli Studi di Padova Padova, Italy

MASSIMO

YIH-HORNG SHIAO, P H D Laboratory of Comparative NCI-FCRDC, NIH Frederick, MD

Carcinogenesis,

MARIA GUIDO, M D Cattedra di Istochimica & Immunoistochimica Patologica Universit/t degli studi di Padova Padova, Italy

DANIELABOVO, P H D

OMBRETTA LUINETTI, M D ROBERTO FIOCCA, P H D LAURA VILLANI, M D

Departimento di Pediatria Universit5 degli studi di Padova Padova, Italy 1. Luinetti O, Fiocca R, Villani L, et al: Genetic pattern, histological structure, and cellular phenotype in early and advanced gastric cancers: Evidence for structure-related genetic subsets and for loss of glandular structure during progression of some tumors. HUMPATHOL29:702-709, 1998 2. Chung YJ, Song JM, Lee JY, etal: Microsatellite instability-associated mutations associate preferentially with the intestinal type of primary gastric carcinomas in a high-risk population. Cancer Res 56:4662-4665, 1996 3. dos Santos NR, Seruca R, Constancia M, et al: Microsatellite instability at multiple loci in gastric carcinoma: Clinicopathologic implications and prognosis. Gastroenterology 110:38-44, 1996 4. Ottini L, Palli D, Falchetti M, etal: Microsatellite instability in gastric cancer is associated with tumor location and family history in a high-risk population from Tuscany. Cancer Res 57:4523-4529, 1997 5. Lin JT, Wu MS, Shun CT, et al: Microsatellite instability in gastric carcinoma with special references to histopathology and cancer stages. Eur J Cancer 31A:1879-1882, 1995 6. Seruca R, Santos NR, David L, et al: Sporadic gastric carcinomas with microsatellite instability display a particular clinicopathologic profile. Int J Cancer 64:32-36, 1995 7. Wu MS, Lee CW, Shun CT, et al: Clinicopathological significance of altered loci of replication error and microsatellite instability-associatedmutations in gastric cancer. Cancer Res 58:1494-1497, 1998 8. Hayden JD, Martin IG, Cawkwell L, etal: The role of microsatellite instability in gastric carcinoma. Gut 42:300-303, 1998

Reply To theEditor:--My c o a u t h o r s a n d I a p p r e c i a t e t h e i n t e r e s t o f Dr. R u g g e a n d coworkers in o u r article o n histological a n d g e n e t i c p a t t e r n s o f gastric c a n c e r ) T h e y c o n f i r m o u r ( a n d o t h e r s ) f i n d i n g t h a t microsatellite instability (MI) is m o r e p r e v a l e n t a m o n g g l a n d u l a r (intestinal) t h a n a m o n g diffuse cancers. However, as they p o i n t out, t h e i r f i n d i n g o f MI in 8% of diffuse c a n c e r s contrasts with o u r negative results. It m u s t b e stressed t h a t i n s t e a d o f t h e classic L a u r e n classification o f gastric cancer, we u s e d a m o d i f i e d classification w h e r e a substantial p r o p o r t i o n o f t u m o r s fits i n a histologically " m i x e d " g l a n d u l a r / d i f f u s e g r o u p . I n o u r study 7% o f early a n d 14% o f invasive mixed cancers showed MI (as opposed to 19% a n d 38%, respectively, of glandular cases). It remains to b e ascertained w h e t h e r R u g g e ' s MI positive " d i f f u s e " t u m o r s w o u l d fit into o u r " p u r e l y diffuse" o r i n t o o u r " m i x e d " t u m o r g r o u p . T h e latter g r o u p has b e e n i n t r o d u c e d after C a r n e i r o et al 2 a n d o t h e r s p r o v i d e d s u b s t a n t i a l e v i d e n c e t h a t it c o r r e s p o n d s to a m o r e aggressive t u m o r entity with a worse clinical o u t c o m e .

PAOLAALBERIZZI,BSc ENPdCOSOLCt~, PHD Department of Human Pathology and Genetics University of Pavia and Pathology Service, IRCCS Policlinico San Matteo PHD Department of Genetics and Microbiology University of Pavia Pavia, Italy N A O ~ RANZANI,

1. Luinetti O, Fiocca R, Villani L, etal: Genetic pattern, histological structure, and cellular phenotype in early and advanced gastric cancers: Evidence for structure-related genetic subsets and for loss of glandular structure during progression of some tumors. HUMPATHOL29:702-709, 1998 2. Carneiro F, Seixas M, Sobrinho-Simoes M: New elements for an updated classification of the carcinomas of the stomach. Pathol Res Pract 191:571-584, 1995 3. Strickler JG, Zheng J, Shu Q, et al: p53 mutations and microsatellite instability on sporadic gastric cancer: When guardians fail. Cancer Res 54:45704755, 1994 4. Renault B, Calistri D, Buonsanti G, et al: Microsatellite instability and mutations of p53 and TGF-BRII genes in gastric cancer. Hum Goner 98:601-607, 1996 5. Buonsanti G, Calisui D, Padovan L, et al: Microsatellite instability in intestinal- and diffuse-typegastric carcinoma.J Patho1182:167-173, 1997 6. Dos Santos, Seruca R, Constancia M, et al: Microsatellite instability at multiple loci in gastric carcinoma. Clinicopathologic implications and prognosis. Gastroenterology 100:38-44, !996 7. Mayer B,JohnsonJP, Leitl F, eta!: E-cadherin expression in primary and metastatic gastric cancer: Down-regulation correlates with cellular dedifferentiation and glandular disintegration. Cancer Res 53:1690-1695, 1993 8. Guilford P, Hopkins J, HarrawayJ, et al: E-cadherin germline mutations in familial gastric cancer. Nature 392:402-404, 1998

Human Papillomavirus in Spontaneous Abortion To the Editor:--We r e a d with i n t e r e s t t h e r e c e n t r e p o r t o f H e r m o n a t a n d colleagues in Human Pathology e n t i t l e d "Trop h o b l a s t s are t h e p r e f e r e n t i a l t a r g e t o f h u m a n p a p i l l o m a virus (HPV) i n f e c t i o n in s p o n t a n e o u s l y a b o r t e d p r o d u c t s o f c o n c e p tion. ''1 U s i n g in situ p o l y m e r a s e c h a i n r e a c t i o n (PCR) amplific a t i o n for HPV-16 E6 g o n e sequences, this study c o m p a r e d f o u r t h i r d t r i m e s t e r p l a c e n t a s with six first t r i m e s t e r s p o n t a n e -

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ous abortions, and c o n c l u d e d that the syncytiotrophoblast of spontaneous abortions may contain HPV DNA. An earlier study by the investigators, using PCR amplification to HPV E 6 / E 7 j u n c t i o n sequences and D N A dot blot hybridization with an HPV-16 internal probe, 2 was the first to suggest that HPV may be a causative factor in s o m e s p o n t a n e o u s abortions. In this study, th e authors r e p o r t e d HPV D N A in 60% (16/25) of spontaneous abortions c o m p a r e d with 20% (3/15) of elective abortions. We w e r e intrigued by these reports, because we are interested b o t h in the pathogenesis of s p o n t a n e o u s a b o r t i o n and in the m o l e c u l a r diagnosis of HPV-related diseases.S 5 We therefore a t t e m p t e d to i n d e p e n d e n t l y verify a possible association between HPV and s p o n t a n e o u s abortion, using r e c e n t surgical pathological material f r o m o u r institution. Thirty consecutive first trimester spontaneous abortion specimens from February, 1998 were assessed by PCR for HPV D N A and the h u m a n globin gene. Paraffin-embedded, formalin-fixed placental tissues were used and the p r o c e d u r e s used a Combination of PCR a n d restriction f r a g m e n t p o l y m o r p h i s m analysis as previously described. 5 A m o n g the 30 cases studied, the globin g e n e was successfully amplified in 70% (21/30) of cases. Amplified sequences characteristic of HPV D N A were not identified in any case. A l t h o u g h the prospect of HPV infection in spontaneous a b o r t i o n is intriguing and worthy of investigation, o u r d a t a do n o t s u p p o r t the conclusions of H e r m o n a t et al that HPV is frequently p r e s e n t in spontaneous a b o r t i o n specimens. O u r findings indicate that HPV does n o t play a role in h u m a n spontaneous abortion. DAVID R. GENEsT, M D DEGIM SUN, M S CHRISTOPHER P. CRUM, M D Division of Wolnen's and Perinatal Pathology Department of Pathology Brigham and Women's Hospital Boston, MA

FIGURE 1. In situ PCR analysis of control and experimental tissue sections. (A) A photomicrograph of a representative section of HPV-positive condyloma specimen (positive control) which was been analyzed by in situ PCR using an L1 gene targeting primer set.~ Note that the upper layer of the tissue shows clear nuclear positivih/. (B) A photomicrograph of a representative section of a spontaneous abortion specimen by in situ PCR for LI sequences. This specimen has previously been shown to be HPV E6/E7 sequence positive. 1,2Note that a significant percentage of the syncytiotrophoblasts show nuclear positivity for L1 DNA.

1. Hermon~it PL, Kechelava S, Lowery CL, et al: Trophoblasts are the preferential target for human papilloma virus infection in spontaneously aborted products of conception. HUNPATHOL29:170-174, 1998 2. Hermonat PL, Han L, Wendel PJ, et al: Human papilloma virus is more prevalent in first trimester spontaneously aborted products of conception compared to electivespecimens.Virus Genes 14:13-7, 1997 3. Joste NE, Kundsin RB, Genest, DR: Histologyand Ureaplasma urealyticure culture in 63 cases of first trimester abortion. AmJ Clin Patho1102:729-732, 1994 4. Genest DR, Roberts D, Boyd T, et al: Fetoplacental histology as a predictor of karyotype: A controlled study of spontaneous first trimester abortions. HUNPATHOL26:201-209, 1995 5. Genest D, Stein L, Cibas E, et al: A binary (Bethesda) system for classifying cervical cancer precursors: Criteria, reproducibility, and viral correlates. HUMPATHOL24:730-736, 1993

Reply To the Editor:--We are pleased that o u r studies have stimulated interest in t h e potential association of h u m a n papillomaviruses (HPV) with s p o n t a n e o u s abortions and the infection of trophoblasts. O f course we are also c o n c e r n e d with t h e knowledge that Genest et al were unable to c o n f i r m o u r data. To briefly review our data, we have used b o t h E6-E7 j u n c t i o n and E6 targeting p r i m e r sets to identify HPV in 60% (15 of 25) of s p o n t a n e o u s a b o r t i o n tissues by polymerase chain reaction ( P C R ) / d o t blot and in situ PCR analysis. 1'2 Both of these studies were in agreement. Recently, however, it has b e c o m e a p p a r e n t that the results as to HPV-positivity

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m i g h t be somewhat d e p e n d e n t on the PCR p r i m e r sets and target region used. 3 It now appears that the LI sequences are the favored target for the PCR analysis of HPVs. Because of c o n c e r n s that we have n o t used the most appropriate HPV p r i m e r sets we have reanalyzed the original specimens using a well-characterized p r i m e r set targeting the L1 gene, 4 as well as using new p r i m e r sets targeting the E1 and E2 genes of our own design. We have g e n e r a t e d p r e l i m i n a r y data which have shown these same spontaneous abortion specimens to be positive for HPV E l , E2, and L1 sequences by in situ PCR. We have also g e n e r a t e d initial preliminary data by P C R / d o t blot hybridization which has shown L1 se%uence positivity in these same specimens. All o f these new data are largely consistent with o u r earlier published studies. An e x a m p l e of L1 positive trophoblasts, as shown by in situ PCR analysis, is shown in Fig 1. N o t only do these data f u r t h e r support o u r two earlier studies, they also suggest that the HPV g e n o m e s are intact and that the HPVs are likely present episomally. T h e r e are also o t h e r laboratories which support our conclusions. 5-s M a l h o m m e et al have, in fact, also ob-